‫المملكة العربية السعودية‬

Kingdom of Saudi Arabia

Imam Mohammad Ibn Saud Islamic University
‫جامعة اإلمام محمد بن سعود‬
‫اإلسالمية‬
College of Science
Department of Biology ‫كليـــــة العــــــــوم‬
‫قسم األحياء‬

The Role of Probiotic Bacteria in Reducing

the Side Effects of Antibiotics

By:

Name ID Number
Aljazi Ali Baharethh 443021485
Sarah Hani Altheban 443019607
Sadeem Saleh Alsharif 441021581
Norah Towayan Altowayan 443019169

A research project submitted for the requirements of the

Bachelor degree of Biology (499 BIO)

Supervised by:
Dr. Shaikha Albatli

I
 First Semester, 2025

II
 ‫بسم الله الرحمن‬
‫الرحيم‬

‫‪II‬‬
 Table of contents
Titel Page
List of Tables VII
List of Figures VIII
Acknowledgement IX
Abstract X
‫الخالصة‬ XII
List of Abbreviations XIII
List of Symbols XV
Chapter 1: Probiotic
Introduction 1
Aims of work 2
1.1 Structural Organization of Probiotic Cell 2
1.1.1 Cell Wall 2
[Link] Peptidoglycan 3
[Link] Teichoic acids 3
[Link] Polysaccharides 3
[Link] Proteins 3
1.1.2 Surface molecules 4
[Link] Surface layer proteins 4
[Link] Flagellin 4
[Link] Pili 4
[Link] Capsular polysaccharides 5
1.1.3 Cytoplasmic structures 5
1.1.4 Extracellular vesicles 5
1.2 Morphological characteristics of probiotics 6
1.3 Types of probiotic microorganisms 7
1.3.1 Lactic acids bacteria 7
1.3.2 non-lactic acid bacteria 8
1.3.3 Yeast 8
1.4 Natural habitats of probiotic microorganisms 9
1.4.1 Human intestines 9

III
 1.4.2 Fermented Foods 9
1.4.3 Biofilms 10
1.4.4 Pharmaceutical applications 10
1.5 Physiological roles and health benefits of probiotics 10
1.5.1 Strengthening the intestinal barrier 11
1.5.2 Competition with pathogenic microorganisms 12
1.5.3 Regulation of immune responses 12
1.5.4 Support of metabolic health 13
1.5.5 Prevention of other health issues 13
1.6 Consequences of probiotic imbalance 14
CHAPTER 2
2.1 Nutritional Factors (Prebiotics and Dietary Components) 16
2.1.1 Prebiotic fibers and oligosaccharides 16
2.1.2 Food matrix and dietary composition 17
2.1.3 Other dietary component 18
2.2 Environmental Factors (Temperature, PH, and Storage Condition) 19
2.2.1 Temperature 19
2.2.2 pH‍‌‍‍‌(Acidity) and digestive environment 19
2.2.3 Host-Related Factors 21
2.3Pharmacological Factors (Effect of Antibiotics and Other Medications) 22
2.3.1 Definition and Chemical Structcres 23
2.3.2 Mechanisms and General Relationship Between Antibiotics and Bacteria 26
CHAPTER 3: Antibiotic-Induced Dysbiosis and the Therapeutic Role of
probiotic in Mitigation Adverse Effects
3.1 The profound impact of Antibiotics on Gut Microbiota homeostasis 29
3.1.1 Antibiotic-induced Dysbiosis and loss of microbial diversity 31
[Link] Coeliac Disorder 32
[Link] Inflammatory Bowel Disease (IBDs) 32
[Link] irritable bowel syndrome (IBS) 33
[Link] Cancer 33
[Link] Neurodevelopmental, neurodegenerative and neurological disorders 34
3.1.2 Reduction of Endogenous Probiotic Populations 34

IV
 [Link] Adhesion to compete with pathogens 35
[Link] Maintenance of intestinal integrity 36
[Link] Tight junction 36
[Link] Mucin expression 37
[Link] Cell proliferation 37
[Link] Cell apoptosis 38
3.1.3 Alteration of Gut microbiome composition 39
[Link] Factors influencing the gut microbiome 40
[Link].1 Age 40
[Link].2 Diet 41
[Link] Functions of the microbiome 41
[Link].1 Metabolic function 42
[Link].2 Structural function 42
[Link].3 Protective function 43
3.2 Clinical Manifestations and Health Implications of Antibiotic-Associated
44
Dysbiosis
3.2.1 Antibiotic-Associated Diarrhea 45
3.2.2 Overgrowth of Opportunistic Pathogens 45
3.2.3 Immunomodulatory and Nutritional Consequences 46
[Link] Immune System Dysfunction 46
[Link] Nutrient Metabolism Alterations 50
3.2.4 Broader Health Implications of Gut Flora Disturbance 51
[Link] General Dysbiosis and Antimicrobial Resistance 52
[Link] Compromised Gut Barrier 53
[Link] Increased Risk of Secondary Infections 54
[Link] Chronic Diseases and Metabolic Disorders 54
[Link] Allergies and Asthma 55
[Link] Neurocognitive and Mental Health Issues 56
3.3 Therapeutic Strategies: The Role of Probiotics in Counteracting Antibiotic
58
Effects
3.3.1 Mechanisms of Probiotic Action in Antibiotic-Challenged Microbiota 58
[Link] Direct Pathogen Modulation and Competition 58
[Link] Restoration of Gut Ecosystem and Barrier Function 60

V
 [Link] Immune System Modulation 61
[Link] Metabolite Production and Cross-Feeding 63
3.3.2 Therapeutic Applications of Probiotics 64
[Link] Antibiotic-Associated and Clostridioides difficile Diarrhea 64
[Link] Eradication of Pathogenic and Antibiotic-Resistant Bacteria 65
[Link] Specific Conditions and Metabolic Influence 66
3.3.3 Factors Influencing Effectiveness and Future Directions 68
[Link] Probiotic Formulations and Administration 68
[Link] Context-Dependent Efficacy and Individual Variability 69
3.3.4 Evidence from Clinical Studies on Probiotic-Antibiotic Co-
70
administration
[Link] Overall Effectiveness and AAD Prevention 70
[Link].1 Specific Conditions and Prevention 71
[Link] Factors Influencing Efficacy 71
[Link].1 Dose and Timing 72
[Link].2 Combined vs. Single Probiotics and Species-Specific Effects 72
[Link].3 Baseline Risk and Age-Related Effects 74
[Link] Mechanisms of Action in Clinical Context 74
[Link].1 Restoring Gut Microbiota and Intestinal Barrier 74
[Link].2 Immune System Modulation and Metabolism Regulation 75
[Link] Impact on Antibiotic Resistance 75
[Link] Safety and Probiotic-Drug Interactions 78
[Link].1 Safety Profile 78
[Link].2 Probiotic-Drug Interactions 78
[Link] Gut Microbiome Diversity and Clinical Relevance 79
Conclusion 81
Recommendation 83
References 84

List of Tables

Table Page

VI
Table 1: shows the beneficial effect of metabolites produced from the gut
43
microbiota on healthy situations.
Table 2. shows Some clinical trials and uses of probiotics in human health 66
Table 3. shows Post-hoc analysis of bacterial cultures 77

List of Figures

Figures Page

VII
 Fig 1. Shows Transmission electron microscopy. Probiotic, were imaged 1
using the TEM technique
Fig2: Shows Rod-shape 6
Fig 3: Shows Cocci shape 6
Fig 4: Shows Role of probiotics in enhancing gut barrier integrity 11
Fig 5: Shows Mechanism of enhanced probiotic delivery using yeast
13
membrane coating
Fig 6: Shows Prebiotic effects for health (Davani-Davari et al., 2019). 16
Fig 7: Shows Classification of major antibiotic classes and representative
compounds (Source: ResearchGate, 2024).
23
Fig 8: shows detailed functions of the human gut microbiota 29
Fig 9: Shows impact of antibiotic resistance on different populations 30
Fig 10: shows multiple disease results from dysbiosis 34
Fig 11: shows the gut microbiota composition and the
40
affected organs by dysbiosis
Fig 12: shows the effect of antibiotic exposure in early age to the gut
microbiome leading to long-term health conditions
44
Fig 13. Shows that in humans, some clinical settings or interventions might
promote intestinal carriage of Enterobacterales. Probiotics might be
beneficial in eradicating gut carriage of pathogenic or antimicrobial-resistant
66
Enterobacterales. This figure adapted from

Acknowledgement
We would like to express our sincere thanks to our
supervisor for the continuous guidance, valuable feedback,

VIII
and constant support throughout the preparation of this
research. Their academic direction and encouragement
played an important role in enhancing the quality of our
work.
We also extend our appreciation to the faculty
members who supported us during our academic journey
and provided us with the knowledge and motivation needed
to complete this study. Special thanks go to our families for
their patience, understanding, and ongoing support during
this challenging period.
This research would not have been completed without
the help and support of everyone who contributed to it,
directly or indirectly, and we are truly grateful to all who
were part of this process.

IX
 Abstract
While antibiotic treatments are valuable in the struggle against disease-
causing pathogens, they often disturb the balance of the gut microbiome,
with significant immunomodulatory and nutritional consequences. This
antibiotic-induced dysbiosis decreases microbial diversity, injures
beneficial probiotic cells, and weakens necessary metabolic and immune
functions. Such perturbations link to disorders including AAD,
Clostridioides difficile infection, impaired gut barrier integrity ("leaky
gut"), reduced SCFAs, and a range of systemic health consequences:
chronic diseases, metabolic disorders, allergic disorders, asthma, and
neurocognitive disorders via the gut-brain axis.
Probiotics are live microorganisms with particular structural
constituents that restore gut homeostasis. They act by strengthening the
intestinal barrier, limiting opportunistic microbes, balancing immune
responses, and supporting metabolic health. For survival and activity, there
are essential elements: prebiotic dietary fiber in adequate quantities, such as
inulin or oligosaccharides; food taken concomitantly to protect against
unfavorable digestive conditions; and proper storage in cold, dry conditions
to maintain viability. The specific characteristics of individual hosts, such
as gut microbiome, mucus quality, immunity, and age, play a major role in
modulating probiotic functions, which might be nullified by concurrent
medication, especially antibiotics.
This research discusses the specific role of probiotics in reducing
adverse side effects. Probiotics apply their therapeutic effects through the
direct modulation of pathogens, restoration of gut ecosystem and barrier,
immune system modulation, and production of useful metabolites. Clinical
evidence supports reduction in AAD incidence, incidence/duration of

X
Clostridioides difficile infection. Optimizing probiotic efficacy requires a
balance in diet and digestion protection, proper storage, and individual host
factors; hence, their potential to support immunity and improve health
during/after antibiotic treatment.

XI
 ‫الخالصة‬
‫بينمــا ُتعــد العالجــات بالمضــادات الحيويــة مهمــة لمكافحــة الميكروبــات‬
‫الضارة‪ ،‬إال أنها ُتخّل بتوازن الميكروبيــوم المعــوي‪ ،‬ممــا يــؤدي إلى تداعيات‬
‫مناعية وغذائية كبيرة‪ .‬يقلل هــذا الخلــل النــاجم عن المضــادات الحيويــة من‬
‫التنوع الميكروبي‪ ،‬ويضـر بالخاليـا البروبيوتيكيـة النافعـة‪ ،‬ويضـعف الوظـائف‬
‫األيضــية والمناعيــة الضــرورية‪ .‬وقــد ارتبطت هــذه االضــطرابات بمجموعــة‬
‫متنوعة من األمراض‪ ،‬منهــا اإلســهال المرتبــط بالمضــادات الحيويــة وعــدوى‬
‫المطثية العسيرة واعتالل الحــاجز المعــوي " األمعــاء المتســربة" وانخفــاض‬
‫إنتاج األحماض الدهنيــة قصــيرة السلســلة‪ ،‬وعــواقب صــحية جهازيــة أوســع‪:‬‬
‫األمــراض المزمنــة واضــطرابات األيض واضــطرابات الحساســية والربــو‬
‫واالضطرابات العصبية المعرفية عبر محور األمعاء‪-‬الدماغ‪.‬‬
‫البروبيوتيك كائنات حية دقيقة ذات مكونات هيكلية محـددة تسـاهم حيويـًا‬
‫في استعادة توازن األمعاء‪ .‬إنهــا تعــزز الحــاجز وتثبــط الميكروبــات الضــارة‪،‬‬
‫وتنظم االستجابات المناعية‪ ،‬وتدعم صحة األيض‪ .‬بقاؤها ووظائفهــا يعتمــدان‬
‫على عوامــل ضــرورية لحيويــة البروبيوتيــك‪ :‬األليــاف البريبايوتيكيــة (مثــل‬
‫اإلينولين‪ ،‬والسكريات القليلــة) كمصــادر غذائيــة أساســية؛ الطعـام المتنــاول‬
‫معها قد يحميها من الظروف الهضمية الضارة؛ وظــروف التحــزين المناســبة‬
‫(كالبرودة والجفاف)‪ .‬العوامل الفردية للمضيف (تكوين الميكروبيــوم‪ ،‬جــودة‬
‫المخاط‪ ،‬المناعة‪ ،‬العمر) حاسمة‪ ،‬بينما قد تكون األدويــة المتزامنــة‪ ،‬بمـا في‬
‫ذلك المضادات الحيوية‪ ،‬مثبطة بقوة‪.‬‬
‫يتناول هذا البحث الدور المحدد الذي تلعبــه البروبيوتك في التخفيــف من‬
‫هذه اآلثار الجانبية السلبية‪ .‬تمــارس البروبيوتك تأثيراتهــا العالجيــة من خالل‬
‫أساليب متعددة‪ ،‬بما في ذلك التعديل المباشر لمسببات األمراض واســتعادة‬
‫النظام البيئي المعوي والحاجز‪ ،‬وتنظيم الجهاز المناعي وإنتــاج المســتقلبات‬
‫المفيدة‪ .‬تدعم األدلــة الســريرية بقــوة اســتخدامها لتقليــل حــدوث اإلســهال‬
‫المرتبــط بالمضــادات الحيويــة ومدته‪ ،‬وعــدوى المطثيــة العســيرة‪ .‬يتطلب‬
‫التحســين النهــائي لفعاليــة البروبيوتك توازًن ا بين النظــام الغــذائي‪ ،‬وحمايــة‬

‫‪XII‬‬
‫الهضــم‪ ،‬والتخــزين الســليم‪ ،‬والعوامــل الفرديــة للمضــيف‪ ،‬ممــا يؤكــد على‬
‫إمكاناتها العالية في دعم المناعة وتحســين الصــحة بشــكل عــام أثنــاء وبعــد‬
‫العالج بالمضادات الحيوية‪.‬‬

‫‪XIII‬‬
 PG Peptidoglycan
CFUs Colony-Forming Units
RH relative humidity
IgA Immunoglobulin-A
NSAIDs nonsteroidal anti-inflammatory drugs
PPIs proton-pump inhibitors
SSRIs selective serotonin reuptake inhibitors
5-HT 5-hydroxytryptamine
GI Gastrointestinal
50S Large subunit of bacterial ribosome
30S Small subunit of bacterial ribosome
COX Cyclooxygenase
LPS Lipopolysaccharide
OCT1 Organic cation transporter1
OCT3 Organic cation transporte3
GLP-1 Glucagon-Like Peptide 1
SGLT2 Sodium-Glucose Co-Transporter 2
GlcNAc N-acetylglucosamine
MurNAc N-acetylmuramic acid
TA Teichoic acid
WTA wall teichoic acid
LTA lipoteichoic acid
PSs Polysaccharides
EPSs Exopolysacharides
CPSs capsular polysaccharides
CWPSs cell wall polysaccharides
SLPs surface layer proteins
PRRs pattern recognition receptors
TLR5 Toll-like receptor 5
LAB Lactic acid bacteria
non-LAB Non-lactic acid bacteria
TNF-α Tumor Necrosis Factor-alpha
CRP C-reactive protein
SCFAs short-chain fatty acids
AAD Antibiotic-Associated Diarrhea
AGA American Gastroenterological Association
AMP Antimicrobial peptide
BV Bacterial vaginosis
CD4+Foxp3 A specific type of T cell (an immune cell) identified by
+ T cells "CD4+" and "Foxp3+" markers.
CDAD Clostridioides difficile–associated diarrhea
EcN E. coli Nissle 1917
ESBL Extended-spectrum beta-lactamase (as in ESBL-
producing bacteria)
GABA Gamma- aminobutyric acid
HGM Human Gut Microbiome
HPV Human Papillomavirus
IBD Inflammatory bowel disease
IBS Irritable bowel syndrome
XIV
IFN-γ Interferon-gamma
 List of symbols

α (alpha) Used in TNF- (Tumor Necrosis Factor alpha).

Used to indicate a value smaller than another, e.g., "< 5
< (less than sign)
kDa".
γ (gamma) Used in IFN- (Interferon-gamma) and Reg3.
Used in NF-B (Nuclear Factor kappa-light-chain-
κ (kappa)
enhancer of activated B cells)
Denotes a positive marker, as in CD4+ and Foxp3+ T
+ (plus, sign)
cells.

XV
 CHAPTER 1

Probiotics
1. introduction
Antibiotics are essential medicines that play a critical role in the
prevention and treatment of bacterial infections. Due to their significant
therapeutic value, antibiotics rank among the most commonly prescribed
and relied-upon medications worldwide. However, while highly effective,
the excessive or inappropriate use of antibiotics can lead to a variety of
adverse effects, including gastrointestinal problems, diarrhea, and
disruption of the host’s normal gut flora. Such imbalances in the intestinal
flora may compromise digestive health, weaken immune defenses, and
increase susceptibility to opportunistic infections (Maftei et al., 2024 (In
recent years, Probiotics have emerged as an effective strategy to avoid any
of these complications associated with antibiotic use. They are defined as a
live microorganism that help restore the natural flora in the stomach, which
in turn contributes to improving human health. For instance, probiotics help
re-establish the organism’s microbial balance, strengthen immune defenses
and prevent opportunistic infections. (Maftei et al., 2024)

Fig 1. Shows Transmission electron microscopy. Probiotic, were imaged using the TEM technique
(Senthil Kumar & Sheik Mohideen, 2024)

As a result, the integration of probiotics into clinical practice and research

has garnered considerable attention. Their role in counteracting the side
effect of antibiotics has become an important topic of interest in healthcare,
medicine, and scientific investigation, highlighting the potential of

1
probiotics as an adjunctive therapy to improve patient outcomes and
maintain gut health during and after antibiotic treatment.

Aims of work:
1- The primary aim of this work is to explore the consequences of
probiotic imbalances.

2- To assess the ways in which probiotic functions-such as adhesion,

mucin modulation, and barrier protection that compromised by
antibiotic exposure and investigate how they contribute to gut integrity.
3- To evaluate the mechanisms and therapeutic efficacy of probiotic
bacteria in mitigating antibiotic-induced gut dysbiosis and its
systemic health impacts.

1.1 Structural Organization of Probiotic Cell

To understand how probiotics exert their beneficial effects, it is necessary
to study their cellular structure and properties. The composition and
organization of their cellular components play an important and
fundamental role in determining their ability to survive under different
environmental and gastrointestinal conditions, as well as their ability to
interact with and affect the host’s body and maintain overall microbial
balance.

1.1.1 Cell wall

The cell wall plays an important role in performing several functions
during bacterial growth, such as maintaining the integrity and external
shape of the cell. It also remains flexible to allow cell division and growth,
and helps the cell resist attacks from bacteriophages or eukaryotic host cells

2
and various environmental stress factors (Chapot-Chartier & Kulakauskas,
2014).

[Link] Peptidoglycan (PG)

Peptidoglycan is one of the main components of the Gram-positive cell
wall. It consists of multiple glycan chains made up of alternating units of
N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)
linked by β-1,4 glycosidic bonds. These chains can be cross-linked, directly
or indirectly, through one or more short amino acid chains (Chapot-Chartier
& Kulakauskas, 2014).

[Link] Teichoic acids (TAs)

The cell wall of Gram-positive bacteria often contains teichoic acids (TAs),
which are negatively charged polymers composed of repeating alditol-
phosphate units. These acids are classified into two groups: wall teichoic
acids (WTAs), covalently attached to the PG layer, and lipoteichoic acids
(LTAs), which are anchored in the cytoplasmic membrane through a
glycolipid moiety (van Dalen et al., 2020).

[Link] Polysaccharides (PSs)

Bacterial cell walls also contain polysaccharides (PSs), which can be
classified into three groups. Firstly, exopolysaccharides (EPSs), which are
loosely associated with the cell surface and secreted into the surrounding
environment. Secondly, capsular polysaccharides (CPSs), which are
permanently attached to the cell, forming a protective capsule around the
bacterium. Thirdly, cell wall polysaccharides (CWPSs), which may or may
not be covalently linked to the cell wall, but they do not form a capsule
(Chapot-Chartier & Kulakauskas, 2014).

3
[Link] Proteins
Surface-associated proteins make up approximately 80% of secreted
proteins. It can be anchored to the bacterial cell surface either covalently,
through sortase-mediated reactions, or non-covalently, via transmembrane
anchor, lipid anchors, or various cell wall binding domains that ensure
stability and functionality (Chapot-Chartier & Kulakauskas, 2014).

1.1.2 Surface Molecules

The probiotic surface molecules consist of various structural components,
including surface layer proteins (SLPs), flagella, fimbriae, and CPSs. These
molecules can be recognized by pattern recognition receptors (PRRs),
thereby playing an important role in maintaining intestinal balance,
promoting gut health, enhancing host-microbe interactions (Liu et al.,
2020).

[Link] SLPs
It is the primary bacterial structure that directly interacts with the host
epithelium. The surface layer is composed of a single, tightly packed, and
regular lattice that attaches to the outer membrane through non-covalent
interactions (Liu et al., 2020). This organized structure provides
mechanical stability and protection against environmental stress.

[Link] Flagellin
It is the main building block of bacterial flagella. It is produced by
pathogenic, symbiotic, or neutral bacteria, including some probiotic strains.
It can be caused an inflammatory response in intestinal epithelial cells
through receptors such as Toll-like receptor 5 (TLR5). However, this effect
depends on contact with the basolateral membrane, and it disappears in the

4
absence of contact with the basolateral membrane of the epithelium (Liu et
al., 2020).

[Link] Pili
These filamentous appendages are accessory structures located on the
bacterial surface that play a vital role in adhesion to the intestinal
epithelium. They facilitate the initial contact between probiotics and host
tissues, strengthening their ability to colonize the gut and exert beneficial
effects. In addition to adhesion, pili can contribute to biofilm formation,
which enhances bacterial persistence and protection within the intestinal
environment. These structures are divided into six types (Type I–Type VI)
based on their shape, quantity, surface distribution, adhesion properties,
genetic locus, and distinct antigenic characteristics (Liu et al., 2020).

[Link] CPS
These polysaccharide structures are homopolymers or heteropolymers
composed of repeating monosaccharide units linked by glycosidic bonds,
and they play a crucial role in adaptation to the intestinal microenvironment
and protection against external stressors, thereby enhancing bacterial
survival and persistence (Liu et al., 2020).

1.1.3 Cytoplasmic structures

The cytoplasmic structure of probiotic bacteria consists of a phospholipid
bilayer plasma membrane associated with proteins, which is responsible for
transport, energy production, metabolism, and cellular protection. It also
contains inclusion bodies that store energy and essential compounds, such
as poly-β-hydroxybutyrate, polysaccharides, phosphate, and metachromatic
granules, which vary depending on the bacterial strain and growth
conditions. The gel-like cytoplasmic matrix suspends all internal cellular

5
components, including 70S ribosomes, metabolic enzymes, and the
nucleoid, which harbors the genetic material (Salton & Kim, 1996).
1.1.4 Extracellular Vesicles (EVs)
Particles composed of cytoplasmic proteins and double-stranded DNA
include mucin-binding factors, such as GroEL and Tal, the possess strong
adhesive properties, thereby supporting bacterial persistence within the
gastrointestinal tract. It also plays essential roles in fundamental cellular
processes (Nishiyama et al., 2020).
1.2 Morphological Characteristics of probiotics
The morphological characteristics of probiotics play a critical role in their
ability to adapt and interact within the gastrointestinal tract (da Cruz
Rodrigues et al., 2019). Cell shape influences adhesion capacity: rod-
shaped strains, such as Lactobacillus, and form short chains that enhance
contact with the intestinal epithelium. On the other hand, cocci, such as
Streptococcus, may aggregate in pairs or longer chains, which helps them
stay attached and stable in the gut environment (Rajab et al., 2020).

Fig 2. Shows rod-shaped (Tallapragada et al., 2018). Fig 3. Shows Cocci shape (Nejadmansouri
et al., 2023)

6
Cell size of cocci represents another essential morphological feature,
typically ranging from 0.5–2 µm in width and 1–10 µm in length,
depending on the species. Smaller cells can move more easily through the
thick mucus layer and reach narrow spaces in the intestines, while larger
cells provide greater cytoplasmic volume, supporting higher metabolic
activity and improved storage of intracellular reserves (Yan et al., 2025). It
also affects resistance to environmental stresses, such as osmotic pressure
and mechanical forces within the gastrointestinal tract (Wendel et al.,
2022).
The shape and size of probiotic cells can change depending on genetic
factors and growth conditions, including nutrient availability, pH, and
oxygen levels. These morphological adaptations enable probiotics survive
in the intestinal environment and compete with pathogenic microbes (Kiepś
et al., 2023).
Understanding these characteristics is essential for selecting provides the
most effective probiotic strains for colonization and health benefits.

1.3 Types of probiotic Microorganisms

Probiotics comprise both bacteria and yeast species, each exhibiting
distinct physiological and functional characteristics that affect how they
work within the host (Shah et al., 2024). These differences influence their
mechanisms of action, including adhesion to intestinal cells, modulation of
immune responses, production of beneficial metabolites, and interaction
with the host’s gut microbiota. Understanding these variations is essential
for selecting appropriate probiotic strains for specific therapeutic and
health-promoting applications.

1.3.1 Lactic acid bacteria (LAB)

7
Gram-positive microorganisms belonging to multiple genera, including
Lactobacillus, can ferment sugars into lactic acid (Chapot-Chartier &
Kulakauskas, 2014), which underpins their extensive use in food
fermentation, particularly in dairy products. LAB also play a critical role in
maintaining and regulating human gastrointestinal health and are employed
in aquaculture to enhance the health, growth, and resilience of aquatic
organisms (Loo et al., 2025).

1.3.2 Non-lactic acid bacteria (non-LAB)

Non-lactic acid bacteria (non-LAB) represent a group that differs from
traditional LAB in their metabolic pathways and ecological niches (Lee,
Jang, & Paik, 2024). Unlike LAB, non-LAB strains exhibit broader
metabolic capabilities, enabling them to produce a wide range of bioactive
compounds, such as short-chain fatty acids, vitamins, and antimicrobial
metabolites (Hill et al., 2014). This group includes several probiotic
species, such as Akkermansia muciniphila, Christensenella minuta, and
Propionibacterium (Lee et al., 2024; Loo et al., 2025). Many of these
species are strictly anaerobic and naturally inhabit the human intestinal
tract, contributing to gut homeostasis and modulation of immune responses.
Despite their promising health benefits, non-LAB probiotics face
challenges in cultivation and formulation due to their oxygen sensitivity
and complex nutritional requirements (Lee et al., 2024).

1.3.3 Yeast
Probiotic yeasts are defined as non-bacterial living microorganisms capable
of providing health benefits to the host when consumed in adequate
amounts, similar to probiotic bacteria. Saccharomyces cerevisiae var.
boulardii is a widely used probiotic yeast due to its ability to survive harsh

8
conditions in the digestive tract, such as low pH and digestive enzymes,
allowing it to reach the intestines alive (Abid et al., 2022). This yeast helps
restore gut microbiome balance after disruptions caused by antibiotics or
microbial infections. It also has antibacterial, antiviral, and anti-
inflammatory properties, supporting immune system regulation and overall
gut health (Staniszewski & Kordowska-Wiater, 2021).

1.4 Natural Habitats of probiotic Microorganisms

Probiotic microorganisms live in diverse environments that enable them to
survive and stay active. They can be found in nature or within the
biological system of living organisms (Abid et al., 2022). These habitats
are essential for understanding their physiological characteristics and
modes of interaction with the environment or the host. Some probiotic
strains naturally reside in the gastrointestinal tracts of humans and animals,
while other strains are isolated from various food sources (Loo et al.,
2025).

1.4.1 Human intestines

The intestine represents one of the most important habitats for probiotic
microorganisms. Traditional strains, such as Lactobacillus and
Bifidobacterium, play a vital role in maintaining the balance of the
intestinal microbiota when consumed in adequate amounts, thereby
contributing to intestinal homeostasis (Chandrasekaran et al., 2024).
Probiotics interact directly with intestinal epithelial cells, enhancing the
integrity of the intestinal barrier, improving epithelial permeability, and
modulating immune responses. The intestinal environment serves as a key

9
site for probiotic activity following their ingestion through functional foods
or dietary supplements (Magalhães et al., 2025).

1.4.2 Fermented Food

Fermented foods are also considered important natural habitats for
beneficial bacteria, as incorporating probiotics into them is regarded as a
promising strategy to enhance intestinal health. The fermentation process
creates a favorable environment that supports the survival, stability and
metabolic activity of probiotics, as it facilitates their stability and biological
activity after being added and utilized in food products (Magalhães et al.,
2025).

1.4.3 Biofilms
Probiotic microorganisms inhabit biofilms, as studies such as Chamignon
et al. (2020) have shown that certain probiotic strains can form biofilms
that enhance their survival and stability in various environments. These
biofilm structures play a crucial role in protecting cells from harsh
conditions, improving their adhesion to surfaces and intestinal epithelial
cells, helping to support their functional activity within the gastrointestinal
tract (Chapot-Chartier & Kulakauskas, 2014). Furthermore, biofilms can
serve as a reservoir for beneficial compounds, allowing probiotics to
release enzymes, antimicrobial substances, and signaling molecules that
positively influence the gut ecosystem.

1.4.4 Pharmaceutical Applications

Probiotics are also present in pharmaceuticals as live therapeutic products.
They are made in various forms such as capsules, tablets, powders, and
nanocapsules. These medicines help in managing gastrointestinal problems
and reduce side effects caused by antibiotics (Loo et al., 2025). According

10
to Baral et al. (2021), encapsulation technologies play a crucial role in
protecting probiotic cells from harsh conditions such as stomach acidity,
oxygen, and humidity, thereby helping to maintain their viability and
stability during processing and storage.

1.5 Physiological Roles and Health Benefits of Probiotics

Probiotics are important biological agents that contribute to supporting and
regulating physiological functions through multiple mechanisms that
influence microbiome balance and intestinal ecological stability, thereby
promoting human health. According to Mercado-Monroy et al. (2025),
probiotics play a pivotal role in maintaining intestinal barrier integrity and
immune homeostasis through enhancing tight junctions and stimulating
mucin production, in addition to competing with pathogenic
microorganisms and limiting their colonization.

11
 Fig 4. Shows Role of probiotics in enhancing gut barrier integrity (Liu et al. 2022).

1.5.1 Strengthening the Intestinal Barrier

Probiotics enhance the resistance and integrity of the mucosal lining,
reducing its permeability to harmful microbes, toxins, and antigens. This
helps prevent the translocation of pathogenic bacteria into the bloodstream
and maintains gut homeostasis (Magalhães et al., 2025). Additionally,
probiotics stimulate the production of tight junction proteins and mucin,
which further reinforce the barrier function, support nutrient absorption,
and protect against inflammatory damage (Rose et al., 2021).

1.5.2 Competition with Pathogenic Microorganisms

Probiotics occupy adhesion sites along the intestinal epithelium, thereby
preventing pathogenic microorganisms from attaching and establishing

12
themselves within the gut environment. In addition, they produce a variety
of antimicrobial substances, such as bacteriocins and organic acids, and
lower the intestinal pH through lactic acid production, creating conditions
that are unfavorable for the growth and proliferation of many enteric
pathogens. Collectively, these mechanisms contribute to a significant
reduction in pathogen colonization and support the maintenance of a
balanced and resilient gut microbiota (Chamignon et al., 2020).

1.5.3 Regulation of Immune Responses

Probiotics help regulate the immune system by modulating key
inflammatory pathways and controlling the production of substances that
either promote or reduce inflammation. They have been shown to reduce
lower levels of inflammatory markers, including Tumor Necrosis Factor-
alpha (TNF-α) and C-reactive protein (CRP), which play critical roles in
the initiation and progression of inflammatory responses. By balancing
these markers, probiotics enhance the body’s ability to fight infections and
minimize tissue damage caused by inflammation. Overall, these effects
contribute to strengthening the immune system and maintaining its proper
balance (Baral et al., 2021).

13
Fig. 5 shows Mechanism of enhanced probiotic delivery using yeast membrane coating (Lin et al., 2021).

1.5.4 Support of Metabolic Health

Probiotics contribute significantly to metabolic health by producing short-
chain fatty acids (SCFAs), including acetate, propionate, and butyrate,
during the fermentation of dietary fibers in the gut. These SCFAs play a
crucial role in regulating various physiological processes, such as
modulating hunger and satiety hormones, influencing lipid metabolism, and
improving glucose homeostasis. In addition, SCFAs can impact energy
expenditure, enhance insulin sensitivity, and reduce systemic inflammation,
thereby supporting overall metabolic balance and reducing the risk of
metabolic disorders such as obesity, type 2 diabetes, and dyslipidemia
(Magalhães et al., 2025).

1.5.5 Prevention of Other Health Issues

Probiotics have been shown to provide benefits beyond gut health,
contributing to the prevention and management of a variety of other health
issues. Several studies, including Probiotics for the Prevention of Vaginal
Infections (2024), indicate that probiotics can help maintain a healthy
vaginal microbiota, thereby reducing the risk of infections such as bacterial

14
vaginosis and candidiasis. In addition to reproductive health, probiotics
also play a role in oral health by helping to alleviate gum inflammation,
reduce plaque formation, and minimize halitosis (bad breath). These effects
are achieved through mechanisms such as competition with pathogenic
microorganisms, production of antimicrobial substances, and modulation of
local immune responses. Collectively, these actions demonstrate the
broader protective potential of probiotics in supporting overall human
health beyond the gastrointestinal tract.

1.6 Consequences of Probiotic Imbalance

Imbalance of probiotics, also known as gut microbiota dysbiosis, can
negatively affect human health. This imbalance occurs when the natural
microbial diversity in the gastrointestinal tract decreases while harmful
microorganisms increase, leading to a weakened intestinal barrier and
increased gut permeability, thereby facilitating the translocation of
pathogenic bacteria and toxins into the bloodstream. This dysbiosis is
associated with a wide range of health problems, including inflammatory
bowel diseases such as ulcerative colitis and Crohn’s disease, metabolic
disorders like obesity and type 2 diabetes, as well as effects on the nervous
system and mood due to its impact on the gut–brain axis. Several factors
contribute to probiotic imbalance, including prolonged use of antibiotics,
poor nutrition, lack of dietary fiber, chronic psychological stress, and
unhealthy lifestyle habits. Additionally, exposure to environmental
pollutants may play a role in altering the gut microbiota balance. Therefore,
maintaining a balanced probiotic population is very important. This can be
achieved through proper nutrition, the use of dietary supplements when
necessary, and adopting a healthy lifestyle that includes regular physical

15
activity, which helps reduce stress, support gut homeostasis, and prevent
chronic diseases (Shreiner et al., 2015; Zmora et al., 2019).

16
 CHAPTER 2

Factors Influencing Probiotic Survival

and Activity
 2.1 Nutritional Factors (Prebiotics and Dietary
Components)
2.1.1 Prebiotic fibers and oligosaccharides:
Non-digestible carbohydrates such as inulin, fructo-oligosaccharides,
and galacto-oligosaccharides act as selective substrates for probiotic
bacteria (Gibson et al., 2017).
These fibers resist host digestion and reach the colon, where they are
fermented by beneficial microbes, enhancing their metabolic activity
and promoting their proliferation (Gibson et al., 2017; Holscher,
2017).

Fig 6. Shows Prebiotic effects for health (Davani-Davari et al., 2019).

For instance,
you et al. (2022) reported that prebiotics enhance probiotic growth in the
human gastrointestinal tract, leading to increased microbial diversity. It has
been revealed in an experiment that plant-based polysaccharides effectively
promote the growth of specific probiotic strains. Growth tests found that
the addition of sources like astragalus or other complex plant polymers

16
significantly increased the number of Lactobacillus and Bifidobacterium
strains (Akhavan et al., 2023). Experimental results on animals have shown
that adding seaweed-derived polysaccharides to the diet significantly
elevates the quantities of probiotic species such as Akkermansia,
Bifidobacterium, and Lactobacillus. In the gut microbiome of mice. From a
practical point of view, the intake of fiber-rich foods (e.g., whole grains,
legumes, fruits) is a source of substrates that support the life and growth of
probiotics in the intestine (You et al., 2022)

2.1.2 Food matrix and dietary composition:

The form and composition of the food that is eaten greatly affect the
survival of probiotics in the digestive process. The food matrix is the
nutrients and the texture of the food that is eaten with the probiotic. Several
recent studies using a standard digestion model demonstrate that survival
varies significantly depending on the carrier (Treven et al., 2024;
Matouskova et al., 2021; Wang et al., 2025).
Treven et al. (2024) reported that probiotic supplements, when simulated
through the gastrointestinal tract and co-digested with a carbohydrate-rich
porridge, showed significantly higher survival rates (~91.8%) compared to
those taken with fruit juice (~79.0%) or water. This enhanced survival is
attributed to the porridge’s buffering capacity and its protein–starch matrix,
which protects bacteria against gastric acidity and provides additional
metabolic substrates. In contrast, acidic beverages offer minimal protection
and result in greater bacterial loss.
The presence of proteins, fats, and fibers in mixed meals can alter digestion
rates and influence probiotic survival as shown in digestion-model studies.
In summary, a diet high in prebiotic fibers, when consumed as part of a
healthy meal rather than on an empty stomach or in acidic drinks, is likely

17
to support the survival of probiotics. For example, one study reported a 1.2-
log decrease of CFUs (Colony-Forming Units) with porridge compared to a
2.5-log decrease with juice (Treven et al., 2024).

2.1.3 Other dietary components:

Besides prebiotics, several dietary components—including proteins, fats,
fibers, and polyphenols—can significantly influence probiotic survival and
activity within the gut ecosystem.
Polyphenols, which are hard-to-digest plant antioxidants, have the ability to
inhibit pathogenic microorganisms and indirectly promote probiotic
proliferation. In contrast, Diets high in simple sugars or saturated fats can
alter gut pH and intestinal transit time, thereby reducing probiotic
persistence. Overall, diets rich in complex carbohydrates and plant-derived
compounds enhance probiotic activity, whereas high-fat, low-fiber diets
contribute to a decline in beneficial bacteria.
Animal feeding trials consistently demonstrate that diets supplemented
with fermentable fibers significantly increase the abundance of
Lactobacillus and Bifidobacterium (You et al., 2022). Similarly, human and
animal studies show that incorporating prebiotics into nutrient-rich meals
alongside probiotics enhances their viability (You et al., 2022; Akhavan et
al., 2023; Treven et al., 2024).

18
2.2Environmental Factors (Temperature, PH, and Storage
Condition)
2.2.1 Temperature:‍‌‍‍‌
Probiotic microorganisms are highly heat-sensitive and are significantly
reduced at elevated temperatures (above room temperature) during the
production and storage of the product (Payne et al., 2025).

A study on spray-dried probiotic coffee showed that Storing the product in

a refrigerator (4°C) preserved high cell counts (>10^7 CFU/g) even after
two years, while viable counts at 30°C fell to the inactivation levels within
~3 months (Jannah et al., 2022).

Usually, cold storage of a product is a way of extending the shelf-life of

probiotics: Many products recommend refrigeration (2-8°C) to slow down
the metabolic activity and autolysis. On the other hand, the heating of a
product (e.g., during a shipment or in a hot place) can drastically lead to
cell death (Noufeu et al., 2025). In particular, probiotic freeze-dried
powders should be kept away from warm and humid places. In their
experimental trials, they found that the accelerated aging at 37°C for only
one week resulted in a very sharp decrease in probiotic viability as
compared to that at 4°C (Jannah et al., 2022). Hence, temperature is one of
the most important factors affecting the stability of probiotics during
storage as lower temperatures slow down harmful reactions and help
maintain probiotic viability

2.2.2 pH‍‌‍‍‌(Acidity) and digestive environment:

The pH was another significant factor, especially referring to the highly
acidic conditions of the stomach (pH ~1.5-3.5) (Stamatopoulos et al.,
2021). Probiotics taken with food have to pass through the harsh acid and

19
bile environment of the stomach before they can reach the small or large
intestine. At a very low pH (high acidity), a large number of bacteria will
be killed (Han et al., 2021). Probiotic strains do not exhibit the same level
of acid tolerance.
For example, some Lactobacillus and Bifidobacterium species can tolerate
an environment with a pH 3 or even pH 2 for a short time. But some of
them can tolerate high pH levels (Ranadheera et al., 2012). Standard in
vitro digestion models present this obstacle; strains must tolerate the
presence of pepsin and the low pH of the stomach, and the antimicrobial
activity of bile salts during gastrointestinal passage (Treven et al., 2024).
In reality, foods or capsules are often coated with a material that protects
probiotics from stomach acids (e.g., enteric-coated capsules) (D’Amico et
al., 2025). However, very low gastric pH during fasting is still unfavorable
for probiotics. Therefore, taking probiotics with food (which naturally
raises the gastric pH) can enhance the survival rate.
Scientists regularly examine strains to see if they can survive at pH 2-3 for
one or two hours; those that have strong acid resistance are the ones that
can deliver enough to the intestine.
Jannah et al. (2022) showed that vacuum packaging at 4 °C enabled a
probiotic instant coffee to maintain >10^7 CFU/g even after 50 days, while
at 30 °C the counts dropped sharply. Min et al. (2017) demonstrated that
storage at 20 °C/20%RH retained ~6–7 log CFU/g of Bifidobacterium on
grains, and storage at 30 °C/50% relative humidity (RH) storage lowered
the counts to ~4–5 log (2–4 log drop). The results of these studies suggest
that the probiotic viability is supported by cold, dry, low-oxygen
conditions, while heat, moisture, and oxygen cause the decline to accelerate
(Acosta et al., 2024). Treven et al., (2024) study shows that low pH is very

20
harmful to unprotected probiotics, thus highlighting the need for protective
matrices‍‌‍‍‌.
2.2.3 Host-Related Factors
Host-related‍‌‍‍‌ factors essentially determine the survival and functioning of
probiotics that are taken orally. Among these, gastric acidity, bile salts, and
digestive pH act as major barriers, whereas co-ingestion of nutrient-rich
foods helps in the viability of probiotics (Treven et al., 2024; Cao et al.,
2021). The attachment of probiotics to intestinal mucus is influenced by the
mucosal and microbiota characteristics of each individual. (Zmora et al.,
2018). The immune system controls colonization by antimicrobial peptides
and Immunoglobulin-A (IgA), which can either limit or support
colonization (Rothschild et al., 2018; Jiang et al., 2023). The resident
microbiota, however, has the most substantial ecological competition, and
antibiotic exposure may disturb this equilibrium (Palleja et al., 2018).
Factors of age, physiology, and host genetics, in turn, influence the results,
although environmental and microbial factors have the leading role (Arrieta
et al., 2015; Yassour et al., 2016). Hence, the effectiveness of probiotics is
an individual matter, and the use of such strategies— such as taking
probiotics with meals, combining them with prebiotics, and tailoring use to
host conditions— can be enhanced (Treven et al., ‍‌‍‌2024).

21
 2.3 Pharmacological Factors (Effect of Antibiotics and Other
Medications)
Pharmacological‍‌‍‍‌ factors involve antibiotics and other drugs that are
frequently used—such as proton-pump inhibitors (PPIs) nonsteroidal anti-
inflammatory drugs (NSAIDs), antidiabetics and psychotropics— which
have the potential to change gut bacteria. This group of drugs is chemically
diverse, comprising compounds like β-lactams with a four-membered
lactam ring, macrolides with large lactone rings, tetracyclines with four
fused rings, and can cause damage to bacterial cell walls, ribosomes, or
DNA replication.
Most NSAIDs, such as ibuprofen and naproxen, are carboxylic acids
(derivatives of propionic or acetic acid) that generally reduce the level of
cyclooxygenases. PPIs comprise benzimidazoles that include a sulfoxide
group, which is the component that blocks activity. Metformin belongs to
the biguanides (a small positively charged guanidine derivative) and is used
in the treatment of type 2 diabetes.

Structurally distinct psychotropic drugs, such as selective serotonin reuptake

inhibitors (SSRIs), including fluoxetine and phenylpropylamine derivatives,
predominantly feature cationic aromatic rings, which may interact with both
human and microbial targets. These chemical traits determine drug
absorption and microbial effects. One of the most important points is that
many of these drugs, including antibiotics, have gut microbes as off-targets.

22
2.3.1 Definition and Chemical Structures
Antibiotics‍‌‍‍‌ are bioactive compounds (either natural or synthetic) that limit
the growth of bacteria or kill them. They are chemically different, with
different molecular structures being the basis for their mechanisms. The
main classes of antibiotics ‍‌‍‌are:

1- β-Lactams‍‌‍‍ ‌ (for

Fig 7. Shows Classification of major antibiotic classes and

representative compounds (Source: ResearchGate, 2024).

membered β-lactam ring in common (a 3-carbon, 1-nitrogen ring),

which is very reactive. To illustrate, penicillins are made of a β-lactam
ring connected to a thiazolidine ring, whereas cephalosporins present a
7-aminocephalosporanic acid nucleus. Carbapenems and monobactams,
are also similar in that they have β-lactam rings (however,
monobactams have an individual lactam) (Zainab et al., 2025). Some β-
lactams are mixed with β-lactamase inhibitors (clavulanate, sulbactam)
to provide a protective shield to the ring against the enzyme attack
(Pandey & Cascella, ‍‌‍‌2023)

23
2- Macrolides‍‌‍‍‌(e.g., erythromycin, azithromycin) are typically described as
large 14– to 16-membered macrocyclic lactone rings that are substituted
with deoxy-sugars (Dinos, 2017). The lipophilic macrocycle interacts
with the large subunit of bacterial (50S) ribosomal subunit and thus
inhibits peptide elongation (Yu & Zeng, 2018). The lipophilicity of
macrolides and the sugar molecules attached to them determines
penetration into the tissue and acid stability in the stomach, thus
influencing the amount of active drug (and consequently off-target
exposure) reaching the intestinal lumen (Ramirez et al., ‍‌‍‌2020).

3- Tetracyclines‍‌‍‍‌ (e.g., doxycycline) are based on four linearly fused

hydrocarbon rings; they chelate divalent cations and inhibit aminoacyl-
tRNA from binding to the small subunit bacterial (30S) ribosomal A-
site. Because of their chelation and metal-binding properties, these
drugs have an impact on interactions with dietary minerals and lumenal
bioavailability (Barrenechea et al., ‍‌‍‌2021).
4- The fluoroquinolone antibiotics, such as ciprofloxacin, have a bicyclic
quinolone core that is typically fluorinated, which results in a high
binding affinity to bacterial DNA gyrase and topoisomerase IV. Thus,
the processes of DNA replication and repair are inhibited. Because of
their relative stability and high oral bioavailability, considerable
intestinal exposure occurs and, consequently, the normal gut
communities undergo marked changes (Millanao et al., ‍‌‍‌2021).
5- Each class of antimicrobial agents (aminoglycosides, glycopeptides,
sulfonamides, polymyxins, etc.) targets specific bacterial features
(ribosome, cell wall precursors, folate pathway, outer membrane
disruption) using a unique chemical structure, which determines
susceptibility among different bacterial taxa.

24
6- Non-antibiotic‍‌‍‍‌ medications that frequently change the gut microbial
ecology also possess equally defining chemotypes that dictate both host
pharmacology and microbial exposure.

2.3.2 Mechanisms and General Relationship Between

Antibiotics and Bacteria
By‍‌‍‍‌ their inherent characteristics, all antibiotics have a considerable
adverse effect on gut microbial ecosystems. For example, Broad-spectrum
antibiotics eradicate beneficial microbes and decrease species diversity to a
great extent (Ramirez et al., 2020). To illustrate, β-lactams and
fluoroquinolones not only kill the pathogens but also the beneficial
Bacteroidetes and Firmicutes; thus, "the niches are left for the opportunists,
such as Clostridioides difficile, allowing them multiply. This leads to
transient dysbiosis after a single course, but repeated or broad-spectrum
courses may cause permanent loss of species like butyrate-producing
Clostridia and changes in metabolism.
At a molecular level, antibiotics disrupt bacterial targets that have been
well-documented: cell-wall inhibitors (β-lactams) lead to lysis, ribosomal
inhibitors (macrolides/tetracyclines) interfere with translation, and DNA
inhibitors (quinolones) induce strand breaks. These operations are carried
out without any selection in the gut lumen, and therefore, the probiotic
strains are also either killed or inhibited. Consequently, antibiotic treatment
markedly decreases the chances of probiotic survival, and colonization is
made difficult.
Essentially, microbiota altered by antibiotics also lose specific functions
(such as short-chain fatty acid production) consequently, the gut barrier and
immune system become less robust at a clinical level, which is the
underlying cause of antibiotic-associated diarrhea and recurrent C. difficile

25
infection (Ramirez et al., 2020). Other‍‌‍‍‌ drugs have similar effects on
microbes, such as -producing Clostridia) and changes in the metabolism.
Antibiotics work on a molecular level by interfering with bacterial targets:
cell-wall inhibitors (β-lactams) cause cell lysis, ribosomal inhibitors
(macrolides/tetracyclines) disrupt translation, and DNA inhibitors
(quinolones) induce strand breaks (PPIs) increase stomach and small
intestine pH; as a result, they eliminate the low-pH barrier that normally
prevents bacterial colonization of the upper digestive tract (Jackson et al.,
2016). Chronically PPI users in large human cohorts have a significantly
lower gut bacterial richness and a shift towards oral or upper.
Gastrointestinal (GI) commensals (Streptococcaceae, in particular, being
greatly increased). This probably indicates the survival of acid-sensitive
organisms that are killed to a large extent. Animal studies showed that,
omeprazole resulted in a significant decrease in commensal
microorganisms: jejunal Actinobacteria and Bifidobacteria levels dropped
by approximately 80% in rats given PPI treatment, allowing
proinflammatory bacteria to proliferate (Rekatsina et al., 2020). The PPI-
induced dysbiosis, such as this, weakens colonization resistance and can,
therefore, make NSAID enteropathy worse (see the following section). To
put it concisely, pH and directly targeting microbial membranes, PPIs may
also decrease the viability of probiotics, affecting many Lactobacilli and
Bifidobacteria, which thrive in acidic environments, and disrupt the gut
ecosystem.

Inhibiting cyclooxygenase (COX) and damaging mitochondria, NSAIDs

cause harm to the gut epithelium, leading to inflammation in the area,
which results from NSAID-induced disruption of the barrier allowing
lipopolysaccharide (LPS) movement and activation of Toll-like receptors,

26
thereby causing neutrophil infiltration and ulceration (Rekatsina et al.,
2020). Bacteria in the gut have a major impact on animals without germs
are quite immune to NSAID enteropathy. Thus, NSAID therapy changes
microbiota composition as well (Zádori et al., 2023).
Antidiabetics such as Metformin are known to consistently enhance
mucosal microbiota. Both animal studies and clinical trials show that
metformin raises the abundance of common probiotic genera. For instance,
metformin treatment results in the enrichment of the gut populations of
Lactobacillus and Bifidobacterium (Petakh et al., 2023).
The presence of more mucus in the intestine is partly responsible for its
impact on mucin-secreting goblet cells, allowing mucin-degraders such as
Akkermansia muciniphila and Bifidobacterium to thrive (Wang et al.,
2024).

Metformin's influence within the lumen, via organic cation Transporter 1

(OCT1)/ organic cation Transporter3 (OCT3) transporters, is thought to
affect bacterial metabolism and bile acid pools directly, resulting in
saccharolytic and mucolytic bacteria thriving. Therefore, metformin, in
contrast to antibiotics/NSAIDs, is usually instrumental in maintaining
microbial diversity as well as acting synergistically with probiotics, hence
facilitating the colonization of beneficial strains.
Metformin's glucose-lowering effects may be partly attributed to the
microbiota, such as the production of short-chain fatty acids and the
reduction of inflammation, suggesting that metformin has a beneficial
impact on the gut's ecosystem. Although other antidiabetic medications,
such as Glucagon-like peptide1 (GLP-1) agonists and sodium-Glucose
Co-Transporter2 (SGLT2) inhibitors, have been less thoroughly researched
and tend to have less pronounced or indirect impacts on the microbiota,

27
changes in gut movement and nutrient distribution can still cause shifts in
microbial balance.
Large-scale human research is now reflecting that the use of SSRIs or
anxiolytics is linked to changes in the gut microbiome (Dilmore et al.,2025)
In the cohort study of depressed and anxious patients, the use of SSRIs led
to such a significant change in microbial β-diversity (which was greater
than the disease effect) that the researchers concluded: microbial
communities in patients treated with SSRIs were characterized by higher
levels of Eubacterium ramulus and lower levels of Turicibacter sanguinis,
besides other changes in microbial composition.
That is important because Turicibacter is one of the cells that take serotonin
inside; SSRIs hinder 5-hydroxytryptamine (5-HT) reuptake to the host, thus
demonstrating a drug–microbe interaction via serotonin transport. In
general, SSRIs and anxiolytics are likely to decrease microbial richness and
change the metabolite outputs (for example, bile acids, indoles).
Antipsychotics, such as olanzapine, also lead to gut dysbiosis, which is
characterized by a marked increase in the Firmicutes/Bacteroidetes ratio
and a notable rise in weight, possibly due to altered production of short-
chain fatty acids. On the other hand, psychiatric patients who are on long-
term psychotropics may develop metabolic syndrome; thus, part of the
reason might be the gut changes, which are revealed clinically. Since
numerous probiotics (mainly Lactobacilli) can either produce or regulate
Neurotransmitters, psychotropic-induced alterations may lead to the
probiotic-host relationship becoming dysfunctional.
In reality, adding probiotics to the broth of patients treated with
psychotropic medications is a method of maintaining gut safety and
reducing the incidence

28
Of GI side effects, which is currently being considered (Dilmore et al., ‍‌‍‌
2025).
To‍‌‍‍‌ most of the extent, pharmacological agents influence probiotics and
microbiota in various ways. Antibiotics eradicate the gut's natural bacterial
population, including beneficial probiotics, thereby reducing its diversity.
PPIs and NSAIDs compromise the mucosal barriers and pH, triggering a
shift in the community's composition and a decrease in populations of
Lactobacillus and Bifidobacterium. Metformin is the only one that supports
the survival of probiotics and the diversity. Psychotropics mostly lead to
dysbiosis and can also inhibit certain bacterial groups. These drug–microbe
interactions have medical consequences: dysbiosis may cause the
worsening of drug side effects (e.g., antibiotic-associated colitis, NSAID-
enteropathy) and influence drug efficacy. Probiotic supplements are often
utilized to treat dysbiosis by replenishing Lactobacilli/Bifidobacteria, but
drug-induced environmental shifts (such as elevated gut pH and
inflammation) can pose a challenge to probiotic colonization. It is therefore
crucial to understand the molecular effects of each medication on the gut
ecosystem in order to refine probiotic co-therapy and preserve the gut
microbiota in patients receiving polypharmacy.

29
 CHAPTER 3

Antibiotic-Induced Dysbiosis and the Therapeutic Role

of Probiotics in Mitigating Adverse Effects
3.1 The Profound Impact of Antibiotics on Gut
Microbiota Homeostasis
The human gut microbiota also known as the “Gastrointestinal microbiota.”
represents a widely dynamic environment containing all kinds of lives:
protozoa, fungi, bacteria, archaea, viruses (Gaia et al.,2025). It can be
classified in six phyla, Firmicutes, Bacteroides, Actinobacteria,
Fusobacteria, Proteobacteria, and Verrucomicrobia (Tadasu et al., 2017).
These microorganisms are anaerobic tolerance and play a critical role in
pathogen defense, metabolism, immunological modifications, extracting
nutrients for energy, maintaining ecological balance, aiding digestion and it
also affect the enteric nervous system (ENS), a Condensed network of
neurons and glial cells controlling the gut movement, secretion and
absorption (Siqi et al.,2025). The gender, geographic location, diet and
delivery mode can affect the microbiota composition (Tadasu et al., 2017).
According toJames Kinross et al.,2018“That the gut microbiome is
complex organization of microbiotic lifeforms and all the things they need
to sustain themselves in the nicheof the body” (James Kinross et al., 2018).

29
 Fig 8: shows detailed functions of the human gut microbiota this figure adapted from Forntiers in
microbiology, 2023.
Antibiotics commonly prescribed to cure and prevent bacterial infection,
they are astaple of modern medicine and rescue millions of lives every year
(Siqi etal.,2025). The process of selecting the infection-causing bacteria
may disrupts the ecosystem of the gut microbiome because the antibiotics
are non-specific to the harming bacteria only, instead they go after all
bacteria in our gut (Jamie et al.,2020). On the other hand, Donatas et
al.,2022 mentioned “think of forest were trying to get rid of one weed
infection, the way deploy antibiotics is to carpet-bomb the forest, killing
the good and the bad” (Donatas et al.,2022). Overall, the relationship
between antibiotics and gut microbiota is complicated but have harming
consequences to the gut health (Allan et al.,2025). Taking high doses or
random intake of antibiotics without medical prescription will be leaded to
long term health problems for example: diarrhea, difficulty in digestion,
immune system imbalance, gut microbiome damage (Dysbiosis) and
recurrent infections or the infection come back stronger or spread to other
parts body damaging other organs especially liver and kidney and colon
inflammation. In the end the biggest global health problems is Antibiotic
resistance meaning the infection become harder to treat (Eric et al 2025).

30
 Fig 9: shows impact of antibiotic resistance on different populations this figure adapted from MDPI
2025.

Bacterial variety and abundance rapidly drop when antibiotics are used.
Hammond et al., 2021 indicated 31 clinical studies to determine how
antibiotics affect gut microbiota. Some people have longer-term
difficulties, lasting two to six months, while the gut microbiota usually
heals in few weeks post treatment. Beneficial genera like
Bifidobacterium and lactobacillus have been demonstrated to be
significantly diminished by some antibiotics, such as Doxycycline and
Clarithromycin, although Amoxicillin and Nitrofurantoin did not have any
effects (Hammond at al.,2021).

3.1.1 Antibiotic-Induced Dysbiosis and Loss of Microbial

Diversity
Dysbiosis arises when the constituents, performance and the diversity of
the gut microbiota destroyed, results in a loss of diversity and lack of
balance between helpful and hurtful bacteria. This lack of balance could
result from different agents involves drug management, dietary habits,
environment, and chemicals these are the extrinsic agents, in contrast
stress, genetic disfunction, getting old and chronic diseases are the intrinsic
agents (Corina et al., 2025). Un regulated dysbiosis can cause conversion
of healable aliment circumstances to the stage of chronicity in the long
period of time, dysbiosis is a risk element for specific diseases found to be
strongly connected with health problems such as digestive tract illnesses
like :colitis, coeliac disorder , inflammatory bowel diseases ( IBDs) ,
irritable bowel syndrome ( IBS ) leaky gut disorder , liver disease , cancer
in colon or rectum ,diabetes and skin conditions such eczema ( Poonam et
al., 2023) .The oral use of antibiotics are the main drugs related with drug-

31
induced dysbiosis and loss of the microbial diversity, and the affects could
be interim or lasting based on the type of medication recommended
(Dahiya et al., 2023). Tetracycline has high impact on reducing abundance
and variety of Bifidobacterium species, important anaerobic bacteria are
responsible in improving the gut health. Board range Penicillin like
amoxicillin lower the Bacteroides and lactobacillus species, which are
promoting the growth of pathogenic species (Corina et al.,2025).

[Link] Coeliac Disorder

People with active coeliac disorder maintain dysbiosis, with huge
amounts of Enterobacteriaceae, Proteobacteria and Staphylococcaceae, in
addition to a minimized amounts of Streptococcus mutans and
Streptococcus anaginosus was detected in patients with effective coeliac
disorder (Aderson et al.,2024). Also have been indicated that the main
reason behind this disorder was the exposure to antibiotics in the first year
of life depending on the dose, medical professionals suggests that people
with coeliac disorder should intake a gluten-free diet across their lifespan
(Francisco et al., 2024)

[Link] Inflammatory Bowel Disease (IBDs)

(IBDs) include in the first place Crohn’s disease (CD) and Ulcerative
Colitis (UC).The primary indicator for IBD (particularly CD) in the fecal
sample is the low quantities of Firmicutes and high proteobacteria and
having abnormal quantities of mucin-degrading bacteria Ruminococcus
gnavus and Cenarcheaum [Link] was surprisingly unexpected
because these two species are also found in the healthy gut , IBD is
believed to be significantly affected by gut dysbiosis , diet ,smoking ,
pollution and genetics that increase intestinal permeability can all affect it

32
(Miguel et al.,2024). Although at any age, dysbiosis can affect UC but
dysbiosis in early age (infants) causes CD (Juan et al.,2024). An increased
risk of CD and occasionally UC has been associated with antibiotics
exposure, especially high doses or frequent uses, which decreasing
beneficial SCFAs, boosting pathogenic bacteria and weakening the gut
barrier which increasing the vulnerability of the gut intestine to
inflammation (Luis et al., 2024).

[Link] irritable bowel syndrome (IBS)

There are findings of dysbiosis in individuals with Functional
Gastrointestinal Disorder (FGIDs), and a link between antibiotics
supplementation and the development of FGIDs, even if IBS dose not
exhibit gut inflammation or tissue damage (Luis et al.,2024). According to
Saffouri et al2020, symptomatic IBS patients had a significantly different
microbial composition of the small intestine, with lower phylogenetic alpha
diversity, richness and evenness as well as decreased in
Prophyromonas, Prevotella and Fussobacterium availability
(Francisco et al., 2024).

[Link] Cancer
A sustainable amount of data indicates that the extend use of antibiotics
is linked to both a relative decrease in the efficiency of cancer therapies
such as chemotherapy, immunotherapy and radiation. The severity of the
relationship between antibiotic exposure and cancer development differs by
cancer type and antibiotic class breast cancer, endocrine gland,
malignancies, pancreatic cancer in a high degree in the other hand a lower
degree of lung, esophagus, gastric and ovarian cancers have all been
involved (Aldo et al.,2024). Colorectal cancer has been well studied in

33
relation to antibiotic exposure, which is not unexpected given to the close
proximity to the gut and the effect on public health. The majority of
researchers have discovered a strong dose dependent correlation with colon
cancer but not with rectal cancer, lastly the low SCFAs level may be
associated with an increased risk of colon cancer (Sylvia and Lucian et al.,
2024).

[Link] Neurodevelopmental, neurodegenerative and

neurological disorders
The rise in the prevalence of multifactorial neurodevelopmental diseases
(NDDs) such as autism spectrum disorder and attention deficit-
hyperactivity disorder. The incidence of ecological prompting the
susceptibility variables like the exposure of antibiotics throughout prenatal,
perinatal, postnatal intervals appear to have a high effect in past few
decades. One recognized characteristic of NDDs is dysbiosis people with
these disorders often have a reduced levels of Bifidobacteria, Veillonella ,
Escherichia, Ruminococceae, Streptococcaeceae, Peptostreptococcaceae
and Erysipelotrichaceae in their feces and have greater levels of
Bacterioidetes and Megamanos (Francisco et al.,2024). On the whole there
is a strong correlation between antibiotic and the exposure and the
likelihood of neurodegenerative disorders according to population-based
research (Aldo et al., 2024).

Fig 10: shows multiple disease results from dysbiosis this figure Adapted from Signal
Transduction and Targeted Therapy 2022.
34
3.1.2 Reduction of Endogenous Probiotic Populations
In the digestive tract (mainly the colon) there are large populations of eco-
friendly bacteria (probiotics), predominantly genera like Lactobacillus and
Bifidobacterium, which helps in digest food, maintain barriers for defense
and signaling the immune system to fight the infections (Hanru et al.,2012).
A decline or a reduction in these populations can be thought of endogenous
probiotic population loss (Feng et al., 2024). To avoid the reduction of
probiotic population there are a proposed mechanisms of probiotic actions
such as: adhesion to the intestine, maintain intestinal integrity, tight
junction, mucin expression, cell proliferation and cell apoptosis (Gordon et
al., 2012).

[Link] Adhesion to compete with pathogens

The adherence to the mucosal surfaces and epithelial cells in the gut by
probiotics is facilitated through large surface proteins and mucus-binding
proteins MUP including Mucin-binding protein Lctococcus spp (MbpL)
and “transaldolase” also other surface proteins which facilitate adhesion to
intestinal cells such as alpha-enolase Elongation factor Tu (EF-Tu),
Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) and Chaperonin
(GroES) ( Gordon et al.,2012). pH and temperature also influence adhesion
characteristics, other properties like antimicrobial activity for example the
probiotics Lactobacillus frementum ,[Link] , Bifidobacterium breve
and [Link] are capable of inhibiting pathogens such as [Link] ,
Salmonella and Listeria either directly or indirectly by inducing the
expression of genes with antimicrobial activity(Hanru et al., 2013).
however not all strains of a given species are active to the same degree,
recent studies have suggested that genetically engineered probiotics that

35
express pathogen-specific adhesion proteins and could be a novel approach
to the prevention of infection (Ross et al.,2012).

[Link] Maintenance of intestinal integrity

Some types of probiotics have been shown to help strengthen the
intestinal barriers by making the gut less preamble and boosting the
epithelial defense system against harmful bacteria, this kind of
improvement mostly happens by changing the mucus layer and tight
junction “proteins that keep the intestines healthy” (Gordon et al.,2012) For
instance, Lactobacillus rhamnosus (LGG) supernatants have shown that
they can stop alcohol from making colorectal adenocarcinoma cell line
(Caco-2cell) monolayers lose their barrier function by keeping epithelial
cells strong and lowering their permeability. The general function of the
LGG is to release a biological active substance that modulates the tight
junction in the intestinal epithelium, over all changes in mucin
composition, intestinal permeability, and the balance between the rate of
apoptosis of damaged enterocytes and the production of new enterocytes all
effect the integrity of the intestinal barrier (Ross et al.,2013). The gut
barrier may become disrupted as o result of condition like IBD and colon
cancer. Certain probiotics may alter the integrity of the gut (Hanru et al.,
2012).

[Link] Tight junction

The intestinal barrier is controlled by tight junction (TJ) proteins like
occluding, zonula occludens-1, claudin-1, claudin-2, claudin-4, junction
adhesion molecules-A and F-actin. These TJ proteins are found in the sub-

36
apical region of the lateralmembranes. They create a physical connection
between cells that keep the intestinal barrier intact (Butler et al.,2012)
However, in conditions like IBD, the breakdown of the intestinal mucosal
barrier can result in increased intestinal permeability due to aberrant
expression of certain TJ proteins in the intestinal epithelium. Although a
number of studies have shown that probiotics can preserve intestinal barrier
integrity via altering TJ proteins expression, in mice with experimentally
produced Crohn’s disease, the probiotic combination has been shown to
control the epithelial TJ proteins, occludin, thereby attenuating increased
gut permeability (Samak et al., 2013).

[Link] Mucin expression

Mucin structure has a role in maintaining the integrity of the gut flora.
The majority of epithelial tissues create highly glycosylated
macromolecules called intestinal mucins, which are the main protein
component of mucus covering the gastrointestinal tract’s epithelium
produce, store, and exude mucins. In order to shield the mucosa from
bacterial overgrowth, mucins also gel and produce a protective mucus
blanket that covers the epithelial surface Goblet cell activity, and the
chemical makeup of intestinal mucus can be affected by luminal toxins and
the changes in the intestinal microbiota (Hanru et al.,2012). Furthermore,
mucin protein overexpression, including MUC1, has been linked to human
malignancies. Additionally, goblet cell secretions and mucins are impacted
by chemotherapy drugs. For example, it has been shown that the
chemotherapeutic drug 5-FU causes rats to have more cavitated goblet cells
and less goblet cells overall (David et al.,2013). The conclusion
reached was that the expression of these mucin genes is
crucial for bacterial adherence. In general, a lot of probiotics have the

37
ability to restore the mucus layer and restore intestinal integrity, when
treating a variety of intestinal conditions and illnesses marked by mucosal
damage, this feature may be therapeutically significant (Gordon et al.,
2013).

[Link] Cell proliferation

By preventing programmed, cell death as well as by encouraging cell
differentiation and cytoprotective processes, probiotics may increase cell
proliferation. After being challenged with pro-apoptotic drug staurosporine
(STS). Lin et al.2012 showed the anti-apoptotic and cytoprotective qualities
of LGG in vivo and in the vitro. By considerably lowering terminal
deoxynucleotidyl transferase (TUNEL) positive, LGG pretreatment
reduced apoptosis in intestinal epithelial IEC-6 and Caco-2 cells produced
by STS-affected newborn animals. More importantly, it has been revealed
that the anti-apoptotic characteristics of LGG were primarily attributed to
the suppression of caspase 3 activity and the control of anti-apoptotic
genes. Additional research revealed that, in contrast to pathogenic
Salmonella tryphimurium, LGG controlled apoptosis-related genes
differently (Hanru et al.,2012). According to reports, LGG controls cellular
migration and proliferation in addition to mitogen-activated protein kinase
(MAPK) pathways, which are essential for cell differentiation, proliferation
and cytoprotection. Moreover, it was shown that [Link] activated
Nuclear Factor kappa-light-chain-enhancer-of activated B cells (NF-KB) to
upregulate anti-apoptotic genes, while LGG induced anti-apoptotic gene
transcription without up-regulating proinflammatory genes, the increased
of cell expression is linked to cell proliferation (Hanru et al., 2012).

[Link] Cell apoptosis

38
 Apoptosis is a cell death mechanism that regulates the quantity of cells
in tissues and gets rid of individual cells. However, unplanned apoptosis in
some certaincells can be harmful. In many disorders, a rise in the ratio of
apoptosis to proliferation causes bacterial invasion and toxin delivery.
Probiotics can stop cell death brough by inflammation (Gordon et al.,
2012). Furthermore, acetaminophen-induced hepatotoxicity and
chemotherapy-induced small and large intestine apoptosis in rats, have
been demonstrated the capacity of probiotics to modify apoptotic and anti-
apoptotic proteins. In the other hand the probiotic can affect the apoptotic
and anti-apoptotic proteins that contribute to effectiveness in response to
cytokine-mediated inflammation and apoptosis in addition to including
gene expression associated with cell proliferation results in boost cell
growth (Hanru et al.,2012).

3.1.3 Alteration of Gut Microbiome Composition and

Function
Every person has a distinct gut microbiota composition that serves a
particular purpose in host nutrition metabolism, immunomodulation,
pathogen, defense and the preservation of the gut mucosal barrier’s
structural integrity (Marco et al.,2019). In contrast to the colon which has
slower flow rates, a gentler pH, and bigger microbial populations,
particularly anaerobic kinds, the small intestine has fast transit times and
high bile concentrations (Kaijian et al., 2022). The constitution of each
person’s gut microbiota is changed in their early years by external variables
including as known the usage of antibiotics and newborn transitions like
gestational date, delivery style, milk feeding techniques, and weaning
phase. (Emanuele et al.,2019). Individual differences in enterotypes, body
mass index (BMI) level, frequency of exercise, lifestyle, and cultural and

39
nutritional habits cause these personal and healthy cores native microbiota
to be largely constant in maturity. As a result, there is not a single ideal gut
microbiota composition because it varies from person to person.
Nonetheless maintaining a healthy host-microorganism balance is
essentialfor optimum metabolic and immunological processes as well as the
prevention of disease development (Antonio et al., 2019).

Fig 11: shows the gut microbiota composition and the affected organs by dysbiosis this figure
adapted from Forntiers microbiology 2022.

[Link] Factors influencing the gut microbiome

Human microbiota development is an evolving process, which
individuals displaying variances in microbial diversity and variety at
different stages of life. The formation of the gut microbiome is influenced
by a number of factors including age, host genetics, food, exercise,
smoking and medications. (Susan et al.,2013). Also, antibiotic usage,
newborn feeding, hospitalization of the infants and gestational age are other
variables that affect the gut microbiota (Paul et al.,2013).

40
[Link].1 Age
the human microbiota is formed at birth when an unstable population
settles in the gut. The environment (such as nursing staff and the air) is a
major source of colonizing bacteria, and these infants have lower intestinal
bacterial counts with less diversity in the early weeks of life. Initially
facultative anaerobes like Enterobacteriaceae, Streptococci, and
Staphylococci dominate more stringent hygienic conditions during delivery
combined with shorter hospital stays, which have reduced bacterial
exposure, leading to changes in the initial colonization pattern (Gerald et
al.,2013). The gut microbiota’s content is largely constant throughout
adulthood and is only momentarily changed by outside disruptions. The
microbiota’s diversity declines as people age, and the significant inter-
individual variance in microbial assemblages persists into old life.
Additionally, the chemistry of the elderly’s gut microbiota may fluctuate
based on the where they live, which is a proxy for drastically varied diets
(Catherine et al.,2013).

[Link].2 Diet
Unquestionably, diet has an impact on the gut microbiota’s composition
for both the host and the bacteria in the gastrointestinal tract receive
nutrients from their food, and it is possible to see variations in the
gastrointestinal microbiota constitute among group of people who follows
various diets, various cultural habits and ethnicity (Giacinto et al., 2019).
The gut microbiota’s metabolic activity may alter as a result of these diet-
related compositional changes, which may then trigger alterations in the
immune and inflammatory system. Despite efforts to alter the gut
microbiota’s makeup through dietary modifications (Paul et al.,2013).

[Link] Functions of the microbiome

41
 It can characterize the host and the microorganisms that live there as
‘superorganisms’ that carries out immunological and metabolic tasks
because of the variety, stability, resilience and symbiotic relationship of gut
microbiota and the host. Bile acid, lipids, amino acids, vitamins, and
SCFAs are just a few of the nutrients and metabolism that are extracted,
synthesized, and absorbed by commensal bacteria, which are major
regulators of digestion along the gastrointestinal system (Pauline et
al.,2018). By preventing the development of harmful bacteria, utilizing
available recourses and generating bacteriocins, gut microbiota plays a
critical immunological role in preventing their colonization. Additionally,
gut microbiota inhibits bacterial invasion by preserving the integrity of the
intestinal epithelium (Francesco et al., 2019). Through a variety of
competitive mechanisms, including food metabolism, pH change,
antimicrobial peptide releases, and their byproducts have been shown to
play a crucial role in controlling the growth, hemostasis, and functionally
of innate and adaptive immune cells, it is paradoxical to observe that while
the activities of the gut microbiota are substantially conserved across
people (Emanuele et al.,2019).

[Link].1 Metabolic function

Dietary fibers that digestive enzymes are unable to break down can be
broken down by the gut bacteria; by breaking down big polysaccharides
and alcohols, the microbiome therefore produces extra energy. The
microbiome generates proteases that can digest many compounds in the
large intestine, according to the MEROPS database. Additionally, the
microbiome’s positive benefits are linked to the synthesis of many vitamins
(Maurizio et al.,2024).

[Link].2 Structural function

42
 Under normal circumstances, the gut epithelium’s integrity is
maintained in part by the microbiome. Cytokines in the gut lumen are
unable to cross the gut epithelium in this case, pathogen including C.
difficile and E. coli may change its function. These bacteria’s dysbiosis
makes it easier for cytokines to diffuse inward (Giuesppina et al.,2024).

[Link].3 Protective function

The microbiota helps to maintain the stability of the gut surface, which
serves as a fundamental barrier. The microbiome’s synthesis SCFAs
fortifies this barrier and gives the epithelium more energy (Maruizio et
al.,2024).
Table 1: this table below shows that the beneficial effect of metabolites
produced from the gut microbiota on healthy situations. This information is

43
displayed in table according to the metabolites, the pathway involved, the
microbial agent in charge and the health advantages generated. Adapted
from National library of Medicine NIH 2024.

44
3.2 Clinical Manifestations and Health Implications of
Antibiotic-Associated Dysbiosis
Dysbiosis or an imbalance of harmful and protective bacteria in the gut,
is linked to several disease conditions. The microbial community in the gut
is influenced by dietary variables, the extensive impact that
microorganisms have on both local and systemic immunity and the changes
of these communities’ structure are followed with long- or short-term
consequences (Yee et al., 2013). Antibiotic-Associated Diarrhea AAD,
CDAD and H. pylori infection are among the short-term consequences of
antibiotic usage, which can last anywhere from a few weeks to many
months. Long-term effects include the development of obesity, allergies,
asthma, atherosclerosis, diabetes, nonalcoholic fatty liver disease as well as
gastrointestinal disorders such as, IBD, IBS and autoimmune diseases
(Marcela et al., 2022).

45
 Fig 12: shows the effect of antibiotic exposure in early age to the gut microbiome leading to long-
term health conditions this figure adapted from eClincal Medicine 2024.
3.2.1 Antibiotic-Associated Diarrhea (AAD)
About 5 to 25% of individuals taking antibiotics get AAD, a frequent
side effect of antibiotic treatment. The type of antibiotic used and the risk
variables in individuals receiving treatment have an impact on the
occurrence of [Link] usual balance of the gut flora is upset by the
antibiotic therapy, the many physiological mechanisms of the normal
microflora that typically contribute to fermentation processes, decreased
generations of SCFAs, and the growth of pathogenic organisms like
[Link] can occur when the ecological balance of the normal gut flora is
disrupted (Leonardo et al.,2022) Diarrhea may be accompanied with colitis,
which is characterized by fever, stomach discomfort, hypoalbuminemia,
and leukocytosis. Many intermediate manifestations have been
documented, from mild diarrhea to fulminant PMC, which manifests as
watery diarrhea, fever ADD has identified two types of predisposing
factors: antibiotic class and host factor which is age and underlying
pathologies (E Bergogne 2000).

3.2.2 Overgrowth of Opportunistic Pathogens

Numerous illnesses might be brought on by an increased in gut flora.
The host genetics, diet and way of life all influence the interaction. The gut
microbiota can change over time during early growth and illness.
Alongside the gut microbiota, a number of host metabolic pathways are
regulated resulting in interactive host-microbiota signaling as well as
metabolic and immune-inflammatory response that physiologically link the
gut, muscle, liver and brain. In order to develop therapeutic techniques that
harness the gut microbiota to fight illness and improvehealth several
species of Bacteroides, Clostridium, Bifidobacterium and non- pathogenic

46
Enterobacteriaceae comprise the most abundant gut bacterial flora
(Farshad et al.,2016). Abscess development, sepsis, CD, toxicity, infection
and malnourishment might all result from an increase in these genera.
However, allergies in newborn, inflammation, malabsorption syndrome,
carbohydrate / fiber intolerance, atopic eczema and IBD accompany a
reduction in these bacteria. Even while the human gut bacterial species are
essential for immunity, nutrition and health in normal intestinal
concentrations, variations in their quantity (increase or decrease) may
contribute to the emergence of serious health conditions. Additionally, even
in healthy condition, gut microbiota can cause illness for example,
inflammation and epithelial invasion might happen. Age, hormonal
fluctuations, and immunological suppression are some of the main causes
of the abnormalities in the gut microbiota community (Ruhleman et
al.,2020).

3.2.3 Immunomodulatory and Nutritional Consequences

Gut microbes are fundamental to the overall health, through their influence
on immune response and metabolism. If disrupted by antibiotics, it can lead
to short-term and long-term damage to the overall health.

[Link] Immune System Dysfunction

Exposure of the gut microbiome to antibiotics can lead to immune
disfunction. Antibiotics affect the gut microbiome through various
mechanisms, including direct and indirect ones (Zhang et al., 2019). Direct
mechanisms include antibiosis through the production of various
metabolites like bacteriocins, organic acids, and antioxidant compounds,
and nutrient-space competition. On the other hand, indirect mechanisms
involve modulation of the host gut microbiota and immune system. These
combined direct and indirect mechanisms effectively reduce dysbiosis and

47
bacterial infections through probiotics. The broader effect on modulation of
the immune system involves the induction of healthy components in the gut
microecology. A balanced microbial community allows the body to
regulate both specific and nonspecific immune responses effectively
(Rabetafika et al., 2023). Although the primary use of antibiotics is to
eliminate harmful microbes, their broad spectrum of action leads to the
non-selective elimination of the microbiome that resides within the gut
(Zhang et al., 2019). This situation is made more serious by the fact that
different antibiotic regimens, whether used with a single type or several,
have different spectrums of action. Thus, causing various changes in the
resident microbiome. Short- or long-term antibiotic treatment can have
multiple effects on the microbiome residing in the gastrointestinal tract. It
includes altered metabolism, decreased homogeneity of microbial
communities, and the emergence of antibiotic-resistant strains (Dahiya et
al., 2023).
The microbial changes will be weakening the immune system, making it
unable to effectively combat pathogens and maintain balance (Dahiya et
al., 2023). SCFAs essential metabolites of gut bacteria, are one of the main
mechanisms causing this immune dysfunction. Antibiotic-induced
deficiency of SCFAs leads to decreased levels of SCFAs and decreased
Th17 and Treg cells (Zhang et al., 2019). Increased intestinal inflammation
has been demonstrated after oral Candida albicans infection in mice (Pan
et al., 2021). This provides a link between microbial metabolic activity and
the regulation of the cellular immune response. The impact of antibiotic
disruption of gut bacteria extends to mucosal pattern recognition receptor
(PRR) signaling and the secretion of AMP in the gut. It also interferes with
the maturation and function of intestinal immune cells. Therefore,
antibiotic-induced changes are the basis for regulating the behavior of the

48
intestinal immune system at the cellular level (Dahiya et al., 2023). In other
words, innate lymphoid cells (ILC3s), which are present in tissues and are
essential for containing microbes in the intestinal lumen and preventing
their translocation via an interleukin (IL)-22-dependent mechanism, are
significantly affected by the loss of gut microbiota caused by antibiotics
(Guarner et al., 2024). Antibiotic-induced disruption of the gut microbiota
significantly reduces IL-22 production, making the body more prone to
invading pathogens. This abnormality further extends into helper T cell
populations beyond innate immunity. More precisely, an imbalance in the
gut microbiota leads to an altered ratio of type 1 to type 2 helper T cells,
impaired differentiation of naive T cells into regulatory T cells (Treg cells),
and reduced numbers of type 17 helper T cells. These changes together
disrupt the intestinal immune homeostasis and predispose toward intestinal
infections (Zhang et al., 2019).
The body's natural defenses, which are typically complemented by the
gut microbiota, are expose to notable influences. Factors that play a part in
determining the content and load of the gut microbiota include gastric acid
and bile, bactericidal compounds in the gastric glands and liver. AMP such
as defensins, lysozymes, and antibacterial lectins (Reg3γ) produced by
Paneth cells, or secretory immunoglobulin A (SIgA) produced by plasma
cells (Kesavelu et al., 2023). All of these factors play a part in keeping the
integrity of the intestinal wall and providing defense against pathogens. In
the complex signaling interaction between the gut microbiota and the host
immune system, gut microbiota molecules can regulate the immune system
through mechanisms such as inflammasome signaling or Toll-like receptor
(TLR) and NOD-like receptor (NLR) signaling. Another process involves
an imbalance between pro-inflammatory and regulatory immune cells
(Hrncir, 2022). Antibiotics reduce gram-negative bacteria by decreasing

49
TLR4- and MyD88-dependent signaling, and inhibit the expression of
Reg3γ, an AMP that acts against gram-positive bacteria. Clinical
significance of this is heightened, as evidenced by the reduction in
clearance of vancomycin-resistant Enterococci (VRE) in mice lacking
gram-negative commensal bacteria (Zhang et al., 2019).
Fortunately, probiotics offer an effective solution to these problems,
such as B. animalis, L. acidophilus, L. casei, L. johnsonii, and L.
rhamnosus. These probiotics shown to enhance innate and nonspecific
cellular immunity by activating macrophages, dendritic cells, TLRs, natural
killer cells, and B or T lymphocytes (Rueda-Robles et al., 2022). Probiotic
mechanisms for suppressing the growth of pathogenic bacteria and their
interaction with the host vary and depend on the strain. These mechanisms
typically involve modifying environmental pH, producing antibacterial
compounds and bacteriocins, competing for nutrients and growth factors, or
directly activating the host immune system (Origüela et al., 2025).
Among the spectrum of antimicrobial metabolites, bacteriocins are
specifically one of the most interesting subjects to research. Drissi et al.
(2015) categorized these metabolites into three categories (I, II, and III),
which exhibit narrow- to broad-spectrum antimicrobial activities (Drissi et
al, 2015). There is proof of their prevalence in gut microbes since scientists
report that 175 bacteriocins occur within Firmicutes, 79 occur within
Proteobacteria, 34 occur within Bacteroidetes, and 25 occur within
Actinobacteria in the Human Gut Microbiome (HGM).The existence of
such bacteriocin-producing bacteria is extremely advantageous as it can
make microbiome resilience possible through remodeling microbial
composition by excluding pathogen colonization., inducing host cell
production of (AMP), inducing tight junction protein expression, and
regulating the host immune system (Safarchi et al., 2025).

50
[Link] Nutrient Metabolism Alterations
In addition to their critical role in immunity, gut microbiota is also a
determinant of effective nutrient digestion; therefore, if they are disturbed
by antibiotics, this represents a great metabolic effect. These microbial
populations, which live at disparate densities in the locations of the gut, are
central to many physiological processes (Dahiya et al., 2023). This
encompasses the help in digestion of food and utilization of energy as well
as the synthesis of vitamins and amino acids. All these ubiquitous
metabolic activities highlight the extensive metabolic contributions of a
normally functioning microbiota (Zhang et al., 2019).
Central to the host physiology, the symbiotic relationship of the gut
microbiome contributes to metabolic homeostasis. These capabilities range
from the prevention of pathogen colonization, modulation of intestinal
permeability, facilitation of metabolism in various kinds of food like fibers
and certain sugars, synthesis of nutrients that are indispensable, such as
vitamins K, B-complex, and folate, and modulating both local and systemic
immune responses (Guarner et al., 2024). It illustrates the wide range of
bioactive compounds, nutrients, and host functions that gut microbes affect
significantly: bioactive compounds, including vitamins such as B6, B9,
B12, or K; enzymes enhancing gastrointestinal metabolic activities, such as
proteases, lipases, amylases, or lactases; and amino acids and peptides
showing established antioxidant and anti-inflammatory activities besides
SCFAs (Origüela et al., 2025).
One crucial factor is the direct interference of antibiotics with key
metabolic processes. Using metabonomic analysis, it can be shown that

51
antibiotics have a strong impact on gut lipids, bile acids, amino acids, and
their derivatives (Dahiya et al., 2023). The most important influence,
however, is on the production of SCFAs, critical bacterial fermentation
products of dietary fiber in the large intestine. SCFAs are generally
considered as having far-reaching effects on enterocytes, such as
maintaining epithelial integrity and controlling Treg differentiation and
accumulation, among other ways of regulating inflammation and immune
responses. Therefore, reduced production of SCFAs directly results in
negative outcomes like compromised epithelial integrity and shifted
inflammatory responses (Zhang et al., 2019).
The disturbance of metabolism is not just limited to the gut but becomes
systemic in implication. Effects on the host metabolic system, especially
glucose and lipid metabolism, are mediated by a number of factors that
include, but are not limited to, changes in bile acid composition, alteration
of production of SCFAs from dietary fiber, and conversion of choline to
Trimethylamine TMA. These pervasive changes together suggest a general
metabolic derangement across the host (Guarner et al., 2024). Moreover,
microbial metabolites hold great promise as biomarkers of health status. In
fact, a variety of gut microbiota metabolites may be acting as biomarkers
for the diagnosis of diseases. For instance, succinate, phenylacetic acid, and
3-(4-hydroxyphenyl)-lactate are some of the metabolites with potential in
diagnosing liver diseases. This therefore opens up potential avenues for
developing diagnostic and prognostic agents rooted in gut health
assessments (Hrncir, 2022).

3.2.4 Broader Health Implications of Gut Flora Disturbance

The derangement of the gut flora has an impact much beyond direct
immune and nutritional roles and initiates a cascade of more general health

52
abnormalities:The consequences of a disturbed gut flora are far from short-
lived immune responses and the metabolism of nutrients, causing a cascade
of broader health [Link] section addresses these more general
implications, showing how dysbiosis of the gut can cause systemic diseases
ranging from antimicrobial resistance to chronic diseases and
neurocognitive disorders (Hrncir, 2022).

[Link] General Dysbiosis and Antimicrobial Resistance

The concept of dysbiosis and its contributions to antimicrobial
resistance perhaps represent the root of many generalized health problems
arising from an imbalance of gut flora. It is suggested that antibiotics, in
particular those with complicated chemical compositions, may alter the
microbial ecology of the gut and thereby induce antimicrobial resistance in
pathogens and other diseases as after-effects of chemotherapy (Holy et al,
2023). This would imply a very important negative feedback loop whereby
antibiotic application can contribute inadvertently to further infection.
Understanding the definition of dysbiosis is important to understand its far-
reaching impact. The term dysbiosis has been defined as the disruption of
the symbiotic balance between microbiota and the host. This imbalance is
not static; rather, dysbiosis implies frequent changes and finally
disturbances in
the ecology of gut microbiota, which may be characterized as an
adaptation in the composition and functioning of the microbiota (Guarner
et al., 2024). Its origins are diverse dysbiosis can be caused by mainly two
factors that could be either environmental or host related. Ultimately, this
microbial imbalance can overwhelm the gut's intrinsic defense mechanisms
as the condition of dysbiosis overpowers the resistance and protection
capabilities of the microbial ecosystem in the gut (Safarchi et al., 2025).

53
The fine balance of the gut microbial community may be disrupted, with
consequences on patient health conditions such as enhanced susceptibility
to other microbial infections as a result of the loss of immunity,
development of gut inflammation, as well as an effect on the mental health
of the patient. This strongly links the health of the gut directly to systemic
immunity and mental health (Dahiya et al., 2023).

[Link] Compromised Gut Barrier

One of the most important consequences of gut dysbiosis is the
disruption of the intestinal barrier, 'leaky gut,' allowing passage for hostile
substances into the bloodstream (Kocot et al., 2022). Gut microbiota
protects the intestinal mucosa, mainly through mucosal-associated
commensals and, in the colon, through butyrate supply as an energy
substrate for colonocytes. This underlines how essential beneficial
microbes and their metabolic products, such as butyrate, are for the
integrity of this barrier. However, dysbiosis may lead to the destruction of
intestinal barrier integrity by itself (Shi et al., 2023). The systemic effects
of this destruction are multifaceted, since the resulting penetration through
pathogenic and non-pathogenic microorganisms promotes antigen
presentation to innate and adaptive immune cells and thus their activation.
This can extend over time to chronic body-wide inflammation (Guarner et
al., 2024).
Consequences of gut barrier disruption are not limited to local
gastrointestinal problems but significantly affect even cognitive functions.
Gut dysbiosis associated with cognitive impairment is characterized by
altered expression of tight-junction-proteins, brain-derived neurotrophic
factor, and the serotonin transporter, etc. This observation underlines the
deep and complex interrelation of the gut-brain axis in which the integrity

54
of the intestinal environment has a direct influence on neural health and
function (Dahiya et al., 2023 and Guarner et al., 2024).

[Link] Increased Risk of Secondary Infections

Antibiotics disturb the microbial balance in the gut, a condition known
as dysbiosis, which creates an environment favorable for the proliferation
of harmful organisms, thus considerably increasing the risk of secondary
infections. One serious and rather common result of this kind of imbalance
is that antibiotic-induced gut dysbiosis can itself trigger diarrhea and
recurrent infections caused by Clostridioides difficile (Dahiya et al., 2023).
Besides this particular risk, several other acute clinical conditions
commonly appear, including (AAD), (CDAD), and Helicobacter pylori
infection. These are some examples of immediate health issues as a result
of changes that antibiotics induce in the gut. It is also important to consider
the direct relationship of some of these conditions to each other: In some
cases, AAD can be caused by an overgrowth of C. difficile, referred to as
CDAD (Kesavelu et al., 2023).

[Link] Chronic Diseases and Metabolic Disorders

Of all the imaginable ramifications emanating from an imbalanced gut
microbiome, it significantly contributes to the development and progression
of chronic and metabolic diseases (Kesavelu et al., 2023). The disturbances
in gut microbiota are increasingly being recognized as being intricately
linked with the origins of many non-communicable diseases. Thus, if left
unattended, such unbridled dysbiosis has the potential to turn those
conditions that otherwise might have been curable into lifelong chronic

55
illnesses (Hrncir, 2022). Chronic gut flora imbalance poses a silent yet
pervasive threat, acting as a risk factor for a wide array of health
conditions. Dysbiosis is closely linked to gastrointestinal disorders
(including colitis, celiac disease, IBS, IBD, and leaky gut syndrome), liver
disease, colorectal cancer, Candida yeast infections, diabetes, and skin
conditions like eczema. This extensive list demonstrates dysbiosis's
systemic influence across various interrelated organ systems throughout the
body. (Dahiya et al., 2023).
More broadly, it has far-reaching implications: changes in the diversity
of the gut microbiota could, over time, correlate with a variety of
conditions, including cardiovascular disease, liver disease, obesity, and
diabetes. Moreover, one of the most critical factors for lifelong health is
early-life exposure to antibiotics. Indeed, studies have linked antibiotic use
during the early development phase of life with acute and chronic
conditions including inflammatory bowel disease, celiac disease, certain
cancers, metabolic disorders such as obesity and type 2 diabetes, allergic
diseases, autoimmune conditions, atherosclerosis, arthritis, as well as
neurodevelopmental, neurodegenerative, and other neurological diseases.
This reiterates that microbial disturbances in early life are not just transient;
this is actually a "long-term programming effect" on health trajectories.
The long-lasting nature of these influences is well highlighted in obesity;
various observational studies have suggested a link between antibiotic use
in early infancy and a higher risk of obesity. The mechanism underlying
this involves a metabolic adaptation whereby, through these changes in
composition, the microbiome of individuals affected by such compositional
changes at critical development phases becomes more adept at gaining
energy from different sources in their diet, hence affecting metabolism and

56
the propensity of the host for obesity (Dahiya et al., 2023 and Guarner et
al., 2024 and Kesavelu et al., 2023).

[Link] Allergies and Asthma

The link between an imbalanced gut microbiome-dysbiosis-and the
development of allergic and respiratory diseases is well established,
especially during infancy. Extensive use of antibiotics in very young
children has raised serious concerns, pointing out that early antibiotic
exposure correlates with the emergence of a variety of distinct health
problems throughout childhood, be they immunological, metabolic, or
neurobehavioral, both in isolation and in combination. In this work Aversa
et al, (2021) the analysis assessed the medical records of 14,572 children
born in Olmsted County, MI, between 2003 and 2011. The investigation
found that the use of antibiotics within the first two years of life, a period
considered critical-or the first 1,000 days-was associated with increased
risk of a variety of conditions such as asthma, allergic rhinitis, atopic
dermatitis, celiac disease, overweight, obesity, attention deficit
hyperactivity disorder, and learning disabilities (Aversa et al, 2021).
Guarner et al, 2024 provides strong epidemiological evidence for the highly
significant and debilitating impact that exposure to antibiotics in early life
may have upon broad aspects of health in childhood (Guarner et al., 2024).

[Link] Neurocognitive and Mental Health Issues

The gut-brain axis is one of the critical ways through which changes in
gut microbiota will have a great effect on the neurological and
psychological conditions of an individual. A great volume of research has
placed huge importance on active participation by gut microbiota in
activities of the brain, including cognition, the overall performance of the
brain, cases of cognitive decline, and even mental health conditions

57
(Guarner et al., 2024). This signifies a direct and huge impact of the gut on
the central nervous system. These can indeed be debilitating symptoms and
may include cognitive weakening manifested by depression, anxiety, and
difficulty thinking or concentrating (Safarchi et al., 2025). In such
cognitive problems, the root etiological mechanism usually points back to
the gut itself, since Prolonged dysbiosis caused by antibiotic intake can
weaken the host's immune response system. This leads to the important
inference that the cognitive weakening could be due to the effect of
dysbiosis on gut microbiota-brain communication, establishing in the
process a direct link between gut health and mental acuity (Dahiya et al.,
2023).

58
3.3 Therapeutic Strategies: The Role of Probiotics in
Counteracting Antibiotic Effects
In the context of gut health management, probiotics serve various
critical functions that are in line with the general goals of probiotic therapy.
As live microorganisms, probiotics are increasingly recognized as natural
candidates to either complement or possibly even replace traditional
antibiotic substances, which most commonly have been used against
bacterial infections that affect both humans and animals, thus offering a
promising therapeutic approach (Rueda-Robles et al., 2022). Their
application is not confined to mere adjunctive purposes; in regard to the
prevention and treatment of diseases, probiotics have earned increasing
recognition for their application against infectious diseases in human and
animal health. Significantly, one of the major applications associated with
probiotics involves their active intervention against the negative effects
induced by antibiotics. If antibiotic treatment has disturbed the microbial
balance within the gut, probiotics are the ones that can help restore good
microbial flora in the gut and eliminate the ensuing dysbiosis, hence
offsetting the unpleasant side effects of antibiotic treatment (Rabetafika et
al., 2023).
3.3.1 Mechanisms of Probiotic Action in Antibiotic-
Challenged Microbiota
They are able to produce antimicrobial metabolites and influence nutrient
metabolism in the process of gut homeostasis restoration.

[Link] Direct Pathogen Modulation and Competition

A basic mechanism by which probiotics exert their effects involves the
direct inhibition of pathogen growth and a keen competition within the

59
intestinal environment. Probiotics actively fight against injurious bacteria
by producing a variety of antimicrobial compounds (Hung et al., 2021).
Indeed, by possessing antagonistic properties, probiotics have been found
to act against the growth of gut pathogens through production of bioactive
metabolites such as bacteriocins, hydrogen peroxide, organic acids,
antioxidants, and AMP. Also, through the competition for nutrients and
attachment sites. Finally, through the modulation of immune system
functions (Holy et al, 2023). In other words, it means that probiotics not
only produce bacteriocins, organic acids, and hydrogen peroxide,
compounds inhibiting the pathogens, but also compete very strongly for the
available nutrition and for important adhesion sites on the intestinal
epithelium, impeding the overgrowth of noxious bacteria (Origüela et al,
2025). Such multimodal competitive and inhibitory action is an integral
constituent of their overall defense strategy against microbial attack
(Rabetafika et al., 2023).
Unlike the relatively less specific actions of organic acids, the probiotic
production of antimicrobial compounds is highly specialized and may be
targeted toward the proliferation of pathogenic bacteria. These bacteriocins
are largely categorized based on their biochemical characteristics: Class I
which consists of heat-stable lantibiotics, smaller peptides less than 5 kDa,
containing polycyclic thioether amino acids such as lanthionine, and which
have either a linear (A-lantibiotics) or globular (B-lantibotics)
conformation (Rabetafika et al., 2023). Class II bacteriocins which are also
heat-stable peptides but do not contain lanthionine and generally have a
molecular weight below 10 kDa (Perez et al., 2014). The final category of
bacteriocins is Class III, which is heatlabile and has a molecular weight of
more than 30 kDa (Rabetafika et al., 2023). A well-known example is the
AMP nisin, which exerts its mode of activity through interaction with

60
membrane-bound protein lipid II. This interaction mediates the formation
of pores within the membrane of the bacterial cell. The process is usually
followed by lysis, or breaking open, of the bacterial cell. One of the most
characterized A-lantibiotics, nisin, is a positively charged AMP produced
by Lactococcus lactis (Rabetafika et al., 2023 and Perez et al., 2014).
One of the probiotic strains, such as E. coli Nissle 1917 EcN, for
example, is known to secrete small protein compounds called microcins,
which exhibit potent antimicrobial activity, especially against pathogenic
Enterobacterales during periods of intestinal inflammation. In addition to
that, some commensals, usually favored by probiotic intervention, establish
an anaerobic environment in the gut by actively consuming the available
oxygen. This may be highly relevant, as many common pathogens,
including Salmonella, depend on good aeration to survive and multiply.
This mechanism is an example of how the commensal microbiota
contributes to colonization resistance by competing directly with pathogens
such as Salmonella enteritidis for oxygen, a key resource necessary for
pathogen expansion and growth (Hung et al., 2021).
Ultimately, these direct probiotic actions culminate in significant
reduction or complete elimination of pathogenic microorganisms and also
effectively mitigate dysbiosis, whether it originates through infections or
antibiotic exposure (Rueda-Robles et al., 2022).

[Link] Restoration of Gut Ecosystem and Barrier Function

probiotics essentially play a crucial role in the restoration of gut
microbiota composition. Most antibiotic treatments eventually deplete the
pool of beneficial bacteria and reduce overall microbial diversity. On the
other hand, probiotics will actively help to repopulate the gut with healthy
microbiota by fostering the growth of advantageous species while co-

61
inhibiting opportunistic pathogens, thus proving their role of paramount
importance in re-establishing microbial balance in a dysbiotic gut
(Rabetafika et al., 2023)
One of the important benefits derived from probiotics includes the
enhancement of intestinal barrier function. Thus, most therapeutic
approaches target the strengthening of this barrier, which is often
compromised either by the use of antibiotics or due to an infection. For
example, some Lactobacillus species have been found to restore gut
barriers damaged by antibiotics through enhanced expression of tight
junction proteins (Shi et al., 2023). The main approaches to the
antimicrobial activity of probiotics include competitive exclusion,
enhancement of intestinal barrier through enhanced expression of mucin
and proteins of tight junctions, secretion of antimicrobial compounds, and
regulation of immune system function (Rabetafika et al., 2023). An
adequate number of probiotic bacteria in the gastrointestinal tract further
promotes competition for nutrients and attachment sites, making conditions
unfavorable for potentially pathogenic bacteria to colonize. In essence,
probiotics strengthen the integrity of the gut barrier by upregulating genes
and proteins that play a critical role in signaling of tight junctions and
modulate the balance between apoptosis/proliferation of intestinal
epithelium cells (Yang et al., 2025).
[Link] Immune System Modulation
Probiotics interact actively with the host's immune system through the
induction and balancing of immune responses, reducing the ability of the
gut to support colonization by pathogens. Such immunomodulation
includes enhancement of phagocytic activity, induction of a balance
between pro-inflammatory and anti-inflammatory cytokines, and increased

62
production of IgA. The mechanism of action is either direct or indirect, as
mentioned before (Rabetafika et al., 2023).
Probiotics have been shown to significantly influence innate immunity,
increasing the cytotoxic activity of NK cells and enhancing the phagocytic
capacity of macrophages. In addition, they affect adaptive immunity by
interacting directly with key intestinal immune cells such as enterocytes,
dendritic cells, and Treg cells. In summary, probiotics modulate immune
responses by reducing inflammation and increasing host immune defense
(Yang et al., 2025). Their effects on host immunity are not limited,
including stimulation of phagocytic activity, precise balance between pro-
and anti-inflammatory cytokines, and enhancement in general cytokine and
immunoglobulin IgA production. One of the most relevant features of this
more general immunomodulatory role may well be the control of T-cell
activity and the maintenance of immune homeostasis. Probiotics influence
immune system balance, especially by acting on T-cell populations, which
are fundamental in gut homeostasis and in carrying out a successful
response against infectious challenges (Rabetafika et al., 2023 and Shi et
al., 2023).
Furthermore, certain Lactobacillus strains have been shown to promote
Treg expression via differentiation of CD4+Foxp3+ T cells and have been
identified as key players in the maintenance of intestinal homeostasis. The
initial antibiotic-induced alterations to immune functions are noteworthy:
microbial ligands under homeostatic conditions activate the transcription
factor Nuclear Factor kappa-light-chain-enhancer of activated B cells NF-
κB and the pro-inflammatory cytokine TNF-α. The depletion of the
intestinal microbiota caused by antibiotics decreases epithelial levels of
Microbe-Associated Molecular Patterns MAMPs, thus causing a

63
subsequent decrease in TLR signaling and resulting in the downregulation
of innate immune functions (Shi et al., 2023).

[Link] Metabolite Production and Cross-Feeding

Among the most important contributions of probiotics, the production of
metabolites plays a significant role, to which SCFAs belong. Probiotics
improve the intestinal environment by producing beneficial SCFAs, such as
butyrate, acetate, and propionate (Yang et al., 2025). These fatty acids, in
particular, become important modulators of immune responses, providing
essential energy for gut epithelial cells and creating an environment that
inhibits pathogen proliferation. Microbial-derived metabolites play a
critical role in colonic regulatory T-cell homeostasis. SCFAs, whether
directly produced by probiotics or induced by other bacteria, have deep-
seated anti-inflammatory properties, very important in an antibiotic-
challenged gut, which is usually prone to inflammation (Guarner et al.,
2024). Aside from influencing immune cells, SCFAs are also a significant
energy source for intestinal cells. Studies reveal these SCFAs participate in
colonic Treg cell development and interact with the G protein-coupled
receptor GPR43 on neutrophils to reduce immune cell infiltration into
tissues and significantly diminish inflammation. SCFAs may also stimulate
the differentiation of naive T cells into Tregs via enhancing FoxP3
expression (Shi et al., 2023).

One important mechanism involves cross-feeding, where metabolic

products from one bacterium are used by another to support its growth. In
accordance with this principle, in vitro culturing of Lactobacillus resulted
in increased amounts of specific molecules-ribose, adenine, and lactate-

64
which, in turn, promoted the in vitro growth of Flavonifractor, a
Clostridiales species known to bloom in vivo following Lactobacillus
administration. These findings provided important clues on how the
metabolomic changes induced by Lactobacillus administration create an
environment inhospitable to the growth of multidrug-resistant Klebsiella
pneumoniae MRKP. Subsequent studies specifically addressed whether
Lactobacillus administration, along with the associated changes in
microbiota-most notably, expansion of Clostridiales-would indeed translate
into gut environmental changes capable of inhibiting MRE growth. The
study concluded that the metabolomic changes induced by Lactobacillus
administration indeed create a hostile environment for the proliferation of
MRKP (Djukovic et al., 2022).

3.3.2 Therapeutic Applications of Probiotics

The recent discovery of the gut microbiome's role in health and disease
has thrust probiotics into the spotlight of current medical interventions.
These beneficial microorganisms will be increasingly effective for
improving a variety of health conditions by offering symptom-specific
support when conventional treatments have failed or may cause adverse
effects. Their application spans a variety of clinical scenarios aimed at
restoring balance and improving physiological functions.

[Link] Antibiotic-Associated and Clostridioides difficile

Diarrhea
Among the main therapeutic uses of probiotics, their efficacy for the
treatment of (AAD) and infections caused by Clostridium difficile is the
most pronounced. Clinical evidence revealed a reduced rate of both AAD
and (CDAD) following treatment with a probiotic cocktail, for example,
one that includes Lactobacillus acidophilus and Lactobacillus casei.

65
Furthermore, the efficiency of individual strains is well documented: hence,
a 28-day course of Lactobacillus reuteri administered to 31 antibiotic-
treated hospitalized patients managed to reduce the rate of AAD (Liao et
al., 2021). At the same time, however, one should consider that the efficacy
of the probiotic treatment may strongly depend on the degree of the
disease. Thus, though Lactobacillus strains distinctly improved the
symptoms of antibiotic-induced moderate or mild dysbiosis of the gut,
these positive effects were less or even completely absented in more
serious cases (Shi et al., 2023).

[Link] Eradication of Pathogenic and Antibiotic-Resistant

Bacteria
One of the most important therapeutic applications of probiotics is the
reduction of gut carriage of pathogenic or antimicrobial-resistant
Enterobacterales (Rabetafika et al., 2023). The principal aim in this regard
is to reduce injurious bacteria, including multidrug-resistant bacteria, in the
intestinal tract. This is mediated through direct inhibition of the growth of
pathogens, competition provided that the probiotics remain viable at the
site of action (Guarner et al., 2024). Probiotics, commonly from fermented
foods, intrinsically promote positive lactic acid-producing bacteria, which
naturally outcompete Enterobacterales. Other forms like multi-strain
probiotics and combined therapies with prebiotics or phytobiotics,
especially in livestock, have been shown to enhance the elimination of gut
Enterobacterales. Reduction of gut Enterobacterales carriage is related to
an increase in beneficial bacteria, for example, Bacteroides,
Bifidobacterium, Lactobacillus, SCFA-producers, and positive changes in
the gut metabolome (Hung et al., 2021). (Figure 13)

66
Fig 13: Shows that in humans, some clinical settings or interventions might promote intestinal carriage of
Enterobacterales. Probiotics might be beneficial in eradicating gut carriage of pathogenic or
antimicrobial-resistant Enterobacterales. This figure adapted from (Hung et al., 2021).

Lactobacillus species, for example, promote restoration of the

Clostridiales genera that inhibit intestinal colonization by
multidrugresistant Klebsiella pneumoniae (MRKP) in mice. However,
experiments using germ-free mice (GFM) showed that Lactobacillus
colonization is insufficient to inhibit MRKP colonization. One reasonable
interpretation is that Lactobacillus does not inhibit growth of MRKP
directly but promotes recovery of other commensals that limit expansion of
MRKP in antibiotic-treated mice (Hung et al., 2021 and Djukovic et al.,
2022).

[Link] Specific Conditions and Metabolic Influence

67
 Probiotics have also begun to be used in the management of a number of
health conditions. Their efficacy is under active investigation for a wide
range of diseases, including Helicobacter pylori infection, irritable bowel
syndrome (IBS), various vaginal infections and even in altering
cardiometabolic profiles (Rabetafika et al., 2023). Complementing their use
as therapeutic agents, evidence suggests that some probiotic strains,
including lactic acidand soil-based bacteria, exhibit both bacteriostatic
(growth-inhibiting) and bactericidal (killing) effects against select
pathogens (Guarner et al., 2024). These include Staphylococcus aureus,
Listeria monocytogenes, Pseudomonas aeruginosa, and Candida albicans,
thus diminishing their ability to colonize the human host. (Rabetafika et al.,
2023) In addition, several clinical trials have demonstrated probiotics to be
effective in treating various diseases showen in (Table no.?) (Rabetafika et
al., 2023)

Table 2. Some clinical trials and uses of probiotics in human health (Rabetafika et al., 2023).

Probiotics might have an important role in the modulation of the risk of

Human Papillomavirus HPV infection by altering the composition of the
vaginal microbiota. Specific species of Lactobacillus are crucial for
maintaining vaginal microbial stability (Rueda-Robles et al., 2022). There
was a drop in the abundance of Lactobacillus after antibiotic treatment,

68
associated with persistent BV and increased risk of HPV. L. crispatus is
closely related to low susceptibility to HPV and a stable cervicovaginal
environment, while on the other hand, L. iners predisposes to BV. Thus,
supplementation with L. crispatus may have a positive effect on reducing
the risk of HPV infection and in preventing the progression to cervical
intraepithelial neoplasia (Rueda-Robles et al., 2022 and Wan et al., 2023).

3.3.3 Factors Influencing Effectiveness and Future Directions

With an increasing awareness of the therapeutic potential of probiotics,
their consistent efficacy across diverse applications is subject to myriad
variables. Understanding such factors will allow the optimization not only
of current probiotic interventions but will also inform future research. This
section discusses the complexities underlying probiotic effectiveness, along
with emerging new areas of development and application. Various "Factors
Influencing Effectiveness & Future Directions" shape the efficacy and
future development of probiotics.

[Link] Probiotic Formulations and Administration

The type of probiotic formulation used is an important consideration.
For both single-strain and multi-strain probiotics-often referred to as
"cocktails"-are used, but multi-strain formulations often exhibit more
widespread effects (Yang et al., 2025). This is emphasized by clinical
applications, especially in antibiotic-induced dysbiosis, for which both
single and cocktail Lactobacillus strains have seen widespread use
(Kesavelu et al., 2023). In fact, probiotic cocktails of Lactobacillus,
Bifidobacterium, and Lactococcus have demonstrated enhanced
microbiological, clinical, and immunomodulatory effects during post-

69
antibiotic gut mucosa reconstitution. The main approach of such
interventions is the rebuilding of microbiota, since antibiotic treatment
directly affects gut microbiota. Thus, the ability of Lactobacillus strains to
promote gut microbiota re-building is a crucial point of their modulation
assessment. Most of the research efforts focus on two main purposes: the
stimulation of beneficial microbes and the inhibition of undesirable bacteria
or pathogens (Shi et al., 2023).
Recent research has pointed out some specific benefits, showing that
Lactobacillus strains can colonize or induce the growth of beneficial
microbes in the anaerobic intestinal tract through biofilm formation and
enhancement of gut mucosa (Yang et al., 2025). Besides that, Lactobacillus
plantarum has been shown to secrete plantaricin EF and maintain gut
barrier integrity by promoting LPS-repairing mechanisms (Shi et al., 2023).
Optimization of delivery methods is also a subject of active exploration.
New routes of administration, including rectal delivery, are under study to
achieve increased efficacy for specific conditions, thus providing
alternative approaches for the eradication of Enterobacterales from the gut
(Hung et al., 2021). Current research is developing genetically modified
probiotics with increased or precisely targeted therapeutic activities.
Indeed, besides natural probiotics, engineered variants specifically
designed to target Enterobacterales are under study to improve eradication
of gut colonization (Hung et al., 2021 and Yang et al., 2025).

[Link] Context-Dependent Efficacy and Individual Variability

The efficacy of probiotics is context-dependent, and this becomes even
more apparent with their strain-specificity and dosage. It seems to suggest
that the outcome of probiotic interventions crucially depends on the exact
strain(s) selected, dose used, and treatment duration (Guarner et al., 2024).

70
Due to such intrinsic complexity, it is hard to predict the eventual optimal
efficiency of some strains, including Lactobacillus, since there is huge
variability in strain-dependence, dosage, and application methodology.
Thus, consideration is warranted that probiotic effects are highly strain-
specific and also context-dependent-they may vary greatly based on the
particular strain involved, its dosage, the unique existing microbiota of an
individual, and the type of co-administered antibiotic (Kopacz et al., 2022).
This is further evidenced by several studies that investigated strain-specific
effects on antibiotic-induced dysbiosis. For instance, one strain, L. casei
CGMCC 12435 (LacC), demonstrated the induction of specific bacterial
taxa, such as Citrobacter and Bifidobacterium, increased the levels of
SCFA. Thus mitigating ampicillin-induced dysbiosis. In contrast, two other
strains tested, LacP and LacG, did not show a similar effect, clearly
indicating strain-specific restoration of the microbial community (Shi et al.,
2023).

3.3.4 Evidence from Clinical Studies on Probiotic-Antibiotic

Co-administration
Clinical trials form a substantial contribution to knowledge on the co-
administration of probiotics and antibiotics in terms of efficacy, variables
that affect efficacy, mechanisms of action, and safety. As a matter of fact,
such studies represent the backbone of present understanding of the roles
played by probiotics in mitigating the adverse effects of antibiotic therapy,
developing optimal strategies for their combined use, and guiding future
development in this important area of health.

[Link] Overall Effectiveness and AAD Prevention

Overall, probiotics demonstrate significant overall efficacy when
administered along with antibiotics to prevent (ADD). Such co-

71
administration decreases the risk of developing AAD in adults by 37%
(Goodman et al., 2021). Indeed, the available evidence strongly supports
the effectiveness of probiotics in preventing AAD in adults and children
receiving antibiotic therapy. The meta-analyses also established that
probiotics have a moderate but significant effect on preventing AAD
among different age groups: children, adults, and elderly. As for
outpatients, data indicate that probiotic use could be of benefit in reducing
AAD incidence (Guarner et al., 2024) and Blaabjerg et al., 2017). Beyond
reducing the incidence of AAD, probiotics also have a beneficial effect on
the course of diarrhea. a shorter duration of diarrhea, a higher 2-day
treatment efficacy, and a shortened length of hospital stay. These findings
all favor that probiotics enhance the overall therapeutic efficacy and
accelerate recovery (Huang et al., 2021).

[Link].1 Specific Conditions and Prevention

Probiotics also prove effective in preventing Clostridioides difficile
diarrhea. At 2017 meta-analysis, with moderate certainty, concluded that
probiotics are beneficial in preventing (CDAD) in patients receiving
antibiotics. Regarding Helicobacter pylori eradication, the 2022 Maastricht
VI/Florence Consensus Report on Helicobacter pylori infection
management reported that certain probiotics can effectively reduce
gastrointestinal side effects resulting from eradication therapies and
therefore result in a favorable treatment outcome. However, this was
qualified by the report stating that the quality of evidence was weak,
resulting in a grade of recommendation of moderate. It appears to indicate
that any improvement in the H. pylori eradication rate seen with probiotics
is due to mitigation of treatment-related side effects, rather than due to
direct antimicrobial activity against H. pylori itself (Guarner et al., 2024).

72
 In fact, compared to antibiotic treatments for other indications,
probiotics exhibited stronger protective effects against H. pylori eradication
regimens. Saccharomyces boulardii significantly improved, whilst
substantially reducing incidence of diarrhea. Lactobacillus strains also
improved eradication rate and substantially reduced incidence of diarrhea
(Liao et al., 2021).

[Link] Factors Influencing Efficacy

Co-administration of probiotics depends on a number of factors, which
include dose, timing, and probiotic strains. All these factors require serious
attention during the design of treatment for the best outcomes because the
appropriateness of selection and application is decisive in view of the fact
that their poor choice may substantially reduce therapeutic efficiency.

[Link].1 Dose and Timing

For any probiotic therapy to succeed, both the dose and the timing of
administration are critical to optimal outcomes. Subgroup analyses
consistently demonstrate that a higher dose of a given probiotic strain has a
more pronounced protective effect compared to a lower dose. Higher doses
of probiotics were also seen to be more effective in preventing AAD,
though this advantage did not translate into significant improvements in
diarrhea duration or other symptoms (Goodman et al., 2021 and Wanyama
et al., 2025).
Timing of Probiotic Administration Timing is critical in the
administration of probiotics. A probiotic duration equal to the length of the
antibiotic course is more effective than continuing the administration for at
least seven days beyond the end of antibiotic treatment. Similarly, starting
the use of probiotics within the first two days of antibiotic treatment is
more effective in preventing diarrhea than starting after this period.

73
Overall, this is somewhat reflected in clinical practice, with the majority of
effective probiotic regimens starting as soon as possible, either before or
within 1–2 days of antibiotic commencement. Specific protocols vary; a
common approach is the administration of probiotics for 7–14 days after
cessation of antibiotics (Liao et al., 2021 and International Scientific
Association for Probiotics and Prebiotics (ISAPP), 2024).

[Link].2 Combined vs. Single Probiotics and Species-Specific

Effects
Single and combined probiotic formulations should be selected
according to their species specificity. Combined probiotic regimens seemed
to shorten diarrhea duration in subgroup analyses compared with single-
strain probiotic administration (Huang et al., 2021). Lactobacillus reuteri
and Saccharomyces boulardii had better therapeutic effects compared to
Lactobacillus rhamnosus or Lactobacillus acidophilus. Some studies
showed that multistrain probiotics have greater protection compared to dual
or single-strain formulas. (Huang et al., 2021 and Wanyama et al., 2025).
Specific strain and species-specific effects further underscore probiotic
variability in efficacy. Trials document that the therapeutic effect for some
strains, such as Lactobacillus rhamnosus and Lactobacillus acidophilus,
did not reach statistical significance. In contrast, most efficacy was
confined within species and mainly to the Lactobacillus and
Bifidobacterium genera. Of all probiotics, Saccharomyces boulardii is
probably the most effective, with moderate evidence to suggest it reduces
the duration of diarrhea (compared with placebo) and the risk of diarrhea
lasting two days or longer (compared with placebo or no treatment) (Huang
et al., 2021 and Goodman et al., 2021 and Li et al., 2021).

74
 Beyond the strains discussed above, there are many other probiotics that
have important benefits in the management of diarrhea. For example, some
individual strains and multispecies blends had significantly reduced
diarrhea duration compared to placebo, including Lactobacillus reuteri,
Bifidobacterium lactis, Saccharomyces boulardii, a mixture of
Lactobacillus spp. + Bifidobacterium spp. + Saccharomyces spp., and a
mixture of Bacillus spp. + Enterococcus spp. + Clostridium spp. In
prevention of prolonged diarrhea ≥ two days, Saccharomyces boulardii and
Lactobacillus reuteri resulted in significantly lower risk than placebo or no
treatment, based on moderate evidence (Li et al., 2021).

[Link].3 Baseline Risk and Age-Related Effects

Characteristics of the patients are an important modifier of the effect of
probiotic interventions. In regard to the baseline risk, the study reports that
low baseline AAD risk resulted in no significant difference with probiotics,
but there was a significant reduction in participants with moderate or high
baseline AAD risk. There are also age-related effects: in the study by
Jafarnejad et al. (2016), probiotics decreased the incidence of AAD in
participants aged 18 to 64, while no benefit could be found in participants
above 65 years (Goodman et al., 2021 and Wanyama et al., 2025).

[Link] Mechanisms of Action in Clinical Context

Probiotics work through a variety of ways in order to execute their
clinical benefits regarding antibiotic co-administration. These range from
direct antimicrobial action against pathogens, modulation of host immune
responses, and reconstitution of gut barrier integrity. All of these actions

75
together help in re-establishing gut homeostasis and mitigating the
dysbiosis caused by antibiotics.

[Link].1 Restoring Gut Microbiota and Intestinal Barrier

Restoration of Gut Microbiota: Probiotics are highly important in the
restoration of gut microbiota and gut homeostasis. They exert beneficial
effects by promoting and re-establishing the delicate balance of gut
microecology, which can be done through a variety of means, including
competing with pathogens for nutrition and receptors, producing
antimicrobial compounds which impede the growth of injurious bacteria,
and improving the immune response of the host. The intestinal barrier is
positively affected; for example, there is reversal of antibiotic-induced gut
barrier disruption after the administration of Lactobacillus GG and
tributyrin. Epithelial cell adhesion molecules play an important role in
reinforcing intestinal tight junctions, a process that probiotics can support
(Yang et al., 2025).

[Link].2 Immune System Modulation and Metabolism

Regulation
Probiotics can modulate immune function. Such as, in ampicillin-treated
mice, the levels of pro-inflammatory cytokines, TNF-α, Interleukin-6 IL-6,
monocyte chemoattractant protein-1, and IFN-γ, were lower in mice treated
with a complex Lactobacilli product, Jup-Y4, compared with a natural
recovery group without probiotic intervention. Besides immunity,
probiotics also contribute to regulating nutrient metabolism, including
SCFAs, bile acids, and glucose, thus blunting negative metabolic effects
elicited by antibiotics. Bidirectional communication of the gut-brain axis is
another point of impact. Research in this direction is ongoing to fully
understand how probiotics affect brain functions, including an

76
improvement in the synthesis pathway of Gamma-aminobutyric acid
GABA within the gut (Yang et al., 2025).

[Link] Impact on Antibiotic Resistance

Probiotics also have a place in the management of antibiotic resistance.
They have shown potential in reducing antibiotic resistance, although a
more profound understanding of the transmission and mechanisms of
antibiotic resistance genes within probiotics themselves is still needed.
However, the combined application of probiotics and antibiotics can
alleviate selection pressure and hence minimize the risk of both the
development and spread of antibiotic resistance (Yang et al., 2025).
Clinical observations further illustrate their impact on Multi-Drug-Resistant
bacteria. For example, in one arm of a probiotic mixture, the colonization
rate of patients with Multi-Drug Resistant MDR bacteria significantly
decreased. The beneficial effect was specific and notably associated with
the decolonization of Pseudomonas (Wieërs et al., 2021).
Not all probiotic effects were uncomplicated: the probiotic mixture arm
had, at first, a rise in colonization with AmpC-producing Enterobacteria,
unlike the minor, non-significant increases observed for the placebo and
Saccharomyces groups. This was followed by a significant decline in the
probiotic mixture arm, while the other groups did not change. Over the
longer term, Extended-spectrum beta-lactamase ESBL-producing bacteria
and Pseudomonas aeruginosa were present in the placebo and
Saccharomyces groups, but notably, ESBL producing bacteria were absent
in the probiotic mixture arm. (Table No.?) (Wieërs et al., 2021).

77
Table 3: Post-hoc analysis of bacterial cultures (Wieërs et al., 2021)

78
[Link] Safety and Probiotic-Drug Interactions
Co-administration of probiotics with therapeutic regimens calls for a
critical evaluation of their safety profile and potential drug interactions. As
their use becomes increasingly popular, the elucidation of these two key
aspects assures responsible clinical application and limits unexpected risks.
This section delves into the overall safety of probiotic coadministration,
and reviews documented cases of probiotic-drug interactions.

[Link].1 Safety Profile

Probiotic co-administration safety is a paramount consideration. No
serious adverse events were reported in the majority of the studies. This is
further confirmed through statistical analyses, showing no statistically
significant increase in adverse events in the probiotic group. A meta-
analysis involving ten trials with 2,363 outpatient participants confirmed
this result and showed no statistically significant differences in the
incidence of any adverse event between probiotic and control. These
findings put together suggest that the use of probiotics is generally safe
(Goodman et al., 2021 and Liao et al., 2021 and Blaabjerg et al., 2017).
In vulnerable populations, however, the use of probiotics should be
undertaken with great caution. Since most probiotic products are developed
for generally healthy people, their application in those with impaired
immunity or in those with serious conditions should be strictly limited to
strains and indications for which there is a firm basis of evidence regarding
safety and efficacy within such patient populations (Guarner et al., 2024)
[Link].2 Probiotic-Drug Interactions
Probiotics interact significantly with drugs on bioavailability and their
effects through various mechanisms of absorption and metabolism, exerting
different clinical responses. Examples are enhancing drug action, such as

79
Amiodarone and Amlodipine, influencing metabolism like Sulfasalazine
and Nitrazepam, or reducing toxicity like Irinotecan and NSAIDs. While
some probiotics inactivate drugs like Digoxin, others necessitate higher
dosing like Tacrolimus. The interactions are very complex and context-
dependent; thus, one has to be very careful in clinical practice. (Purdel et
al., 2023).

[Link] Gut Microbiome Diversity and Clinical Relevance

The composition and diversity of the gut microbiome are increasingly
recognized as central to human health, and it's, therefore essential to
understand how interventions, especially probiotics during antibiotic
therapy, influence this delicate ecosystem. While there is extensive use of
probiotics, their precise impact on the restoration of diverse microbial
communities is still under active scientific investigation. Research shows
that probiotics administered alongside antibiotic treatment do not
commonly alter diversity indices of the gut microbiome significantly. The
minimal and temporal nature of these effects, along with the gut's natural
tendency for restoration within 3-8 weeks independent of probiotic
supplementation, provides the premise that routine probiotic use for
maintaining microbial balance and diversity in itself is probably not
justified. Thus, the changes in composition were just as likely to happen in
the intervention group as in the control group. In consequence, any benefits
in the forms of routine probiotic supplementation during antibiotic
treatment would, therefore, be very questionable (Elias et al., 2023).
Most importantly, clinical relevance is key, especially where strain-
specific probiotic supplementation alongside antibiotics may prevent
Clostridioides difficile infection or (ADD) in vulnerable groups. This
recommendation is similar to those currently given by the American

80
Gastroenterological Association AGA and World Gastroenterology
Organisatio WGO. However, from these, no additional benefits are found
regarding microbiome composition, and further studies need to be done to
delineate the complex interrelation among clinical manifestations,
microbial diversity indices, and taxonomic composition necessary to
explain the gut microbes' role in human health and its modulating factors.
(Elias et al., 2023).

81
Conclusion
1. Probiotic bacteria play a key complementary role in
maintaining gut microbial balance during antibiotic
therapy.
2. Using probiotics with antibiotics can help the body
tolerate the treatment better and speed up the recovery
of gut microbiota.
3. Probiotic survival depends heavily on diet, storage
conditions, and the digestive environment, which
together determine how many probiotic cells reach the
gut alive.
4. Combining probiotics with proper prebiotics, suitable food
matrices, and minimal disruptive medications supports
their activity and helps them function more effectively in
the intestine.
5. Antibiotics-induced Dysbiosis highly effect microbial
diversity and the fundamental functions of probiotics to
maintain gastrointestinal homeostasis.
6. The result ensures the importance of evidence-based
strategies to recover the microbial balance and limits the
long-term clinical health conditions liknes to disrupted
gut microbiota.
7. Proven Efficacy in Mitigating Side Effects: Clinical
evidence strongly supports that probiotics are highly
effective in reducing the negative impacts of antibiotic
treatment, most notably in preventing (AAD) and

82
 Clostridioides difficile-associated diarrhea (CDAD). They
actively help restore the gut's microbial balance and
overall health.
8. Varied Mechanisms of Action: Probiotics exert their
beneficial effect against antibiotic-induced damage by
several important mechanisms, which include the
restoration of intestinal barrier integrity, regulation of
host immune responses, direct competition with
pathogenic bacteria, and altering nutrient metabolism
toward re-establishing gut homeostasis.
9. Specificity and Safety: Although generally regarded as
safe for co-administration with antibiotics, the efficacy of
probiotics is highly strain-specific, dose-dependent, and
timing-sensitive.
10. Nuanced Impact on Microbiome Diversity: Although
probiotics are effective for specific clinical outcomes,
current research suggests that their routine use may not
significantly alter or be justified solely for broadly
restoring the overall diversity of the gut microbiome,
which often recovers naturally after antibiotic treatment.

83
Recommendations
How to increase probiotics
1. Nourish Existing Probiotics with Prebiotics: The Probiotics population
can be significantly increased in the gut by feeding the good bacteria
already existing there. Prebiotics come in dietary fibers and compounds
not digested by your body. They travel to the large intestine, where the
gut's good microbes ferment them and obtain all the nutrients they need
to thrive and multiply.
2. Prebiotics enhance the growth and metabolism: Another role of these
special nutrients is that they play a significant role in encouraging and
stimulating the growth, reproduction, and metabolic activity of
probiotics present in one's intestine. This fermentation process also
produces other beneficial byproducts such as (SCFAs), which is
critically important for maintaining a healthy intestinal environment and
overall well-being.

84
3. Introduce New Probiotics Through Supplements: By directly taking
probiotic supplements, these contain live, beneficial microorganisms
that can colonize the gut if taken in sufficient amounts. They support a
direct rebalancing of your intestinal flora and an inhibition of harmful
bacteria, thus contributing to various health benefits.
4. Supplements vs. Nourishment: Prebiotic supplements feed the
probiotics that already exist, while probiotic supplements are the
probiotics taken to introduce new strains. Both approaches are of value
in a healthy gut. In combining prebiotic-rich foods or supplements with
targeted probiotic intake, it creates a synergistic environment for

optimal balance of the gut microbiota.

85
References
Abid, R., Waseem, H., Ali, J., Ghazanfar, S., Muhammad Ali, G., Elasbali,
A. M., & Alharethi, S. H. (2022). Probiotic yeast Saccharomyces: Back
to nature to improve human health. Journal of Fungi, 8(5), 444.
Afzaal, M., Saeed, F., Shah, Y. A., Hussain, M., Rabail, R., Socol, C. T.,
… & Aadil, R. M. (2022). Human gut microbiota in health and disease:
Unveiling the relationship. Frontiers in Microbiology, 13, 999001.
Akhavan, N., Hrynkiewicz, K., Thiem, D., Randazzo, C., Walsh, A. M.,
Guinan, K. J., O’Sullivan, J. T., & Stadnicka, K. (2023). Evaluation of
probiotic growth stimulation using prebiotic ingredients to optimize
compounds for in ovo delivery. Frontiers in Microbiology, 14, 1242027.
Arrieta, M.-C., Stiemsma, L. T., Amenyogbe, N., Brown, E. M., & Finlay,
B. (2015). The intestinal microbiome in early life: Health and disease.
Frontiers in Immunology, 6, 427.
Arsic, B., Barber, J., Čikoš, A., Mladenovic, M., Stankovic, N., & Novak,
P. (2018). 16-membered macrolide antibiotics: A review. International
Journal of Antimicrobial Agents, 51(3), 283–298.
Aversa, Z., Atkinson, E. J., Schafer, M. J., Theiler, R. N., Rocca, W. A.,
Blaser, M. J., & LeBrasseur, N. K. (2021). Association of infant
antibiotic exposure with childhood health outcomes. Mayo Clinic
Proceedings, 96(1), 66.
Baral, K. C., Bajracharya, R., Lee, S. H., & Han, H. K. (2021).
Advancements in the pharmaceutical applications of probiotics: Dosage
forms and formulation technology. International Journal of
Nanomedicine, 16, 7535–7556.

84
Barrenechea, V., Vargas-Reyes, M., Quiliano, M., & Milón, P. (2021). A
complementary mechanism of bacterial mRNA translation inhibition by
tetracyclines. Frontiers in Microbiology, 12, 682682.
Bergogne-Berezin, E. (2000). Treatment and prevention of antibiotic
associated diarrhea. International Journal of Antimicrobial Agents,
16(4), 521–526.
Blaabjerg, S., Artzi, D. M., & Aabenhus, R. (2017). Probiotics for the
prevention of antibiotic-associated diarrhea in outpatients—a systematic
review and meta-analysis. Antibiotics, 6(4), 21.
Cao, C., Kwok, L.-Y., Zhang, M., Zhang, H., Zhang, W., & Yao, G.
(2021). Lactobacillus casei Zhang exerts probiotic effects to antibiotic-
treated rats by rebalancing gut microbiota and metabolome.
Computational and Structural Biotechnology Journal, 19, 5888–5897.
Chai, X., Liu, L., & Chen, F. (2024). Oral nitrate-reducing bacteria as
potential probiotics for blood pressure homeostasis. Frontiers in
Cardiovascular Medicine, 11, 1337281.
Chandrasekaran, P., Weiskirchen, S., & Weiskirchen, R. (2024). Effects of
probiotics on gut microbiota: An overview. International Journal of
Molecular Sciences, 25(11), 6022.
Chapot-Chartier, M. P., & Kulakauskas, S. (2014). Cell wall structure and
function in lactic acid bacteria. Microbial Cell Factories, 13(Suppl 1),
S9.
Cohen, C. R., Wierzbicki, M. R., French, A. L., Morris, S., Newmann, S.,
Reno, H., … & Hemmerling, A. (2020). Randomized trial of lactin-V to
prevent recurrence of bacterial vaginosis. New England Journal of
Medicine, 382(20), 1906-1915.

85
Cusumano, G., Flores, G. A., Venanzoni, R., & Angelini, P. (2025). The
impact of antibiotic therapy on intestinal microbiota: Dysbiosis,
antibiotic resistance, and restoration strategies. Antibiotics, 14(4), 371.
da Cruz Rodrigues, V. C., da Silva, L. G. S., Simabuco, F. M., Venema, K.,
& Antunes, A. E. C. (2019). Survival, metabolic status and cellular
morphology of probiotics in dairy products and dietary supplement after
simulated digestion. Journal of Functional Foods, 55, 126–134.
Dahiya, D., & Nigam, P. S. (2023). Antibiotic-therapy-induced gut
dysbiosis affecting gut microbiota—brain axis and cognition:
Restoration by intake of probiotics and synbiotics. International Journal
of Molecular Sciences, 24(4), 3074.
D’Amico, V., Cavaliere, M., Ivone, M., Lacassia, C., Celano, G., Vacca,
M., … & Lopedota, A. A. (2025). Microencapsulation of probiotics for
enhanced stability and health benefits in dairy functional foods.
Pharmaceutics, 17(2), 185.
D’Argenio, V., & Salvatore, F. (2015). The role of the gut microbiome in
the healthy adult status. Clinica Chimica Acta, 451, 97–102.
Dilmore, A. H., Kuplicki, R., McDonald, D., Kumar, M., Estaki, M.,
Youngblut, N., … Knight, R. (2025). Medication use is associated with
distinct microbial features in anxiety and depression. Molecular
Psychiatry, 30(6), 2545–2557.
Djukovic, A., Garzón, M. J., Canlet, C., Cabral, V., Lalaoui, R., García-
Garcerá, M., … & Ubeda, C. (2022). Lactobacillus supports
Clostridiales to restrict gut colonization by multidrug-resistant
Enterobacteriaceae. Nature Communications, 13(1), 5617.
Drissi, F., Buffet, S., Raoult, D., & Merhej, V. (2015). Common occurrence
of antibacterial agents in human intestinal microbiota. Frontiers in
Microbiology, 6, 441.

86
Duan, H., Yu, L., Tian, F., Zhai, Q., Fan, L., & Chen, W. (2022).
Antibiotic-induced gut dysbiosis and barrier disruption and the potential
protective strategies. Critical Reviews in Food Science and Nutrition,
62(6), 1427–1452.
Elias, A. J., Barna, V., Patoni, C., Demeter, D., Veres, D. S., Bunduc, S.,
… & Lenti, K. (2023). Probiotic supplementation during antibiotic
treatment is unjustified in maintaining the gut microbiome diversity: A
systematic review and meta-analysis. BMC Medicine, 21(1), 262.
Goodman, C., Keating, G., Georgousopoulou, E., Hespe, C., & Levett, K.
(2021). Probiotics for the prevention of antibiotic-associated diarrhoea:
A systematic review and meta-analysis. BMJ Open, 11(8), e043054.
Guarner, F., Bustos Fernandez, L., Cruchet, S., Damião, A., Maruy Saito,
A., Riveros Lopez, J. P., … & Valdovinos Diaz, M. A. (2024). Gut
dysbiosis mediates the association between antibiotic exposure and
chronic disease. Frontiers in Medicine, 11, 1477882.
Guarner, F., Sanders, M. E., Szajewska, H., Cohen, H., Eliakim, R.,
Herrera-deGuise, C., … & Melberg, J. (2024). World gastroenterology
organisation global guidelines: Probiotics and prebiotics. Journal of
Clinical Gastroenterology, 58(6), 533–553.
Han, S., Lu, Y., Xie, J., Fei, Y., Zheng, G., Wang, Z., Liu, J., Lv, L., Ling,
Z., Berglund, B., Yao, M., & Li, L. (2021). Probiotic gastrointestinal
transit and colonization after oral administration: A long journey.
Frontiers in Cellular and Infection Microbiology, 11, 609722.
Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., …
& Sanders, M. E. (2014). The International Scientific Association for
Probiotics and Prebiotics consensus statement. Nature Reviews
Gastroenterology & Hepatology, 11(8), 506–514.

87
Howarth, G. S., & Wang, H. (2013). Role of endogenous microbiota,
probiotics and their biological products in human health. Nutrients, 5(1),
58–81.
Hrncir, T. (2022). Gut microbiota dysbiosis: Triggers, consequences,
diagnostic and therapeutic options. Microorganisms, 10(3), 578.
Huang, R., Xing, H. Y., Liu, H. J., Chen, Z. F., & Tang, B. B. (2021).
Efficacy of probiotics in treating acute diarrhea in children.
Translational Pediatrics, 10(12), 3248.
Hung, Y. P., Lee, C. C., Lee, J. C., Tsai, P. J., Hsueh, P. R., & Ko, W. C.
(2021). The potential of probiotics to eradicate gut carriage of
pathogenic Enterobacterales. Antibiotics, 10(9), 1086.
International S. (2024). Probiotic use alongside antibiotics: A guide to
clinical FAQs. ISAPP Guide.
Jackson, M. A., Goodrich, J. K., Maxan, M.-E., Freedberg, D. E., Abrams,
J. A., Poole, A. C., … Spector, T. D. (2016). Proton pump inhibitors
alter the composition of the gut microbiota. Gut, 65(5), 749–756.
Jafarnejad, S., Shab-Bidar, S., Speakman, J. R., Parastui, K., Daneshi-
Maskooni, M., & Djafarian, K. (2016). Probiotics reduce risk of
antibiotic-associated diarrhea in adults. Nutrition in Clinical Practice,
31(4), 502–513.
Jannah, S. R., Yanti, R., Suroto, D. A., & Wikandari, R. (2022). Viability
and shelf life of probiotic instant coffee. International Journal of Food
Science, 2022, 1663772.
Jiang, S., Zhang, C., Han, Z., Ma, W., Wang, S., Huo, D., … & Zhang, J.
(2023). Native microbiome dominates over host factors in shaping
probiotic evolution. NPJ Biofilms and Microbiomes, 9, 80.

88
Kesavelu, D., & Jog, P. (2023). Understanding antibiotic-associated
dysbiosis and management approaches. Therapeutic Advances in
Infectious Disease, 10, 20499361231154443.
Kiepś, J., Juzwa, W., & Dembczyński, R. (2023). Imaging flow cytometry
of probiotic bacteria. International Journal of Molecular Sciences, 24(7),
6841.
Kim, M., Park, S. J., Choi, S., Chang, J., Kim, S. M., Jeong, S., … & Park,
S. M. (2022). Association between antibiotics and dementia risk.
Frontiers in Pharmacology, 13, 888333.
Kocot, A. M., Jarocka-Cyrta, E., & Drabińska, N. (2022). Importance of
biotics in gut barrier integrity. International Journal of Molecular
Sciences, 23(5), 2896.
Konstantinidis, T., Tsigalou, C., Karvelas, A., Stavropoulou, E., Voidarou,
C., & Bezirtzoglou, E. (2020). Effects of antibiotics upon the gut
microbiome: A literature review. Biomedicines, 8(11), 502.
Kopacz, K., & Phadtare, S. (2022). Probiotics for preventing antibiotic-
associated diarrhea. Healthcare, 10(8), 1450.
Krishna Rao, R., & Samak, G. (2013). Protection of gut barrier by
probiotics. Current Nutrition & Food Science, 9(2), 99–107.
Lathakumari, R. H., Vajravelu, L. K., Satheesan, A., Ravi, S., &
Thulukanam, J. (2024). Antibiotics and the gut microbiome. Medicine
in Microecology, 20, 100106.
Lee, N. K., Jang, H. J., & Paik, H. D. (2024). Non-lactic acid bacteria
probiotics. Food Science and Biotechnology, 33(9), 1997–2007.
Li, Z., Zhu, G., Li, C., Lai, H., Liu, X., & Zhang, L. (2021). Which
probiotic is most effective for treating acute diarrhea in children?
Nutrients, 13(12), 4319.

89
Liang, B., Yuan, Y., Peng, X. J., Liu, X. L., Hu, X. K., & Xing, D. M.
(2022). Future perspectives for Helicobacter pylori treatment. Frontiers
in Cellular and Infection Microbiology, 12, 1042070.
Liao, W., Chen, C., Wen, T., & Zhao, Q. (2021). Probiotics for preventing
antibiotic-associated diarrhea in adults. Journal of Clinical
Gastroenterology, 55(6), 469-480.
Liu, J., Tan, Y., Cheng, H., Zhang, D., Feng, W., & Peng, C. (2022).
Functions of gut microbiota metabolites. Aging and Disease, 13(4),
1106.
Liu, Q., Yu, Z., Tian, F., Zhao, J., Zhang, H., Zhai, Q., & Chen, W. (2020).
Surface components and metabolites of probiotics. Microbial Cell
Factories, 19(1), 23.
Loo, J. S., Oslan, S. N. H., Mokshin, N. A. S., Othman, R., Amin, Z.,
Dejtisakdi, W., … & Tan, J. S. (2025). Lactic acid bacteria production
and enhancements. Fermentation, 11(5), 241.
Ma, Y., Yang, J. Y., Peng, X., Xiao, K. Y., Xu, Q., & Wang, C. (2020).
Best probiotics for preventing C. difficile diarrhea. Journal of Digestive
Diseases, 21(2), 69–80.
Maftei, N. M., Raileanu, C. R., Balta, A. A., Ambrose, L., Boev, M.,
Marin, D. B., & Lisa, E. L. (2024). Probiotics health-promoting
properties. Microorganisms, 12(2), 234.
Magalhães, K. T., da Silva, R. N. A., Borges, A. S., Siqueira, A. E. B.,
Puerari, C., & Bento, J. A. C. (2025). Functional probiotic
microorganisms. Fermentation, 11(9), 537.
Maseda, D., & Ricciotti, E. (2020). NSAID–gut microbiota interactions.
Frontiers in Pharmacology, 11, 558924.
Matouskova, P., Hoova, J., Rysavka, P., & Marova, I. (2021). Stress effect
of food matrices on probiotic viability. Microorganisms, 9(8), 1625.

90
Mercado‐Monroy, J., Falfán‐Cortés, R. N., Muñóz‐Pérez, V. M., Gómez‐
Aldapa, C. A., & Castro‐Rosas, J. (2025). Probiotics and intestinal
barrier integrity. Journal of the Science of Food and Agriculture.
Millanao, A. R., Mora, A. Y., Villagra, N. A., Bucarey, S. A., & Hidalgo,
A. A. (2021). Biological effects of quinolones. Molecules, 26(23), 7153.
Min, M., Bunt, C. R., Mason, S. L., Bennett, G. N., & Hussain, M. A.
(2017). Effect of non-dairy food matrices on probiotic survival.
Microorganisms, 5(3), 43.
Nejadmansouri, M., Eskandari, M. H., Yousefi, G. H., Riazi, M., &
Hosseini, S. M. H. (2023). Probiotic microorganisms as stabilizers.
Scientific Reports, 13(1), 15915.
Nishiyama, K., Takaki, T., Sugiyama, M., Fukuda, I., Aiso, M., Mukai, T.,
… & Okada, N. (2020). Extracellular vesicles produced by
Bifidobacterium longum. Applied and Environmental Microbiology,
86(19), e01464-20.
Nojoomi, F., & Ghasemian, A. (2016). Effect of overgrowth or decrease in
gut microbiota on health. Arch Pediatr, 4(2), e34558.
Noufeu, T., Li, Y., Toure, N. F., Yao, H., Zeng, X., Du, Q., & Pan, D.
(2025). Overview of glycometabolism of lactic acid bacteria during
freeze-drying. Foods, 14(5), 743.
Oh, J. H., Jang, Y. S., Kang, D., Chang, D. K., & Min, Y. W. (2019).
Efficacy and safety of new lactobacilli probiotics. Nutrients, 11(12),
2887.
Origüela, V., & Lopez-Zaplana, A. (2025). Gut microbiota: Dysbiosis and
probiotics. Microorganisms, 13(5), 1084.
Pan, C. H., Lo, H. J., Yan, J. Y., Hsiao, Y. J., Hsueh, J. W., Lin, D. W., …
& Chen, Y. C. (2021). Candida albicans colonizes the gastrointestinal
tract. Frontiers in Microbiology, 11, 619878.

91
Pandey, N., & Cascella, M. (2023). Beta-lactam antibiotics. In StatPearls.
StatPearls Publishing.
Palleja, A., Mikkelsen, K. H., Forslund, S. K., Kashani, A., Allin, K. H.,
Nielsen, T., … & Pedersen, O. (2018). Recovery of gut microbiota after
antibiotic exposure. Nature Microbiology, 3(11), 1255–1265.
Payne, J., Bellmer, D., Jadeja, R., Spring, S., Holcomb, B., & Holt, B.
(2025). Impact of relative humidity on Bacillus probiotic viability.
Applied Food Research, 101045.
Perez, R. H., Zendo, T., & Sonomoto, K. (2014). Novel bacteriocins from
lactic acid bacteria. Microbial Cell Factories, 13(Suppl 1), S3.
Petakh, P., Kamyshna, I., & Kamyshnyi, A. (2023). Effects of metformin
on the gut microbiota. Molecular Metabolism, 77, 101805.
Power, S. E., O’Toole, P. W., Stanton, C., Ross, R. P., & Fitzgerald, G. F.
(2014). Intestinal microbiota, diet and health. British Journal of
Nutrition, 111(3), 387–402.
Purdel, C., Ungurianu, A., Adam-Dima, I., & Margină, D. (2023). Impact
of probiotic use on drug metabolism. Biomedicine & Pharmacotherapy,
161, 114468.
Rabetafika, H. N., Razafindralambo, A., Ebenso, B., & Razafindralambo,
H. L. (2023). Probiotics as antibiotic alternatives. Encyclopedia, 3(2),
561-581.
Rajab, S., Tabandeh, F., Shahraky, M. K., & Alahyaribeik, S. (2020).
Effect of lactobacillus cell size on probiotic characteristics. Anaerobe,
62, 102103.
Ramirez, J., Guarner, F., Bustos Fernández, L., Maruy, A., Sdepanian, V.
L., & Cohen, H. (2020). Antibiotics as major disruptors of gut
microbiota. Frontiers in Cellular and Infection Microbiology, 10,
572912.

92
Ranadheera, C. S., Evans, C. A., Adams, M. C., & Baines, S. K. (2012).
Gastrointestinal tolerance of probiotics in goat’s milk ice cream and
yogurt. Food Research International, 49(2), 619–625.
Rekatsina, M., Paladini, A., Cifone, M. G., Lombardi, F., Pergolizzi, J. V.,
& Varrassi, G. (2020). Influence of microbiota on NSAID enteropathy.
Advances in Therapy, 37(5), 1933–1945.
Rinninella, E., Raoul, P., Cintoni, M., Franceschi, F., Miggiano, G. A. D.,
Gasbarrini, A., & Mele, M. C. (2019). What is the healthy gut
microbiota composition? Microorganisms, 7(1), 14.
Rogers, M. A. M., & Aronoff, D. M. (2016). Influence of NSAIDs on the
gut microbiome. Clinical Microbiology and Infection, 22(2), 178.e1–
178.e9.
Rose, E. C., Odle, J., Blikslager, A. T., & Ziegler, A. L. (2021). Probiotics,
prebiotics and tight junctions. International Journal of Molecular
Sciences, 22(13), 6729.
Rueda-Robles, A., Rodríguez-Lara, A., Meyers, M. S., Sáez-Lara, M. J., &
Álvarez-Mercado, A. I. (2022). Effect of probiotics on bacterial
infections. Pathogens, 11(9), 986.
Safarchi, A., Al-Qadami, G., Tran, C. D., & Conlon, M. (2025).
Understanding dysbiosis and resilience in the gut microbiome. Frontiers
in Microbiology, 16, 1559521.
Salvadori, M., & Rosso, G. (2024). Update on the gut microbiome in health
and diseases. World Journal of Methodology, 14(1), 89196.
Salton, M. R. J., & Kim, K.-S. (1996). Medical Microbiology (4th ed.).
University of Texas Medical Branch at Galveston.
Senthil Kumar, S., & Sheik Mohideen, S. (2024). Chitosan-coated probiotic
nanoparticles. Scientific Reports, 14(1), 21182.

93
Shah, A. B., Baiseitova, A., Zahoor, M., Ahmad, I., Ikram, M., Bakhsh, A.,
… & Al-Zharani, M. (2024). Probiotic significance of Lactobacillus
strains. Gut Microbes, 16(1), 2431643.
Shi, Y., Luo, J., Narbad, A., & Chen, Q. (2023). Lactobacillus restoration
after β-Lactam antibiotic-induced dysbiosis. Microorganisms, 11(1),
179.
Shreiner, A. B., Kao, J. Y., & Young, V. B. (2015). The gut microbiome in
health and disease. Current Opinion in Gastroenterology, 31(1), 69–75.
Stamatopoulos, K., O’Farrell, C., Simmons, M., & Batchelor, H. (2021). In
vivo models to evaluate ingestible devices. Advanced Drug Delivery
Reviews, 177, 113915.
Staniszewski, A., & Kordowska-Wiater, M. (2021). Probiotic yeasts—
Characteristics and application. Foods, 10(6), 1306.
Treven, P., Pavelšek, D., Bogovič Matijašić, B., & Lorbeg, P. M. (2024).
Effect of food matrix on probiotic survival. Foods, 13(19), 3135.
van Dalen, R., Peschel, A., & van Sorge, N. M. (2020). Wall teichoic acid
in Staphylococcus aureus host interaction. Trends in Microbiology,
28(12), 985–998.
Vinueza, A. M. Z. (2024). Probiotics for preventing vaginal infections.
Cureus, 16(7), e11234.
Wan, B., Wei, L. J., Tan, T. M., Qin, L., & Wang, H. (2023). Mechanism
of Lactobacillus crispatus on cervical precancerous cells. Infectious
Agents and Cancer, 18(1), 5.
Wang, G., & Feng, D. (2019). Therapeutic effect of Saccharomyces
boulardii. Experimental and Therapeutic Medicine, 18(4), 2653–2659.
Wang, Y., Jia, X., & Cong, B. (2024). Mechanism of metformin on
intestinal microbiota. Frontiers in Microbiology, 15, 1396031.

94
Wanyama, H., Akhtar, T. S., & Abbas, S. (2025). Probiotic use reduces
antibiotic-associated diarrhea. Gastroenterology Review, 20(1), 5–16.
Weinbreck, F., Bodnár, I., & Marco, M. L. (2010). Can encapsulation
lengthen probiotic shelf-life? International Journal of Food
Microbiology, 136(3), 364–367.
Wendel, U. (2022). Assessing probiotic viability and stress tolerance.
Frontiers in Microbiology, 12, 818468.
Wieërs, G., Verbelen, V., Van Den Driessche, M., Melnik, E., Vanheule,
G., Marot, J. C., & Cani, P. D. (2021). Do probiotics prevent
colonization with multi-drug-resistant bacteria? Frontiers in Public
Health, 8, 578089.
Yan, S. Q., Shi, Y. Y., Yang, R., Li, R., Hang, F., & Zhang, H. (2025).
Boosting Lactobacillus acidophilus biomass. Fermentation, 11(10), 564.
Yang, S., Qiao, J., Zhang, M., Kwok, L. Y., Matijašić, B. B., Zhang, H., &
Zhang, W. (2025). Prevention and treatment of antibiotic-associated
adverse effects through probiotics. Journal of Advanced Research, 71,
209–226.
You, S., Ma, Y., Yan, B., Pei, W., Wu, Q., Ding, C., & Huang, C. (2022).
Promotion mechanism of prebiotics for probiotics. Frontiers in
Nutrition, 9, 1000517.
Yu, T., & Zeng, F. (2024). Chloramphenicol interferes with 50S ribosomal
subunit maturation. Biomaterials, 14(10), 1225.
Zádori, Z. S., Király, K., Al-Khrasani, M., & Gyires, K. (2023).
Interactions between NSAIDs, opioids and the gut microbiota.
Pharmacology & Therapeutics, 241, 108327.
Zhang, S., & Chen, D. C. (2019). Adverse effects of antibiotics on gut
microbiota. Chinese Medical Journal, 132(10), 1135-1138.

95
Zhou, P., Chen, C., Patil, S., & Dong, S. (2024). Probiotics, prebiotics, and
postbiotics in gut-immune harmony. Frontiers in Nutrition, 11,
1355542.
Zmora, N., Suez, J., & Elinav, E. (2019). You are what you eat: Diet,
health, and gut microbiota. Nature Reviews Gastroenterology &
Hepatology, 16(1), 35–56.
Zmora, N., Zilberman-Schapira, G., Suez, J., Mor, U., Dori-Bachash, M.,
Bashiardes, S., … Elinav, E. (2018). Personalized gut mucosal
colonization resistance to empiric probiotics. Cell, 174(6), 1388–
1405.e21.

96