Critical Reviews in Biotechnology, 28:173–212, 2008

Copyright c Informa UK Ltd.

ISSN: 0738-8551 print / 1549-7801 online
DOI: 10.1080/07388550802262197

Microbial Biosynthesis and Applications of Gentamicin:

A Critical Appraisal
C. Ganesh Kumar
Centre for Chemical Biology, Indian Institute of Chemical Technology, Uppal Road, Hyderabad, India

M. Himabindu and Annapurna Jetty

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Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology,

Uppal Road, Hyderabad, India

Gentamicin is an aminoglycoside antibiotic produced by various species of the genus Mi-

cromonospora and has received much attention in the recent years as a broad-spectrum antibiotic
for treatment of various infections. It exists as a complex of closely related aminoglycoside struc-
tures and the clinically significant one is the gentamicin C complex. This review article focuses
attention on the present status of knowledge and the main advancements achieved in the last few
decades on the subject of gentamicin with regard to its production, biosynthetic pathway, mode
of action, and uses. The various nutritional and environmental parameters affecting gentamicin
production and the factors affecting the release of bound gentamicin are discussed. Further,
strain improvement using UV and/or chemical mutagenesis can be applied to augment the ef-
ficiency of the producer strain and a number of case studies are presented. Different detection
For personal use only.

and quantitative methods for gentamicin estimation and the mode of action of gentamicin are
discussed in detail. This antibiotic finds extensive use in combination chemotherapy and as a
drug for different delivery agents for treatment of osteomyelitis and other recent applications
in gene therapy.

Keywords actinomycetes, aminoglycosides, antibiotics, combination chemotherapy, drug deliv-

ery, gentamicin, Micromonospora

INTRODUCTION antagonism between living organisms. The close examination

Antibiotics are metabolic intermediates or end products of of the literature provides an evidence of wide variation on the
different species of microorganisms and other living systems. usage of the term “antibiotic” by various researchers. Bentley
They are chemical substances produced in small concentrations and Bennett (2003) recently published an excellent review on
that are capable of killing or inhibiting competing microorgan- the origin and definition of antibiotics.
isms. Although few developments are acknowledged in the his- The first extensive therapeutic application of the active prin-
tory of medicine, nevertheless, this resulted in a greater impact ciple was carried out by Emmerich in 1889. He extracted a
on human life and society for the control of infectious diseases substance termed pyocyanase from the culture of Bacillus py-
caused by microorganisms. The history on the use of antibiotics ocyaneus which killed the bacilli of anthrax, diphtheria, typhoid
in folk medicine dates back some 2,500 years ago when the fever, and bubonic plague. Unknown at that time, the prod-
Chinese reported beneficial medicinal effects of the moldy bean uct actually contained two substances, now identified as antibi-
curd. Even the Egyptians had the practice of applying a poultice otics. Forty years later, in 1928, Alexander Fleming discovered
of mouldy bread to treat infected wounds. In the 19th century a contaminating Penicillum isolate with remarkable antibacte-
when Pasteur founded the science of bacteriology, it became fea- rial properties. He noted that this contaminating mold could lyze
sible to study these substances systematically. A few years later staphylococci colonies, and later he showed that the filtrate of the
in 1889, Vuillemin coined the term “antibiotics” to represent pure mold culture killed a number of microorganisms. He named
the active antibacterial principle “penicillin” since the mold was
a strain of Penicillum notatum (Fleming, 1929). This formed the
Address correspondence to C. Ganesh Kumar, Centre for Chem- archetype for all the discovered antibiotics. The importance of
ical Biology, Indian Institute of Chemical Technology, Uppal Road, Fleming’s discovery was realized a decade later by the German-
Hyderabad 500 007, India. E-mail: cgkumar1@[Link] British biochemist Ernst Chain and the Australian pathologist Sir
173
 174 C. GANESH KUMAR ET AL.

TABLE 1
Timeline for discovery of various antibiotics

Antibiotic Producer strain Discoverer Year

Penicillin Penicillium notatum Sir Alexander Fleming 1928

Gramicidin Streptomyces venezuelae Rene Dubois 1939
Actinomycin A Streptomyces sp. Selman Waksman and colleagues 1940
Streptothricin Streptomyces lavendulae Selman Waksman 1942
Streptomycin Streptomyces griseus Selman Waksman and Albert Schatz 1943
(graduate student)
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Chloromycetin (also called Streptomyces venezuelae John Ehrlich and Quentin Bartz 1947
Chloramphenicol)
Tetracycline (available as Streptomyces aureofaciens Benjamin Minge Duggar (patented by 1948
Aureomycin) Lloyd Conover of Pfizer)
Neomycin Streptomyces fradiae Selman Waksman and Hubert Lechevalier 1949
Actinomycin C Streptomyces chrysomallus Hans Brockmann 1949
Nystatin Streptomyces noursei Elizabeth Lee Hazen and Rachel Fuller 1950
Brown
Erythromycin Saccharopolyspora erythraea Robert Bunch and James McGuire (Eli 1952
(earlier Streptomyces erythraea) Lily)
Vancomycin Amycolatopsis orientalis E.C. Kornfeld (Eli Lily) 1952
Dactinomycin (Actinomycin D) Streptomyces parvullus Selman Waksman 1953
Hygromycin B Streptomyces hygroscopicus McGuire and Pettinger 1953
Sarcidin Streptomyces achromogenes Takeuchi and colleagues 1953
For personal use only.

Amphotericin B Streptomyces nodosus Squibb Institute for Medical Research 1955

Kanamycin Streptomyces kanamyceticus Hamao Umezawa 1957
Rifamycin Amycolatopsis mediterranei Grazia Beretta (Italy) and Pinhas 1957
Margalith (Israel)
Bleomycin Streptomyces verticillus Hamao Umezawa 1962
Daunorubicin Streptomyces peucetius Di Marco and colleagues 1962

Howard W. Florey who purified sufficient amounts of penicillin Gram-positive bacteria with high G+C (>55%) content and
antibiotic and showed that it would protect mice from infection. a unique group having different morphological, cultural, bio-
They subsequently developed this purified antibiotic prepara- chemical, and physiological characteristics (Goodfellow and
tion for human use. Further, with the assistance from the U.S. Williams, 1983; Lechevalier and Lechevalier, 1967). Actino-
government, Chain and Florey manufactured penicillin in 1941 mycetes are noteworthy producers of antibiotics and other “sec-
and made it commercially available. This episode marked the ondary metabolites” with biological activity, accounting for
dawn of the antibiotic era. The timeline of some early discov- nearly three-quarters of all the known products. Approaches
ered antibiotics is shown in Table 1. Following these discoveries, for the search and discovery of new antibiotics are generally
the pace for search and identification of new antibiotics acceler- based on screening of naturally occurring actinomycetes. Within
ated in the coming years. Today, over 100 different antibiotics the actinomycetes, members of the genus Streptomyces are pro-
are known and almost 90% of them are derived from different lific in nature and produced nearly 70–80% of antibiotics and
microorganisms and others are produced synthetically, either in other secondary metabolites with smaller contributions from
whole or in part. other genera, such as Saccharopolyspora, Amycolatopsis, Mi-
cromonospora, and Actinoplanes (Challis and Hopwood, 2003).
The genus Micromonospora is one of the groups of rare acti-
ACTINOMYCETES AS SOURCE OF ANTIBIOTICS nomycetes isolated on a massive scale and encompasses the
Majority of actinomycetes are free living, heterotrophic, and second largest group of culturable actinomycetes in soil. M.
saprophytic in nature with ubiquitous distribution in various nat- purpurea was first proposed by Ørskov (1923) as a genus be-
ural environments like soil, water, colonizing plants, and other longing to Actinomycetales that has attracted considerable at-
man-made environments. They are one of the major groups of tention as a useful source for antibiotics since the discovery of
soil population. Actinomycetes are filamentous prokaryotes and gentamicin C complex (Weinstein et al., 1963a, 1963b). The
form aerial hyphae at some stage of their growth. They are isolation of gentamicin-producing Micromonospora strains are
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 175

OH OH
6 4
OH 5 OH
HO HO
NH2 NH2 NH2
NH2 2
OH 1 3

Streptamine 2-Deoxystreptamine

HO
OH
HO
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HN NH
OH
H2N NH2
NH HN

Streptidine

FIG. 1. Aminocyclitol components of aminoglycosides. Numbering of the ring atoms are shown for 2-deoxystreptamine, begins
with the nitrogen-bearing carbon atoms having the R configuration (Stead, 2000).

reported from some of the unusual habitats such as mud and wa- fermentation products from different actinomycetes and re-
ter samples (Szabo, 1984), meadows (Zenova and Zvyaginstsev, cently a few semisynthetic derivatives were prepared and put
2002), peat swamp forest (Thawai, Tanasupawat, Itoh, Suwan- into clinical practice. This class of compounds possesses an
borirux, and Kudo, 2004), river mud (Shibata et al., 1980), and extended antibacterial spectrum with proven efficacy for treat-
For personal use only.

plant rhizosphere (Shirokikh and Merzaeva, 2005). For routine ment of serious infections, usually in combination with other
isolations, the soil, water, and sediment samples are often pre- drugs (Carbon, Collatz, and Humbert, 1986). However, the use
treated using different methods like heat treatment at 70◦ C in of aminoglycosides has decreased for some time due to their
a water bath for 10 min or chlorine treatment with 4 ppm am- perceived toxicity and effect of newer antimicrobial agents,
monia followed by 2 ppm chlorine (as sodium hypochlorite) for such as the third-generation antibiotics, cephalosporins and flu-
30 min (Rowbotham and Cross, 1977), microwave irradiation oroquinolones, which possess significant activity against many
(Bulina, Alferova, and Terekhova, 1997), electric pulses (Bulina, bacterial pathogens of medical importance (Janknegt, 1990;
Terekhova, and Tyurin, 1998), and extremely high-frequency Spelman, McDonald, and Spicer, 1989).
radiation (Li, Terekhova, and Gapochka, 2002) followed by Since resistance to these newer agents has emerged, there was
dilution plate technique. The obtained isolates are inoculated a recent surge in the use of aminoglycoside antibiotics especially
subsequently onto a suitable isolation agar medium like starch- for the treatment of nosocomial infections mainly due to their
casein (Küster and Williams, 1964), colloidal chitin (Hsu and potent bactericidal activity and ability to interact synergistically
Lockwood, 1975), M3 medium (Rowbotham and Cross, 1977), with other antimicrobial agents.
and Kidota’s cellulose benzoate (Sandrak, 1977). For selective Aminoglycosides are a large and diverse class of small,
isolation, the incorporation of several antibiotics in the isolation polycationic molecules closely related to carbohydrates whose
medium like novobacin (25–50 μg ml−1 ) (Sveshnikova et al., backbone structure consists of a six-membered aminocycli-
1976), gentamicin (1–10 μg ml−1 ) (Ivanitskaya et al., 1978) and tol ring saturated with amine and hydroxyl substitutions. The
tunicamycin (25–50 μg ml−1 ) (Wakisaka et al., 1982), nystatin aminocyclitol nucleus is linked by glycosidic bonds to two or
(25–50 μg ml−1 ) (Zenova and Zvyaginstsev, 2002) or deleteri- more aminosugars (aminoglycosides), and it is these specific
ous agents such as phenol and chlorhexidine gluconate solutions aminosugars which are a characteristic feature to distinguish
(Hayakawa et al., 1991), and sodium propionate (1 μg ml−1 ) the different drugs (Ristuccia and Cunha, 1982). The cyclitol is
(Zenova and Zvyaginstsev, 2002) were found effective. 2-deoxystreptamine in most cases with one exception of spec-
tromycin, which has a streptidine moiety (see Figure 1). The
AMINOGLYCOSIDES AND THEIR IMPORTANCE aminoglycosides are usually classified according to the pattern
Aminoglycoside antibiotic are valuable therapeutic agents of substitution of the cyclitol ring and the structure of their
and among the earliest discovered antibiotics. Streptomycin respective aminocyclitol unit as follows: (i) The aminoglyco-
was the first aminoglycoside discovered by Waksman and col- sides whose aminocylitol is streptidine ring (streptamine with
leagues in 1944, which initiated the era of aminoglycoside an- two guanidine functions). The only clinically important repre-
tibiotic therapy (Schatz, Bugie, and Waksman, 1944). Since sentatives are streptomycin and dihydrostreptomycin. (ii) Most
then many aminoglycosides were produced and identified as aminoglycosides having a 2-deoxy streptamine ring which is
 176 C. GANESH KUMAR ET AL.

sometimes N-substituted. Two of the hydroxyl groups of the ring the aminoglycoside chemotherapy for the treatment of several
are linked to amino sugar moieties either in position 4 and 5 (for Gram-negative bacterial infections (Weinstein et al., 1963a). It
example, neomycin, paromomycin, lividomycin and butrocin) was introduced for therapeutic use in 1969 in the United States
or in position 4 and 6 (for example, kanamycin, tobramycin, (Korzybski, 1978). Gentamicin is a complex of numerous de-
gentamicin and sisomicin). (iii) Certain compounds do not con- oxystreptamine antibiotics which are unique since they were
tain either aminosugars or the glycosidic bonds while often con- the first aminoglycosides to be isolated from the genus Mi-
sidered as aminoglycosides, for example spectinomycin. It con- cromonospora purpurea and/or M. echinospora, a source other
tains the aminocyclitol N ,N  -dimethyl-2-epi-streptamine linked than the genus Streptomyces from which most aminoglycoside
at both the 4- and 5-positions to a single carbohydrate moiety. antibiotics were identified. Production of gentamicin from re-
When compared with other agents, the optimal use of amino- lated species like M. sagamiensis (Okachi et al., 1974), M.
glycosides has contributed to less emergence of drug resis- purpurea var. nigrescens (Gado et al., 1975), and M. purpurea
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tance. By convention, most aminoglycosides originating from var. violaceae (Abrasimovskii, Valdimirov, and Bartoschevich,
the genus Streptomyces are labeled -mycins, whereas those orig- 1990) has also been reported. Commercial gentamicin consists
inating from the genus Micromonospora are labeled -micins. mainly of different gentamicin C components. Although gen-
Most of the aminoglycosides are naturally occurring substances tamicin C1 , C2 , and C1a are the most important members of
produced by actinomycetes of either the genus Streptomyces or the gentamicin family, they show striking structural similarities
Micromonospora. However, five exceptions to this regard exist, between the compounds of kanamycin family. In the gentam-
namely, amikacin (derived from kanamycin A), arbekacin and icin structure, the amino sugar is 3-methylamino-3-deoxy-4-C-
dibekacin (from kanamycin B), isepamicin (from gentamicin methyl-β-L-arabinose (garosamine) attached to the 6-position
B), and netilmicin (from sisomicin), which are semisyntheti- of deoxystreptamine unit (see Figure 2). These compounds
cally derived from these natural products. differ from the neomycins, kanamycins, and paromomycins,
which contain both C-methyl and N-methyl substituents (Rein-
GENESIS OF GENTAMICIN hart and Stroshane, 1976). Gentamicin is manufactured by dif-
The discovery of gentamicin by Marvin Weinstein’s group ferent companies and marketed with different brand names
For personal use only.

at Schering Plough in 1963 was a major breakthrough in (see Table 2).

R2 NHR1
R3 Purpurosamines
O
Gentamines
NH2
NH2 O

HO NH2
O
Deoxystreptamine
Garamine
O
HO CH3

Garosamine NH OH
H2C

Gentamicin R1 R2 R3
C1 CH3 H CH3
C1a H H H
C2 H H CH3
C2a H CH3 H
C2b CH3 H H

FIG. 2. Structure of gentamicin C complex with its components and subunits (Stead, 2000).
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 177

TABLE 2
Various brand names of gentamicin manufactured by different companies

Brand names Companies


R 
R 
R
Cidomycin , Cidomycin Intrathecal Injectable, Cidomycin Sterile Roussel, UK

R
Powder, Cidomycin Topical
R R 
R
Genticin , Genticin Paediatric Injection, Gentamicin L – BDH, Nicholas, UK
R 
R  R
Genticine Pure Powder, Gentisone HC , Genticin HC
Lugacin Lagap, UK
R R 
R 
R 
R
Duracoll , Garacol , Garalone , Garamycin , Garamycin Paediatric Kirby-Warrick Pharmaceuticals Ltd.,
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R 
R
Injection, Gentalline , Refobacin (Schering-Plough Corp.), UK
[Link]

R 
R
Garasol , Gentocin Topical Spray, MometamaxTM Otic Suspension, Schering-Plough Animal Health Corporation,
R
Otomax (Veterinary products for otic or topical use in dogs) UK
R 
R R
Gentamicin BDH, Gentamicin L – BDH, Gentigan E. Merck, UK

R
Minims Gentamicin S & N Pharma, UK
R
Alcomicin Alcon, Farillon, UK

R
Gentamicin Gentamicinum Chinoin Pharmaceutical and Chemical Works
Co. Ltd., Budapest, Hungary.

R 
R 
R 
R
PangramTM (vet.), G4 (vet.), Genta (vet.), Gentadog (vet.), Gentacat Virbac SA, France
(vet.)
PangramTM 5% Injection (vet.) Bimeda, Anglesey, UK; Virbac Ltd.,
Cambridge, UK
For personal use only.


R
G-Mycin Neofarma, Finland

R
G-Myticin Pedinol, Mexico

R
Gentamicin “Tyrol Pharma” Tyrol Pharma GmbH, Kundl, Austria
R
Garatec Technilab, Canada

R
Gentasporin M/s PCI Pharmaceuticals Pvt. Ltd., Mumbai,
India
Gentamycin M/s Themis Chemicals Ltd., Valsad, Gujarat,
India

R 
R
Genticyn eye drops, Genoptic eye drops (0.3%, w/v) Allergan India Pvt. Ltd., Bangalore, India

R
Gentacip (injection) Cipla Ltd., Mumbai, India
Gentamycin M/s Themis Chemicals Ltd., Valsad, Gujarat,
India
Bactigen (Eye or ear drops, 0.3%, w/v) FDC Limited, Mumbai, India
Garamycin (injection, 20 mg per 2 ml and eye/ear drops, 3 mg ml−1 ) Fulford (India) Limited, Mumbai, India
Enderm GM cream Leben Laboratories Pvt. Ltd., India
Clop-G Cream (gentamicin, 0.1%, w/w) Liva Health Care Ltd, Mumbai, India

R 
R
Gentamycin cream, Lyramycin (cream and injection) Lyka Labs Ltd., India

Lupigenta (Injection, 40 mg ml−1 ),
R
Lupin Ltd., Mumbai, India
Lupiderm GM (Cream, 0.1% w/w)
R
Genticyn B ear/eye drops (0.3%, w/v) Nicholas Piramal (India) Limited, Mumbai,
India

BIOSYNTHETIC PATHWAY OF GENTAMICIN initial studies on gentamicin production through submerged fer-
The gentamicin C complex produced by different species of mentation using M. purpurea revealed the presence of several
the genus Micromonospora is the clinically significant one and major components like gentamicin C1 , C1a , and C2 , and mi-
consists of gentamicin C1 , C1a , C2 , and C2b (sagamicin). The nor microbiologically active components in the culture broth
 178 C. GANESH KUMAR ET AL.

(Weinstein et al., 1963a, 1963b, 1965). The major components 2-deoxystreptamine (DOS) to which different amino sugar anal-
differed from each other by the degree of methylation at 6 -C ogous aminocyclitol moieties are appended (Benveniste and
position. The minor component (Cooper et al., 1971) closely Davies, 1973; Llewellyn and Spencer, 2006). The gentamicin
related to the gentamicin C’s was gentamicin C2b , a 6 − N - gene cluster in M. echinospora ATCC 15835 was recently in-
methylgentamicin C1a . Subsequently, several of these minor vestigated by three independent groups, namely a South Korean
components were identified (Daniels et al., 1975; Nagabhushan group (Kharel et al., 2004b), an English group (Unwin et al.,
et al., 1975; Wagman et al., 1972) as gentamicins A, A1 , A2 , 2004), and in M. echinospora DSM 43036 by a German group
A3 , D, X2 , antibiotics like Sch 14342 (Waitz et al., 1972), and (Aboshanab et al., 2006), and established the putative functional
intermediates like JI-20A and JI-20B (Ilavsky et al., 1975), role of the different genes involved in the gentamicin biosyn-
which showed structural relationship to the gentamicin C’s. thetic pathway using a cosmid library approach. The strategy
Further evidence gained by Testa and Tilley (1976) based on employed for the elucidation of the biosynthetic pathway in-
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radioscanning paper chromatography indicated that these mi- volved the use of DOS biosynthetic gene-specific probes con-
nor components generally remained near the origin and were structed based on the conserved region of the M. echinospora
present in the whole fermented medium to a smaller extent, ATCC 15835 chromosome that encoded homologues of the
suggesting that these may be the precursors to the gentamicin aminoglycoside biosynthesis genes. It has been verified from the
C’s. feeding experiments with S. fradiae that glucose is the source
The biosynthetic pathway for gentamicin production has been of DOS (Reinhart and Stroshane, 1976). The 2-deoxy-scyllo-
proposed by Testa and Tilley (1976) based on the initial studies inosose (DOI) synthase catalyzed the intramolecular carbocy-
carried out using M. purpurea Paro 346, an idiotrophic mu- clization of D-glucose-6-phosphate to 2-deoxy-scyllo-inosose
tant which produced paromamine disaccharide and blocked in (DOI) in the presence of NAD+ and Co2+ cofactors, which is
gentamicin production. It was noticed that gentamicin C1 was a key step in DOS biosynthesis (Kudo et al., 1997). The gene
not transformed by the mutant into any detectable active com- involved in this bioconversion step is either gtmA (Kharel et al.,
ponent, while gentamicin C2 was transformed into gentamicin 2004b) or gntB (Unwin et al., 2004). This multistep process
C1 . However, gentamicin C1a was transformed into gentamicin included the first oxidation step at the C-4 position of glucose-
C2b . Both these steps required an N-methylation step at the 6 -
For personal use only.

6-phosphate followed by elimination of a phosphate group from

position, suggesting the presence of two different pathways for the activated ulose moiety forming an enol or enolate interme-
the production of different gentamicin C’s. The chromatographic diate. Subsequent reduction at C-4 resulted in the opening of
data obtained from these studies provided evidence for the bio- the hemiacetal ring, and the last aldol-type intramolecular con-
transformation of different substrates added in the fermentation densation between C-1 and C-6 gave rise to DOI (Iwase et al.,
medium. Moreover, the N-methylation step at the C-6 amino 1998; Kudo et al., 1997, 1999; Yamauchi and Kakinuma, 1992,
group was identified as the key reaction in the conversion of 1995).
gentamicin C2 to C1 . It was also demonstrated in case of M. L-glutamine:keto-scyllo-inositol aminotransferase was puri-
rhodorangea NRRL 5326 that sisomicin can be biotransformed fied from the cell-free extracts of [Link] which catalyzed
to gentamicin C2b . The plausible mechanism involved in this the transamination reaction at the carbonyl carbon of 2-deoxy-
biotransformation is via 6 -N-methylation followed by (4 -5 )- scyllo-inosose to form 2-deoxy-scyllo-inosamine (Lucher et al.,
reduction activity (Lee et al., 1977). 1989; Tamegai et al., 2002). It was later identified that the en-
Based on these observations, Testa and Tilley (1976) sug- zyme L-glutamine:keto-scyllo-inositol aminotransferase was a
gested that different enzymatic reactions, such as transmethy- product of gene gtmB (Kharel et al., 2004b) or gntA (Unwin
lation, transamination, deoxygenation, and transglycosylation, et al., 2004) from M. echinospora ATCC 15835, which partici-
were possibly involved in the biosynthesis of gentamicin com- pated in this transamination reaction and utilized L-glutamine
ponents. These enzymatic reactions may irreversibly be depend- as an amino source and pyridoxal phosphate as a cofactor.
ing upon some unknown conditions which may contribute to the The functional role of the gene gtmB was similar to that
formation of the minor components. Further, these researchers identified in different genes from various microorganisms like
suggested an alternate possibility that some or all of the methyl tbmB from Streptomyces tenebrarius (Kharel et al., 2005), stsC
groups of the gentamicin’s major components were demethy- (45.5 kDa) from Streptomyces griseus N2–3-11 (Ahlert et al.,
lated, via oxidation mechanisms, and these methyl groups were 1997), spcS2 from the spectinomycin producer, Streptomyces
reused biosynthetically in the de novo synthesis of the minor spectabilis (Hyun et al., 2000), and brtR (50 kDa) from Bacillus
components. circulans (Huang et al., 2002). Further, it was identified that the
Various genetic and biochemical studies have recently gene product of either gtmC (Kharel et al., 2004b) or gntC (Un-
demonstrated the functional role of several genes and enzymes win et al., 2004) from M. echinospora ATCC15835 catalyzed
involved in the gentamicin biosynthetic pathway (see Figure 3). the dehydrogenation of the cyclitol ring to yield a suitable keto
Further, many aminocyclitol antibiotics such as neomycin, intermediate, 1-keto-2-deoxy-scyllo-inosamine, for the second
kanamycin, tobramycin, ribostamycin, and sisomycin synthe- aminotransfer reaction which is mediated by the dehydrogenase
sized by Streptomyces and Micromonospora share a common enzyme.
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 179

The gene product of either gtmD (Kharel et al., 2004b) or (Kelemen, Cundliffe, and Financsek, 1991). Further, the gene
gntF (Unwin et al., 2004) has been recognized to catalyze the product of gtmJ was found to exhibit significant homology to
conversion of 1-keto-2-deoxy-scyllo-inosamine to DOS which aminoglycoside 3 -phosphotransferases from different amino-
is supposed to be mediated via another L-glutamine-dependent glycoside producers: 46% with S. ribosifidicus (Hoshihiko et al.,
aminotransferase. This particular observation was based on the 1988) and about 44% with S. fradiae (Bibb, Findlay, and John-
earlier studies on the in vitro transamination of scyllo-inosose son, 1984) and M. chalchea (Salauze and Davies, 1991). These
and 2-ketoglutarate by the protein purified from M. echinospora proteins shared a common mechanism which involved the enzy-
ATCC15835 in both the transamination steps of the DOS biosyn- matic transfer of the γ -phosphate from ATP (phosphorylation)
thesis (Lucher et al., 1989). Further, two glycosyltransferases to the 3 -hydroxy moiety of the acceptor aminoglycoside. When
which are the gene products of gtmE and gtmG (Kharel et al., the aminoglycoside gets phosphorylated it can no longer func-
2004b) or gntD and gntZ (Unwin et al., 2004) within the gentam- tion as an efficient chemotherapeutic agent, and so the organisms
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icin cluster were predicted to exhibit the two glycosyl transfer were able to survive in the presence of aminoglycosides.
reactions in gentamicin biosynthesis which included the addition
of D-glucosamine to DOS to form the pseudodisaccharide inter- ANALOGS AND/OR DERIVATIVES OF GENTAMICIN
mediate paromamine and the subsequent addition of D-xylose to
paromamine to form gentamicin A2 . These two glycosyltrans- Geneticin
ferases also exhibited significant similarity with the B. circulans Geneticin (G418 sulfate) is a DOS antibiotic produced by
btrM protein. M. rhodorangea and is structurally related to gentamicin with
Further, the conversion of gentamicin A to X2 occurs by a C- a broad spectrum of inhibitory activity against bacteria, yeasts,
methylation step which involves the replacement of the H-group fungi, algae, and plant and animal cells (Davies and Jimenez,
with the methyl group at the 4 -carbon of the garosamine analog 1980; Jimenez and Davies, 1980; Ursic, Kemp, and Helge-
of gentamicin A. Another C-methylation step enables the con- son, 1981; Waitz et al., 1974). It has been tested as an an-
version of gentamicin X2 to G418 via the addition of methyl and tiparasitic agent, specifically with potent antiamoebic activity
hydroxyl groups at the 6 -carbon of the purpurosamine analog (Griffiths et al., 1998; Loebenberg, Counelis, and Waitz, 1975).
Geneticin interferes with the function of 80S ribosomes and
For personal use only.

of gentamicin X2 (Unwin et al., 2004). The N- and C-methyl

groups in the garosamine and purpurosamine subunits derived blocks protein synthesis in eukaryotic cells (Bar-Nun, Shnegow,
from L-methionine were verified earlier through feeding experi- and Beckmann, 1983; Eustice and Wilhelm, 1984). It finds use
ments (Lee et al., 1975). These steps were analogous to the gene as a drug marker for the selection of DNA-transfected cells and
product of Fms7 as observed in case of fortimicin biosynthesis has become a key factor in the successful development of human
from M. olivasterospora. Moreover, the gntK (Unwin et al., gene therapies. The activity of geneticin and hygromycin against
2004) or gtmI (Kharel et al., 2004b) protein in the gentamicin eukaryotic cells has been widely exploited for the design of
biosynthetic gene cluster shared about 57% homology with the recombinant DNA cloning vectors for use in the genetic manip-
Fms7 gene product in fortimicin KK1 biosynthesis (Dairi et al., ulation of yeasts, fungi, plants, and mammalian cells; a cloned
1992). The gntK or gtmI gene product(s) may possibly be in- aminoglycoside modifying enzyme (resistance) gene, usually an
volved in the C-methylation of the garosamine or purpurosamine aminoglycoside phosphotransferase (APH) gene (aph 3 ) which
subunits of gentamicin (Kharel et al., 2004b). provided as a counterselective marker (Jauert, Jensen, and Kirk-
As a first step to understand the genetic organization of patrick, 2005; Kim, Song, and Choi, 2004; Rodriguez-Saiz et al.,
the gentamicin production genes in Micromonospora, the resis- 2004).
tance genes from M. purpurea and M. rosea were cloned (Kele-
men, Cundliffe, and Financsek, 1991). In general, the antibiotic Isepamicin
biosynthetic genes were clustered with the corresponding an- In recent years, there has been an emergence of resistance
tibiotic resistance genes (Kharel et al., 2004b). Moreover, in towards existing aminoglycosides in certain geographical areas
antibiotic-producing microorganisms it is well recognized that and in hospitals, where isepamicin finds place as a prospec-
they confer resistance to the antibiotics they produced to prevent tive alternative aminoglycoside due to its potential antimicro-
self-death by three different mechanisms, namely (1) by pump- bial properties. Isepamicin (formerly Sch 21420 or 1-N-HAPA-
ing out the antibiotics, (2) by destroying the antibiotic warheads, gentamicin B) is a novel broad spectrum aminoglycoside syn-
and (3) by modification of the target structure (Walsh, 2000). All thesized from gentamicin B (Waitz et al., 1972), which is copro-
these three mechanisms existed in S. kanamyceticus as well as duced during fermentation of gentamicin. It was first developed
in M. echinospora. It has been demonstrated that two putative in 1978 by the company Schering-Plough via a semisynthetic
genes such as gtmF and gtmJ that conferred resistance existed route (Miller et al., 1978; Nagabhushan et al., 1978). Isepam-
in the gentamicin biosynthetic gene cluster in M. echinospora. icin was synthesized by N-acylation of gentamicin B with (S)-3-
The gene product of gtmF was observed to exhibit 100% ho- amino-2-hydroxypropionic acid (Nagabhushan et al., 1978) and
mology with the previously reported gentamicin-resistance ri- later launched in the market in 1988. The side chain is believed
bosomal RNA methyltransferase (Grm) from the same organism to block the access of a variety of aminoglycoside-modifying
 180 C. GANESH KUMAR ET AL.

enzymes to their target sites. Subsequently, an improved process It was earlier noticed that sisomicin is ionically bound to
for converting gentamicin B to isepamicin was patented (Tann, the cell walls of the producing strain. During routine cultiva-
Chiu, and Green, 1991). An alternate semisynthetic method for tion, as the concentration of the cell-bound sisomicin increased
isepamicin production by fragmentation was recently reported to high levels, the production rates gradually decreased due to
(Moon et al., 2005). product inhibition resulting in low yields of sisomicin (Schmidt-
A fluorescence polarization immunoassay (Uematsu et al., Kastner and Reimann, 1976; Shin et al., 1988). The strategy of
1988) and HPLC method using precolumn derivatization with MgSO4 (25–100 mM) addition to an air-lift fermenter allevi-
1-fluoro-2,4-dinitrobenzene and ultraviolet detection (Dionisotti ated feedback inhibition and facilitated the effective release of
et al., 1991) were developed for the measurement of isepam- the cell-bound sisomicin resulting in improved sisomicin yields
icin. A simple extraction procedure of isepamicin in plasma (Han and Shin, 1992; Shin and Han, 1995; Shin et al., 1994).
and dilutions of urine and dialysate samples using commer- Kim and his colleagues (1998) later confirmed the fact that si-
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cially available Cyano solid-phase cartridges was developed. somicin molecules ionically bind to the carboxyl groups of the
The assay has a quantitation limit of 100 ng ml−1 isepamicin peptidoglycan of the cell walls of the producing strain.
in plasma and 50 ng ml−1 in urine and dialysate samples (Mal- Further, extensive microbiological and chemical modifica-
oney and Awni, 1990). Later, a simple reversed-phase HPLC tions of sisomicin were carried out by Schering Corporation,
method was developed for the separation of the isepamicin from New Jersey, USA, to produce mutamicins (Testa et al., 1974)
the structurally similar related compounds (Vogel, DeFillipo, and netilmicin (Sch 20569 or 1-N-ethylsisomicin) (Fu and Neu,
and Reif, 2001). Isepamicin also possesses a high level of sta- 1976). The addition of exogenous DOS analogs like streptamine
bility to aminoglycoside inactivating enzymes and exhibits low and 2,5-dideoxystreptamine as precursors to the DOS requiring
levels of nephro- and oto-toxicity (Jones, 1995). Clinical stud- idiotrophic mutant of M. inyoensis 1550F resulted in the gener-
ies have revealed that isepamicin is equally effective and safe as ation of new sisomicin analogs (mutamicins) like Mutamicin 1
amikacin for the treatment of adult patients with complicated uri- and Mutamicin 2 (Sch 22591 or 5-episisomicin), respectively.
nary tract infections (Lee et al., 1999; Sturm, 1995), neutropenic Sch 22591 is a 5-epimer of sisomicin produced by the modifica-
and non-neutropenic paediatric patients with various bacterial tion at the 5-position of the deoxystreptamine ring of sisomicin
For personal use only.

infections (Vigano and Principi, 1995), nosocomial pneumonia (Testa et al., 1974; Waitz et al., 1978). Netilmicin (Sch 20569)
and septicaemia (Beaucaire, 1995), respiratory tract infections is a semisynthetic derivative of sisomicin and has an ethyl group
(Covi and Velluti, 1995), intra-abdominal infections (Leal Del located at the 1-N position of the deoxystreptamine moiety of
Rosal, 1995), pyelonephritis in children (Kafetzis et al., 2000), sisomicin. This antibiotic closely resembles the C1a component
leptospirosis (Kobayashi, 2001), and cancer (Kadar and Ilona, of the gentamicin complex (Fu and Neu, 1976; Phillips, Smith,
1980). and Shannon, 1977). The synthesis of 1-N -ethylsisomicin was
also reported by Wright (1976).
Sisomicin
Sisomicin (antibiotic Sch 6640, formerly known as rick- PRODUCTION OF GENTAMICIN
amicin) is an aminoglycoside antibiotic reported to be
produced by fermentation using a DOS-requiring idiotrophic Optimization of Fermentation Conditions
Micromonospora inyoensis mutant 1550F (Testa et al., 1974), Gentamicin is produced conventionally by submerged fer-
derived originally from M. inyoensis NRRL 3292 (Wagman, mentation and there is no solid state fermentation process de-
Testa, and Marquez, 1970; Weinstein et al., 1970). The id- signed for producing this antibiotic. In commercial practice the
iotrophic mutant was generated by blocking the biosynthetic optimization of medium composition is carried out to maintain
pathway step leading to 2-DOS; as a result it was unable to a proper balance between the various medium ingredients, thus
produce sisomicin without the exogenous supplementation of minimizing the amount of unutilized ingredients at the end of
2-DOS. Sisomicin is an analog of gentamicin and is character- fermentation. Medium design is often considered an empirical
ized by an unsaturated aminosugar in the purpurosamine subunit science and many medium formulations used for large-scale
with amino groups and a positive charge in its structure (Testa production employ complex and undefined cocktails of nutri-
and Tilley, 1975). Chen and coworkers (2006) reported that the ents supplemented as undefined sugars and peptones (Stanbury,
methylation step of the precursor gentamicin A in sisomicin Whitaker, and Hall, 1995). In general, many commercial produc-
biosynthesis was stimulated by the supplementation of 6–10 ers of fermentation products encounter a major issue of inherent
mg l−1 cobaltous chloride or 1–2 g l−1 methionine, while in variations in peptones and sugar sources often compounded by
the presence of high phosphate concentrations, sisomicin yields seasonal variation. The medium component variability has a
decreased. Based on these observations, it was suggested that significant impact on the fermentation product titers. To over-
methionine addition should be in the pre-middle stage, while come these variations it is necessary to adopt chemically refined
the phosphate concentration needs to be controlled below 3.14 recipes with established and precise functions and interrelation-
mmol l−1 in cell growth phase and below 0.10 mmol l−1 during ships between all the medium nutrients. Considering these as-
the sisomicin biosynthesis phase. pects, research efforts are mainly directed towards (1) evaluation
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 181

of the effect of various carbon and nitrogen nutrients as cost- Recently, M. purpurea MP3–12 was reported to consume
effective substrates on the gentamicin yield; (2) requirement of crude vegetable oils as carbon source and produced large
divalent metal ions, amino acids, vitamins, and nucleosides in amounts of gentamicin (Choi et al., 2005). In the follow-up
the fermentation medium; (3) optimization of environmental and study, an initial crude soybean oil concentration of 40 g l−1
fermentation parameters such as pH, temperature, aeration, and produced increased gentamicin yields of 0.75 g l−1 . In ad-
agitation. dition, when 0.4 g l−1 linolenic acid was added, the maxi-
mum gentamicin concentration achieved was 0.82 g l−1 , which
was 3-fold higher as compared to the absence of linolenic
Carbon Sources acid (Shin et al., 2008). Utilization of crude fish oils can also
It is well documented that the type and concentration of the be suggested as an alternate oil source for reducing the pro-
carbon source has a marked effect on the production yields of duction costs of gentamicin, since they are cheap and readily
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several antibiotics (Hu et al., 1984). In general, it is observed available.

that in fermentation media containing a mixture of a rapidly as-
similated carbon source and a slowly assimilable carbon source,
the former is used first for cell growth but contributes to little Nitrogen Sources
or no secondary metabolite formation. After the rapidly me- In a fermentation process, the organic and inorganic forms
tabolizable carbon source is depleted, the “second-best” carbon of nitrogen are metabolized by the microorganism to produce
source is used for the idiophase. Different polysaccharides such amino acids, nucleic acids, proteins, and cell wall components.
as starches, dextrins (Wagman and Weinstein, 1966), sugars like Although complex organic nitrogen sources favor gentamicin
fructose and sucrose (Abou-Zeid et al., 1974; Escalante et al., production, the requirement for specific nitrogen source differs
1992), and oils such as soybean oil (Shin et al., 2008) were re- from strain to strain. The optimal use of organic nitrogen sources
ported to support good growth and contribute to high gentamicin like fodder yeast (at 50% total nitrogen, 2 g l−1 ), yeast extract
yields. These carbohydrates are broken down to glucose subunits (8 g l−1 ), and tryptone (8 g l−1 ) has been reported to stimulate
which are readily available as an assimilable carbon source for growth and gentamicin production as compared to the addition
For personal use only.

the growing culture. In case of M. purpurea, an optimal gentam- of inorganic nitrogen sources (Abou-Zeid et al., 1974). Further,
icin yield of 0.8 g l−1 was achieved with the addition of glucose Shin et al. (2008) reported that soybean meal (15 g l−1 ) and
(Abou-Zeid et al., 1974) at 8 g l−1 , and 0.19 g l−1 of gentamicin corn steep liquor (15 g l−1 ) were favorable nitrogen sources for
was observed on supplementation of fructose (Escalante et al., gentamicin production (1.2 g l−1 ) from M. purpurea MP3–12.
1992) at 10 g l−1 , while the gentamicin yields decreased with the Supplementation of rapidly metabolizable nitrogen sources
addition of mannose, lactose, and maltose. A gentamicin yield of such as ammonium salts and amino acids in the medium was
0.43 g l−1 was noticed in case of M. echinospora with addition of also reported to have positive effect on growth and gentam-
starch at a maximum concentration of 7.5 g l−1 and gentamicin icin synthesis (Abou-Zeid et al., 1976a; Gonzalez et al., 1995;
production lowered with higher starch concentrations. However, Laznikova, Gracheva, and Orlova, 1977). The effect of different
gentamicin synthesis was repressed with the addition of glucose, inorganic forms of reduced and oxidized nitrogen on gentam-
lactose, sucrose, and dextrin (Himabindu and Jetty, 2006). Fur- icin biosynthesis was studied by Laznikova et al. (1977). They
ther, different low-cost carbon sources like black strap molasses observed that both the ammonium and the nitrate nitrogen were
(treated with 0.2–0.3 g l−1 EDTA), corn steep liquor, and whey consumed by the antibiotic-producing organism. Among all the
powder were demonstrated as potential substrates for gentam- salts tested, only ammonium sulfate stimulated the biosynthe-
icin production (Abou-Zeid et al., 1974, 1978; El-Sokkary et al., sis of gentamicin. The positive role of this salt was due to both
1995a). the ammonium and sulfo groups, since the presence of the sulfo
Glucose being an excellent and rapidly metabolizable car- group alone in sodium sulfate, magnesium, or sulfuric acid re-
bon source for growth often exhibited repression in antibiotic sulted only in partial stimulation of gentamicin biosynthesis.
biosynthesis (Satoh, Ogawa, and Satomura, 1975). A decrease However, simultaneous addition of sulfuric acid and ammonium
in gentamicin production due to catabolite repression exerted nitrate produced stimulatory effect on the gentamicin biosyn-
by D-glucose and D-xylose has been reported (Escalante et al., thesis when used alone as compared to supplementation of only
1992). This effect was confirmed by the use of a nonutilizable ammonium sulfate.
glucose analog, 2-deoxyglucose (DOG), which affected growth Similarly, in case of M. purpurea NRRL-2953 it was re-
but not gentamicin formation. They suggested that d-glucose in- ported that addition of ammonium chloride stimulated gen-
fluences antibiotic formation only if added during the stage of tamicin formation due to its conversion to glutamine, which
active protein synthesis and needs to be metabolized to exhibit played an active role in the formation of various gentamicin
its negative effect on gentamicin synthesis. Further, addition of precursors including 2-deoxystreptamine, aminodeoxyinosose,
different organic acids as carbon sources like succinic, malonic, and D-glucosamine (Gonzalez et al., 1995). Dimchev (1989b)
citric, acetic, propionic, and formic acids has been reported to reported that the addition of D-glucosamine (10 mM) at the
decrease gentamicin productivity (Abou-Zeid et al., 1976a). start of the fermentation or after 24 h showed 40% increase
 182 C. GANESH KUMAR ET AL.

in gentamicin productivity and exhibited lower yields when Metal Ions

D-glucosamine was added after 48 h of the fermentation which Abou-Zeid et al. (1976a) reported that the addition of differ-
possibly may be due to the extension in the duration of the ent trace elements such as Co, Mo, Fe, Cu, Zn, and Mn has a stim-
tropophase. ulatory role on the biosynthesis of gentamicin. It was observed
Amino acids contain amino groups which are essential com- that lower concentrations of these trace elements were more ef-
ponents for both microbial growth and gentamicin biosynthesis. fective than higher concentrations. However, Co2+ ions had a
Abou-Zeid et al. (1976a) reported that certain amino acids such major effect on the antibiotic synthesis. The amount of gentam-
as D-phenylalanine, DL-isoleucine L-lysine, DL-methionine are icin yield produced in the synthetic medium supplemented with
known to favorably contribute to the fermentative production different concentrations of cobalt decreased with an increase in
of gentamicin, while some amino acids like L-valine, L-serine, cobalt concentration. Further, a gentamicin producing M. pur-
L-glutamic acid, and L-tyrosine were found to be neutral in na- purea strain fermented in soybean-dextrin medium with added
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ture and did not favor gentamicin synthesis. It was suggested cobalt resulted in normal production of gentamicin C complex
that gentamicin molecules contained amino groups and these (C1a , C2 , and C1 ), while a 10-fold decrease in gentamicin titers
were possibly derived from the amino acids supplemented in was observed in the absence of added cobalt (Testa and Tilley,
the fermentation medium. Further, in M. echinospora ATCC 1979). Further, addition of an optimal Co2+ concentration of 6
15837, it was demonstrated that amino acids supplemented in mg l−1 to the medium was reported to produce good gentamicin
the growth medium played an important role in glucose up- yields (Lee and Ryu, 1979).
take (Hoskisson, Sharples, and Hobbs, 2003). They observed The supplementation of cobalt to the fermentation medium
that asparagine supplemented in a defined growth medium was containing either soybean meal or gelatin impeded the mycelial
consumed more preferentially as a carbon source by the mi- growth with 6-fold increase in gentamicin titers. It was observed
croorganism and exhibited a downregulation phenomenon in the that gentamicin biosynthesis by cobalt was inversely propor-
uptake rate of glucose. Further, they suggested that a relation- tional to the time of its addition. This cobalt effect was dependent
ship existed between the amino acid metabolism and glucose on several fermentation parameters like aeration conditions, in-
uptake rate and it was necessary to maintain an optimal level organic phosphate level in the medium, and the physiological
For personal use only.

of free amino acids in the medium that would prevent the in- conditions of the mycelium (Krasnova, Laznikova, and Orlova,
hibition of sugar transport (Hoskisson, Sharples, and Hobbs, 1977). In a later study, Krasnova et al. (1978) demonstrated that
2003). addition of cobalt or vitamin B12 to the fermentation medium re-
sulted in accumulation of high levels of gentamicin. The relative
content of the most methylated components, C1 and C2 , in the
Phosphate Ions gentamicin complex was higher, while a decrease in the con-
Potassium phosphate has been used as a source of phosphate tent of the least methylated components, C1a , and other minor
in most studies and was found essential for both growth and components was observed. This observation was confirmed by
antibiotic production. It also contributes to the buffering action the addition of sulphodimezine inhibitor which lowered the gen-
of the production medium. The addition of phosphate in the tamicin biosynthesis rate and the relative content of C1 and C2
medium at a concentration of 1.2 g l−1 was found optimal for gentamicins. It was suggested that cobalt ions stimulated the
gentamicin production with a titer of 0.45 g l−1 . However, addi- vitamin B12 -dependent synthesis of methionine, which is the
tion of excess phosphate resulted in the inhibition of gentamicin source of methyl groups for biosynthesis of methylated C1 and
production (Himabindu and Jetty, 2006). The phosphate regu- C2 components of gentamicin complex.
lation for gentamicin synthesis in M. echinospora subsp. echi- In a continued study, Krasnova, Bukin, and Orlova (1979b,
nospora was studied by the use of phosphate-trapping agents 1979c) observed 2.5 times increase in gentamicin biosynthesis
(Omura and Tanaka, 1985). Further, Abrasimovskii et al. (1990) on supplementation of cyanocobalamine, methylcobalamine, L-
studied that addition of high levels of inorganic phosphate in the methionine, or L-serine in the synthetic medium. Glycine and
medium led to growth inhibition and gentamicin biosynthesis dihydrofolate increased gentamicin yield on an average by 1.5 to
in all the tested isogenic strains of M. purpurea var. violaceae. 1.8 times. When L-methionine concentration was optimum in the
However, in case of M. purpurea, high inorganic phosphate lev- medium, the production titer of gentamicin by cobalt-deficient
els (up to 5.75 mM) did not inhibit cell growth but repressed mycelium markedly increased on addition of extra amounts of
gentamicin synthesis (Obregon et al., 1994), a phenomenon sim- cobalamin into the medium. Similarly, high gentamicin biosyn-
ilar to that observed in non-aminoglycoside antibiotics. They thetic levels were observed in the absence of L-methionine by
established that phosphate ions transiently repressed antibiotic supplementing a combination of either dihydrofolate and L-
formation by preventing the synthesis of 2-deoxystreptamine serine or cyanocobalamine, dihydrofolate, and glycine to the
from 2-deoxyinosose, a nonphosphorylated compound, but not medium. Their study suggested that the folate-dependent neo-
by repressing the dephosphorylation reactions as observed with genesis of methyl group and the methyl-B12 -dependent resyn-
other aminoglycosides. thesis of L-methionine played an important functional role in
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 183

gentamicin biosynthesis. Further, a poorly active mutant was case of M. purpurea, the initial pH level of the growth medium
developed which utilized a lesser amount (by 40%) of ammo- must be maintained between pH 7.0 and 7.5 for increased gen-
nium nitrogen and synthesized higher amounts of gentamicin tamicin titers and the final pH of production medium shifted
C1a and lower amounts of C1 and C2 as compared to the active toward the alkaline side (pH 8.2) at the end of the fermenta-
strain (Krasnova et al., 1979a). tion period (Abou-Zeid, Eissa, and Salem, 1974). In another
Cobalt was also demonstrated to stimulate the two C- study, the maximum pH for growth and gentamicin production
methylation steps at the stages of the conversion of gentamicin for M. purpurea was noticed at pH 6.8–7.2 and 7.0, respectively
A to gentamicin X2 and the conversion of gentamicin X2 to (Grachev, Laznikova, and Orlova, 1976). Lee and Ryu (1979)
G418 in the gentamicin biosynthetic pathway (Kase, Odakura, reported an optimal pH of 6.8 for achieving good gentamicin
and Nakayama, 1982). Further, it was demonstrated that supple- yields.
mentation of MnSO4 (50 mg l−1 ) or Tween 20 (0.02 ml l−1 ) to
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the fermentation medium resulted in accumulation of increased

levels of gentamicin (Chen et al., 2001). Consequently, in the Aeration and Agitation
process of gentamicin fermentation, the interaction between the During fermentation, the aeration rate indirectly signifies the
gentamicin and cell wall should not be ignored. It was observed dissolved oxygen (DO) level in the fermentation medium. Dif-
that cell wall of M. echinospora var. 49–925 absorbed consider- ferent dissolved oxygen profiles can be achieved with (1) vari-
able amounts of gentamicin and the gentamicin content bound ations in the aeration rate, (2) variations in the agitator type
to wet cell wall and wet mycelium was approximately 20,000 and agitation speeds of the bioreactor, and (3) use of oxygen-
and 4,776 units g−1 , respectively, which suggested that the cell rich or oxygen-deficient gas phase (appropriate air–oxygen or
wall contributed mostly to the accumulation of gentamicin (Li air–nitrogen mixtures) as a source of oxygen. The variation in
et al., 1998). the agitation speed influences the extent of mixing in the shake
flask or the bioreactor, which affects the nutrient uptake and
bioavailability and the shear stress on the mycelia. It also affects
Other Factors the mass transfer phenomena (i.e., oxygen transfer rates). Op-
For personal use only.

The addition of certain vitamins, purine, and pyrimidine timum yields of gentamicin at shake flask level were achieved
bases to the fermentation medium for gentamicin production using different Micromonospora strains when cultivated at dif-
was earlier reported. Among the different vitamins tested, such ferent agitation speeds of 175 rpm (Gonzalez et al., 1995), 200
as cobalamin (B12 ), folic acid, riboflavin, and vitamin B1 , rpm (Abou-Zeid et al., 1976a; Himabindu and Jetty, 2006), 230
cobalamin exhibited stimulatory action for higher gentamicin rpm (Chu et al., 2002), and 240 rpm (Chu et al., 2004). In
production (Laznikova et al., 1978). Further, among the supple- a bioreactor, M. echinospora var. 49–925 produced increased
mented purine and pyrimidine bases like uracil, adenine, and gentamicin titers when agitated at 400 rpm and aerated at 1 vvm
guanine, uracil proved favorable and induced higher antibiotic (Chu et al., 2004). The type of agitators used in the bioreac-
titers (Abou-Zeid et al., 1976a, 1976b). It is believed that the tor also contributed to the oxygen transfer rate and mixing of
physiology of the inoculum also plays a significant impact on the culture (Losev et al., 1981a). In another study, M. purpurea
gentamicin production. Losev and coworkers (1981b) devised a MP3–12 produced higher gentamicin concentration of 1.45 g
method for testing the quality of the inoculum which is based on l−1 when the strain was grown for 5 days with agitation at 400
methylene blue decolorization that characterizes the respiration rpm in a 5-liter bioreactor. Later in a fed-batch process, the same
level of the culture and its oxidation–reduction (dehydrogenase) strain produced maximum gentamicin concentration of 3.5 g l−1 ,
activity. When the seed mycelium was treated with methylene which was about 4.9-fold higher than that achieved in the glu-
blue, it got decolorized within two minutes which suggested that cose medium when cultivated for 8 days with agitation at 400
the seed inoculum was suitable and highly productive for gen- rpm in a 10-liter bioreactor (Shin et al., 2008). Further, M. echi-
tamicin biosynthesis irrespective of the growth period, biomass nospora Me-22, a mutant derived by mutagenic treatment with
volume, cultivation temperature, and volume of the inoculum ethidium bromide, produced maximum gentamicin concentra-
transferred from one stage to the other. tion of 4.1 g l−1 when cultivated in a stirred tank reactor (STR)
with an agitation of 200 rpm and aeration of 2 vvm. Under opti-
mal culture conditions in STR, a 3-fold increase in gentamicin
pH production could be achieved when compared with shake flask
The pH of the culture medium is one of the most critical en- conditions (Himabindu et al., 2008).
vironmental parameters affecting both growth and gentamicin It was observed that the use of a Rushton turbine agitator
biosynthesis in submerged cultures. However, the effect of pH caused high shear rates as compared to the use of an arrow turbine
on the biosynthesis of gentamicin and cell growth varies within agitator. The shear rate had a remarkable effect on the mycelial
different strains of Micromonospora and the medium composi- morphology and biosynthesis of gentamicin. With high shear
tion. The culture pH also affects many enzymatic processes and rate, the mycelial hyphae gets mechanically broken and short-
transport of various components across the cell membrane. In ened, which resulted in lower gentamicin production. However,
 184 C. GANESH KUMAR ET AL.

Guo et al. (2005) suggested that when arrow turbines were used based on RSM were employed for optimization of production
instead of Rushton turbines, the potency of gentamicin increased conditions for antibiotics (Bapat and Wangikar, 2004; Dasu and
by 49.0% and the yield was 1.3 g l−1 . Further, Chen (1990) ob- Panda, 2000; Elibol, 2004), enzymes (Bernal, Diaz, and Coello,
served that using a six-blade turbine-like stirrer resulted in higher 2006; Francis et al., 2003), and other metabolites (Carla and
gentamicin yields. Roberto, 2001; Rodrigues et al., 2006), An RSM strategy using
Aeration also had a significant effect on the production of a 24 full-factorial central composite design was applied to op-
gentamicin. When the DO level was about 40%, higher gentam- timize the gentamicin production from M. echinospora ATCC
icin titers were observed (Zhang et al., 2002). However, lowering 15838. Based on the RSM analysis, soybean meal and CoCl2
the aeration rate caused a drastic reduction in the mycelium pro- were observed as critical factors affecting the yield of gen-
ductivity with respect to gentamicin yields (Laznikova et al., tamicin. The optimized production medium comprised starch
1978). This suggests that reduction in oxygen supply is a criti- (9 g l−1 ), soyabean meal (3 g l−1 ), K2 HPO4 (0.9 g l−1 ), and
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cal limiting factor for growth as well as gentamicin synthesis. It CoCl2 (0.01 g l−1 ) which produced 850 mg l−1 of gentamicin,
was observed in lab-scale experiments using M. purpurea var. which was 2-fold higher than the unoptimized medium consist-
violacea strain VNIIA 7R that the medium redox potential (Eh) ing of medium constituents like starch, soybean meal, FeSO4 ,
had a significant effect on gentamicin biosynthesis. When the Eh K2 HPO4 , and CoCl2 (Himabindu et al., 2006).
increased from 108 to 168 mV, it stimulated gentamicin biosyn-
thesis, while a further increase in Eh to more than 170 mV caused Factors Affecting the Release of Bound Gentamicin
an inhibition in gentamicin biosynthesis (Vainshtein et al., Gentamicin, a product of submerged fermentation, is bound
1989). to the M. purpurea mycelium which needs to be released using
various physical and chemical methods. The ribosome, actin,
tubulin, phospholipids, and cell wall teichoic acids were con-
Medium Design sidered to bind aminoglycosides in both prokaryotic and eu-
The optimization of fermentation conditions is of prime im- karyotic cells (Kusser et al., 1985). It was earlier recognized
portance in the development of any fermentation process owing that low amounts of gentamicin were accumulated in the fer-
For personal use only.

to their impact on the economy and practical feasibility of the mentation broth and peptidoglycan has been considered as the
process. During this phase, an understanding of the relation- main binding site for gentamicin in the cell wall of M. echi-
ship between sugar and nitrogen metabolism is important from nospora. In case of M. echinospora var. 49–925, the cell wall
a commercial perspective, which provides a judicial strategy for absorbed considerable amounts of gentamicin and the content
fermenter medium design and feeding regimes. The classical of gentamicin bound to wet cell wall and wet mycelium was
“one factor at a time” (OFAT) approach is a well-accepted and approximately 20,000 and 4,776 U g−1 , respectively, suggest-
routinely employed strategy; however, it is practically impos- ing that cell wall contributed significantly to the accumulation
sible to achieve an appropriate optimum with a finite number of gentamicin. Some of the physical methods that were adopted
of experiments. Further, single-variable optimization methods for releasing gentamicin from mycelial cell wall include online
are not only tedious and time-consuming, but also result in mis- ultrasonic treatment and single or multiple supplements of peni-
interpretation of results, since they ignore the combined inter- cillin (Chu et al., 2000, 2002; Li et al., 1997a, 1997b). With
actions among various physicochemical parameters (Haltrich online ultrasonic treatment of 40- to 50-min duration, the gen-
et al., 1994; He et al., 2004). tamicin titer increased by 1.5 times (Li et al., 1997b), while in a
Statistical experimental designs can be adopted at various follow-up study (Chu et al., 2000) with online ultrasonic treat-
phases of an optimization strategy, such as during screening ex- ment of five consecutive cycles with 7-min duration each during
periments or when looking for optimal conditions for targeted 80–90 h after inoculation, it was found that the secretion and
responses (Box et al., 1978). Fermentation optimization using total gentamicin titer increased by 170%. The use of multiple
a statistically planned experiment involves the initial screening microwave treatments (Xiong et al., 2002) with an intensity of
of a large number of factors (typically in the range of 5–12), 127.5 watts for 40 s at a 12-h interval increased the gentamicin
which enables one to eliminate the insignificant ones in order to secretion ratio from 24.5 to 47.5%. Himabindu and coworkers
obtain a smaller, more manageable set of factors. The remain- (2007a) demonstrated that using a microwave treatment with an
ing factors could later be optimized using a response surface intensity of 800 watts for 40 s resulted in an improved gentamicin
modeling methodology. Finally, after the model building and yield of 983 mg l−1 as compared to the untreated culture.
optimization, the predicted optimum is verified experimentally Chemical methods for releasing cell-bound gentamicin, such
(Haaland, 1989; Strobel and Nakatsukasa, 1993). The response as treatment with antibiotics like ampicillin, penicillin, and bac-
surface methodology (RSM) comprises factorial design and re- itracin, have been reported. Addition of either ampicillin or
gression analysis, which helps in evaluating effective factors cephotaxime at a sub-bacteriostatic concentration increased the
and building models to study the interaction and select the opti- gentamicin yields from 52.5 to 83.2 and 150 μg ml−1 , respec-
mum conditions of variables for a desirable response (Haaland, tively, for free cells and from 83.2 to 150 and 260 μg ml−1 ,
1989). Recently, a number of statistical experimental designs respectively, for immobilized cells (El-Sokkary et al., 1995a).
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 185

Different studies showed that supplementation of penicillin (Li regulatory proteins and/or the inactivation of repressor proteins.
et al., 1997a) at the rate of 20 U ml−1 enhanced the gentamicin Mutational biosynthesis is another technique applied for pro-
potency by 117%, while the addition of bacitracin (Xiong et al., ducing idiotrophic mutants which are derived by chemical muta-
2002) at the rate of 0.6 mg ml−1 increased the gentamicin po- gens like N -methyl-N  -nitro-N -nitrosoguanidine (MNNG), and
tency by 18.2%. The function of the antibiotic used in the above requires the supplementation of 2-DOS for increased produc-
studies was due to the interference of the cell wall synthesis tion of the desired aminoglycoside antibiotic analogs (Nagaoka
resulting in enhanced secretion of gentamicin. and Demain, 1975). The increased yields resulting from the use
Moreover, it was observed that supplementation of MgSO4 of these mutagenic agents are advantageous from an economic
(6 mM) to the shake flask culture facilitated M. echinospora perspective.
var. 49–925 to release cell bound gentamicin effectively with Lee and Ryu (1979) reported that a high gentamicin-
an increase in gentamicin productivity (Chu et al., 2004). High producing strain was produced by UV-mutagenesis of a wild
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concentrations of MgSO4 were also used for streptomycin pro- type strain, M. purpurea ATCC 15835. Another mutant strain
duction (Ochi, 1987). Himabindu et al. (2007a) showed that M814–455 reported by Huan et al. (1987) was obtained by UV
the addition of NaCl (0.6%) to M. echinospora MTCC708 cul- mutagenesis (at 365 nm) in the presence of 8-methoxysopralen
ture caused the release of cell-bound gentamicin with an in- (100 μg ml−1 ), which produced gentamicin yields of 1380 U
creased gentamicin production (1,115 mg l−1 ) as compared to ml−1 as compared to the wild type (980 U ml−1 ). The C2 com-
the control (880 mg l−1 ). It was suggested that the cell wall of- ponent of gentamicin produced by this mutant strain was 51%.
fers common binding sites for the metal ions and gentamicin, Further, a mutant strain of M. purpurea JY3−5 was derived by
and all charged groups in the cell wall seem to be involved in UV irradiation of a wild strain, M. purpurea JY1−12 which ex-
the direct interaction with metal ions, thus causing the desorp- hibited an increase in the gentamicin potency by 34% in a shaker
tion of the cell-bound gentamicin. It was established that the and 19% in a 30 m3 fermenter as compared to the wild type M.
release and secretion of the bound gentamicin by metal ions purpurea JY1−12 strain (Yang, 1999). One mutant M. purpurea
played a significant role in alleviating the feedback repression strain 4–2 derived by neutron irradiation (14 MeV) produced
of gentamicin biosynthesis (Chu et al., 2004; Reiblein et al., gentamicin yields in the range of 42 to 49% as compared to that
For personal use only.

1973). Further, Tris and acrylamide were reported to favor the of the parent strain N-21 (Zhang et al., 1985). Moreover, when
release of gentamicin from the cell wall to a certain extent; how- M. purpurea KR 960796 was neutron-irradiated (30 KeV), 4
ever, their effects on gentamicin secretion were not considered mutants, namely B0020, B0030, B0017, and B0013, were gen-
appropriate for metal ions (Niu et al., 2003; Reiblein et al., erated, which produced high gentamicin yields of 30, 22, 17
1973). and 15% as compared to that of the parent strain. However,
only one mutant strain B0017 showed a 46% increase in gen-
Strain Improvement tamicin yield under optimized fermentation medium conditions
Strain improvement plays a major role in the commercial de- (Dai et al., 1999). Another wild M. purpurea strain 23–18 was
velopment of a microbial fermentation process. As a rule, the irradiated with a 12 C ion beam which generated five mutants.
wild-type strains isolated from nature usually produce limited Based on the production studies, two mutants were selected
quantities (usually 0.1–20 mg l−1 ) of the desired metabolite that which showed >10% overall increase in the gentamicin yield as
can find suitable commercial application. A great deal of effort compared to the wild strain (Liu et al., 2003). Several mutants
and resources are needed to improve antibiotic-producing strains were generated for effective production of gentamicin from a
to meet commercial requirements. Current practices of improv- wild M. purpurea strain using protoplast mutagenesis with UV
ing the fermentation productivity of industrial microorganisms irradiation and spore mutagenesis with a combination of 60 Co
range from a classical random approach to the application and UV irradiation. On selection, one mutant M. purpurea MP3–
of highly rational methods, for example, directed evolution, 143 produced about 16-fold higher gentamicin titer as compared
metabolic engineering, and ribosome engineering. Although to that of the parent strain (Choi et al., 2005).
classical methods are still effective even without using genomic MNNG is a commonly used chemical mutagen with effec-
information or genetic tools to obtain highly productive strains, tive performance to develop mutants in a variety of microorgan-
these methods are time- and resource-intensive (Vinci and Byng, isms. The advantage of using MNNG is that a high frequency
1999; Zhang et al., 2002). However, these wild strains need to of mutants can be induced with comparatively low lethal ef-
be manipulated to obtain strains that are superior and produce fect to the cells. Idiotrophic mutants of M. purpurea were de-
hundreds or thousands of times more than that produced by the rived by mutagenesis of cells with MNNG and produced in-
originally isolated strain. The strain improvement programs tra- creased levels of new gentamicin analogs only on exogenous
ditionally employ mutagenesis followed by screening or selec- supplementation of 2-DOS (Daum et al., 1977; Rosi et al.,
tion; this concept is known as “brute-force” technology (Demain 1977). In another study, increased yields of sagamicin and gen-
and Adrio, 2008). The use of classical UV or chemical muta- tamicin were produced from two M. purpurea mutant strains
genesis programs for strain improvement may enable screening 374 and 46 which were derived by irradiation with 60 Co fol-
or selection of mutants with increased production of positive lowed by mutagenesis with MNNG. The cell fusion of the
 186 C. GANESH KUMAR ET AL.

two above selected strains resulted in a mutant producing high were isolated in clusters. Recently, Ochi and coworkers (2004)
yields of sagamicin (Zhou et al., 1987). Increased yields of proposed a new approach termed “ribosome engineering” for the
gentamicin of more than 8- to 10-fold were obtained from a overproduction of antibiotics. Using this strategy, the antibiotic
M. purpurea var. violacea strain mutagenized for 60–180 min production can be increased by several-fold in an antibiotic-
with 1 mg ml−1 MNNG plus chloramphenicol (50 μg ml−1 ) as producing strain by modulating the ribosomal components (ri-
compared to the mutant derived with MNNG alone (Dimchev, bosomal proteins or rRNA), that is by sequential introduction of
1989a). multiple drug resistance mutations in the gene conferring resis-
Mutant strains such as 79 and 168 were derived by muta- tance to different drugs. This approach has several advantages
genesis of M. echinospora var. 23–18 by initial treatment with such as the ability to screen for drug-resistant mutations by sim-
penicillin and ultrasonics to increase the spore cell wall perme- ple selection protocols on drug-containing plates, even if the
ability and later again mutated with 60 Co to increase the gen- mutation frequency is extremely low (e.g., <10−10 ), the ability
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tamicin titers by 20% with shortening of the fermentation period to select for mutations lacking prior genetic information and it
by 10 h as compared to the parent strain 23–18 (Liao, 2002). requires less time, cost, and labor than other methods (Ochi,
Kheira and El-Sokkary (2003) subjected the original strain of 2007). It was confirmed in Streptomyces coelicolor A3(2) by
M. purpurea DSM 43036 with different doses of antibiotics like the sequential introduction of three drug resistance mutations
streptomycin and amikacin up to 1 mg ml−1 and β-lactam an- (str, gen, and rif, which conferred resistance to streptomycin
tibiotics such as ampicillin (400 μg ml−1 ) and cephalosporin (20 (Sm), gentamicin (Gen), and rifampin (Rif), respectively), three
μg ml−1 ) and subsequently selected mutants with increased gen- rounds of selection resulted in a triple mutant, Streptomycin-
tamicin productivity. Dramatic increases in gentamicin yields of Gentamicin-Rifampin, which showed hierarchical increments
1.37, 1.45, and 2.75 times were recorded with cephalosporin-, in the polyketide antibiotic, actinorhodin, production (Hu and
streptomycin-, and ampicillin-resistant mutants, respectively. Ochi, 2001). The dramatic overproduction in antibiotic yields
The advantage imparted by β-lactam antibiotic mutagenesis is is due to the acquisition of mutant ribosomes with aberrant
that the mutant strain generated has an altered cell wall murein protein and guanosine tetraphosphate (ppGpp) synthesis activ-
composition which transports the antibiotic outside the cell con- ity which was demonstrated using in vitro protein synthesis as-
For personal use only.

tributing to higher gentamicin production. Four mutant strains says. It was suggested that this aberrant protein synthesis abil-
(SF-01–1, -11, -14, and -68) were selected through rational ity in the multiple-drug-resistant mutant, which is a result of
screening of the parent strain producing gentamicin C1a sub- increased stability of the 70S ribosome complex, is responsi-
jected to antibiotic mutagenesis with fosfomycin. The produc- ble for the remarkable enhancement in antibiotic production
tivity of these four mutants increased in a range of 75–101% (Okamoto-Hosoya et al., 2003). This approach was success-
as compared to the parent strain (Mingqi et al., 1998). Further, fully demonstrated in an industrial Streptomyces albus strain
Himabindu and coworkers (2007a) demonstrated that a wild for enhancement of salinomycin production (Tamehiro et al.,
strain, M. echinospora subsp. pallida MTCC 708, mutagenized 2003), in Planobispora rosea for improvement of thiazolylpep-
with ethidium bromide (200 μg ml−1 ) plus penicillin (50 μg tide GE2270 antibiotic production (Beltrametti et al., 2006), and
ml−1 ) resulted in a mutant strain, M. echinospora EtBr-22, that to activate the synthesis of dormant antibiotics (Hu et al., 2002;
exhibited increased gentamicin production (1354 mg l−1 ) and Inaoka et al., 2004). Very recently, this strategy has also been
the yields were about 1.5-fold higher as compared to that of the applied to S. coelicolor A3(2) to improve the production of acti-
wild type. norhodin by 180-fold as compared to the wild type (Wang et al.,
The advent of genetic and protein engineering technologies 2008).
for production of new metabolites has gained renewed interest
in the recent years. New constructions were made by the trans-
fer of genes between organisms to produce high yielding vari- Protoplast Transformation
ants. Further, the advancements in metabolic engineering have Genetic recombination among Streptomyces and Mi-
deciphered the important biosynthetic pathways and resulted cromonospora strains finds major application for strain improve-
in the overproduction of several limiting enzymes within the ment and genetic analysis in antibiotic-producing strains. Bio-
biosynthetic pathway, thus increasing the production of novel chemical mutants were derived from different Micromonospora
metabolites (Nielsen, 1998). The isolation and characterization strains like chalcea, M. purpurea, and M. echinospora by muta-
of the numerous genes involved in the primary and secondary genesis with UV and MNNG. Genetic recombination was per-
metabolism were carried out for better understanding of the an- formed by crosses between complementary nutritional mutants
tibiotic biosynthetic pathway (Deng and Bai, 2006). All or most of the same species that exhibited a positive genetic interaction,
of the genes responsible for the production of different antibi- while interspecific genetic recombination lacked evidence for
otics, such as erythromycin (Stanzak et al., 1986), streptomycin any form of genetic interaction (Beretta et al., 1971). In many
(Distler et al., 1987), lincomycin (Peschke et al., 1995), gentam- Streptomyces members, it is well established that protoplasts
icin (Aboshanab et al., 2006; Kharel et al., 2004b; Unwin et al., generated by artificial fusion with polyethylene glycol treatment
2004; Yanai et al., 2004), and tobramycin (Kharel et al., 2004a), and subsequent regeneration on nonselective medium exhibited
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 187

efficient genetic recombination (Hopwood et al., 1977). Ogawa fected by the diffusional limitation (Gosmann and Rehm, 1986).
et al. (1983) developed an improved protoplast formation The rationale for choosing whole cell immobilization is that it
methodology for M. purpurea or Micromonospora sp. SF-1854 provides higher antibiotic yields after immobilization, higher
by treatment of cells with a mixture of lysozyme and achro- operational stability, greater resistance to environmental pertur-
mopeptidase, which produced protoplasts at a high frequency bations, and lower effective operational costs.
rate of 1.1 to 3.0 × 108 ml−1 . Further, a mutagenesis protocol was Calcium alginate beads have been widely applied for immobi-
formulated for protoplasts or mycelial fragments of M. rosaria lization purposes and the parameters that influence the viability
NRRL 3718 to produce auxotrophs with high-sporulation ef- of bacteria and which needs optimization include alginate con-
ficiency (Kim et al., 1983). The genetic transformation of M. centration, calcium chloride concentration, hardening time of
rosaria with a plasmid pIJ702 derived from S. lividans was gel beads in calcium chloride, bead diameter, and cross-linking
found to evaluate the host-range of Streptomyces plasmid in agents, if used. El-Sokkary et al. (1995a) noticed that when
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nonrelated genera and further to produce hybrid antibiotics. It whole M. purpurea cells were immobilized in calcium alginate
was demonstrated that the use of mannitol instead of sucrose gels and repeated batch fermentation was carried out in casein-
as an osmotic stabilizer improved the regeneration efficiency glucose and molasses media, it resulted in maximum gentamicin
of cells from the protoplasts (Matsushima and Blatz, 1988). yield of 83 and 52 μg ml−1 after 5 days of incubation and the
Moreover, an efficient transformation protocol for producing process could be operated continuously in two cycles for a period
protoplasts with pIJ702 plasmid showing high transformation of 10 days. In another batch fermentation process using calcium
frequency was developed, and the optimal initial temperature alginate beads immobilized with M. echinospora cells, the max-
of 26◦ C was found essential for the efficient protoplast regen- imum gentamicin yield obtained was 1096 μg ml−1 after 4 days
eration and transformation of M. purpurea MNG 00209 (Jarai of incubation and in a repeated batch fermentation process, the
et al., 1982; Kelemen, Financsek, and Jarai, 1989). The treat- maximum gentamicin yield of 1549 μg ml−1 was achieved after
ment of protoplasts of M. purpurea with an intercalating dye, 3 days of operation when the fermenter was operated continu-
acriflavine, resulted in the loss of gentamicin-producing abil- ously for 18 days and six cycles (Himabindu, Potumarthi, and
ity due to chromosomal mutation or rearrangement. However, Jetty, 2007b).
For personal use only.

this property was reversed when the gentamicin-nonproducing

strains were supplemented with streptamine or deoxystrep- Recovery and Detection Methods of Gentamicin
tamine (Kim et al., 1990). This protoplast formation technique In general, antibiotics are nonvolatile in nature and conven-
promises to be extremely useful for the development of recom- tional methods like distillation, stripping with carrier gas, and
bination studies in new species, probably being capable of ex- pervaporation cannot be applied on antibiotics for their recov-
tension to members of other genera and perhaps to interspecific ery. Some of the important in situ recovery methods include ad-
crosses. sorption, solvent extraction, and crystallization. Adsorption is
the widely applied method and different cation-exchange resins
Whole-Cell Immobilization for Gentamicin Production that are commonly employed for gentamicin recovery include
Cell immobilization technology is gaining renewed interest Amberlite IRC-50, CG-50, and CG-120. These cation-exchange
in the recent past for use in various biotechnological processes resins selectively bind only gentamicin from the neutralized cul-
for the production of antibiotics (Sallam et al., 2005; Teruel ture filtrate which is subsequently eluted with ammonium hy-
et al., 1997), enzymes (Kokubu, Karube, and Suzuki, 1981; droxide (Lee et al., 1977, 1973; Luedemann and Weinstein,
Sutar et al., 1986), amino acids (Bodalo et al., 1996; Morikawa, 1963; Maehr and Schaffner, 1967; Testa and Tilley, 1976).
Karabe, and Suzuki, 1980), steroids (Hocknull and Lilly, 1990), Anion-exchange resins used for gentamicin recovery include
alcohol (Sanchez et al., 1996), and bioremediation processes Amberlite IRA-400 (Luedemann and Weinstein, 1963), Dowex
(Jianlong, Ping, and Yi, 1997). Different matrices were em- 1×2 (Lee et al., 1977), 1×4 (Maehr et al., 1980), and Dionex
ployed, such as agar, alginate, carrageenan, gelatin, polyacry- Carbopac PA-1 (Kaine and Wolnick, 1994). Gentamicin is an-
lamide, collagen, chitosan, polyurethane, foam, etc., which act ionic in nature at high pH and therefore binds to anion-exchange
as supporting materials for gel-entrapment, carrier binding, and columns. The Dowex 1×2 resin has been employed for the
cross-liked immobilization. removal of salts and other impurities (Lee et al., 1978). The
Immobilization offers several potential advantages in the fer- gentamicin recovered as a concentrate after ion-exchange chro-
mentation systems from the standpoint of process engineering matography is further extracted in methanol and then dried un-
which includes ease of handling and cell separation, as well der vacuum to obtain purified gentamicin (Luedemann and We-
as the obvious benefits of continuous operation at high dilu- instein, 1963). Three major gentamicin components, C1 , C1a ,
tion rates with retention of high-cell density in the bioreactor, and C2 , were isolated on cellulose phosphate columns (Thomas
avoiding their washout. Moreover, immobilized cells can ex- and Tappin, 1974; Wagman, Marquez, and Weinstein, 1968),
hibit modifications in physiology and biochemical composition whereas Craig counter-current distribution was employed to sep-
as compared to suspended cells (Duran and Bailey, 1987). The arate the other C compounds (C2a and C2b ) and represented only
physiological activity of cells entrapped in a gel matrix is also af- 4% of the total gentamicin C complex (Bryne et al., 1977). Silica
 188 C. GANESH KUMAR ET AL.

gel column chromatography was employed for the isolation of from the biosynthetic mixture using di(2-ethylhexyl) phosphoric
minor gentamicin components of the gentamicin complex like acid (D2EHPA) dissolved in dichloromethane. The selectivity of
A, B, B1 , and X (Wagman et al., 1972). However, the silica extraction was observed to be the maximal at pH 8 and with 10 g
gel chromatography method was found not satisfactory for the l−1 D2EHPA. This was followed by the recovery of gentamicins
separation of gentamicin C components (Claes, Busson, and C1a , C2 , and C2a from the extracts by re-extraction with sulphuric
Vanderhaeghe, 1984). acid. The mixture resulting from re-extration contained 92.6%
Gentamicin exists as a complex mixture of four major com- gentamicins C1a , C2 , and C2a .
ponents, C1 , C1a , C2 , C2a , and one minor component, C2b . Since
it is a fermentation-derived product, it can also contain several Quantitative Methods for Estimation of Gentamicin
structurally related constituents like gentamicin B1 , sisomicin, Microbiological Assays
dihydroxygentamicin C1a , JI-20B, degradation products like
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Microbiological assay is the conventional method of choice

garamine, 2-deoxystreptamine, and several other unknown com- for the routine assay of gentamicin and other aminoglycoside
pounds, produced in smaller amounts. The differences in antimi- antibiotics to estimate their biological potency. Microbiological
crobial potency and sometimes toxicity demands to limit and assays are preferred for primary screening since they are easy
control the amount of impurities in commercial samples. Hilde- and inexpensive to perform, do not require sophisticated equip-
brand (2002) earlier reported 66 deaths in addition to hundreds ment, and are used for handling large number of samples. This
of patients suffering from severe side effects caused by admin- method was prescribed by U.S., British, and European Pharma-
istration of gentamicin sulphate. It was assumed that these cases copoeias for estimation of gentamicin and other aminoglycoside
were caused by impurities due to faulty manufacture. This fact antibiotics which can be performed either by agar diffusion or by
demands in the pharmaceutical industry the need of a power- turbidimetry (British Pharmacopoeia, 1998; European Pharma-
ful separation technique combined with a sensitive detector to copoeia 1997; United States Pharmacopeia XXIII, 1995). Use of
separate and detect impurities, even if they are present in lower redox indicators like Nitroblue tetrazolium and triphenyltetra-
concentrations. zolium chloride enabled the measurement of the inhibition zones
Gentamicin is highly polar, nonvolatile, and lacks a UV- (Urban and Jarstrand, 1979a, 1979b). Recently, a microbiolog-
For personal use only.

absorbing chromophore. These properties posed a great chal- ical assay based on the cleavage of fluorescein diacetate (FDA)
lenge in the analysis of this antibiotic for many years. Rosselet was reported (Wanandy et al., 2005). The quantitative estimation
et al. (1963) first reported the isolation of gentamicin complex of gentamicin by microbiological assay or bioautography was re-
using ion-exchange chromatography. Since then, ion-exchange ported earlier by several researchers in fermented culture filtrates
procedures were used extensively for the separation and purifi- (Bryne et al., 1977; Wagman et al., 1968; Weinstein et al., 1963b,
cation of gentamicin. The different techniques commonly em- 1967) and in different biological matrices like blood and other
ployed for the separation of different gentamicin components body fluids (Faine and Knight, 1968; Rosner and Aviv, 1980a),
in the fermented culture filtrates include paper chromatogra- serum (Alcid and Seligman, 1973; Jankovic and Ninkovic,
phy (Wagman et al., 1968), thin-layer chromatography (TLC) 1998), milk (El-Sayed, Hatem, and El-Kmay, 1989), etc.
(Bryne et al., 1977; Kadar and Ilona, 1980; Wilson, Richard, and
Hughes, 1973), Dowex 50×8 type resin coated TLC (Kadar,
1972), ion-exchange column chromatography (Thomas and Spectrophotometric Methods
Tappin, 1974), radioimmunoassay (Rosner and Aviv, 1980b), A spectrophotometric assay was developed for estimation of
high-performance TLC (Jork and Kunz, 1988; Prasad et al., gentamicin levels in blood serum, which used a purified en-
1998), high-performance liquid chromatography (HPLC) (Al- zyme derived from Escherichia coli JR88/C600 that acetylated
bracht and De Wit, 1987; Anhalt, Sancilio, and McCorkle, 1978; gentamicin in the 3-amino position with the production of coen-
Claes, Busson, and Vanderhaeghe, 1984), reversed-phase HPLC zyme A, which later reacted with a sulphydryl reagent, 5,5 -
(Kraisintu, Parfitt, and Rowan, 1982; Lesniak et al., 2003), and dithiobis-(2-nitrobenzoic acid) (DTNB), to produce a sensitive
capillary electrophoresis (Curiel et al., 2007). The ratio of the chromophore in stoichiometric amounts that could be quanti-
gentamicin C components in the mixture was determined using fied at 412 nm (Williams, Langer, and Northrop, 1975). Wang
HPLC, which varied from one biosynthetic product to another; and coworkers (1993) used p-dimethylaminobenzaldehyde as
the average values of their concentrations were: gentamicin C1 , a coloring reagent, which reacted with gentamicin to form a
35%; gentamicin C1a , 25%; and gentamicin C2 (including gen- yellow colored complex that could be detected at a maximum
tamicin C2a ), 40% (Claes, Busson, and Vanderhaeghe, 1984). absorbance of 405 nm. However, the sensitivity of both these
Very recently, a selective and sensitive method of profiling methods was observed to be too low. Further, a rapid luciferase
the DOS-containing aminoglycoside derivatives involved in the bioassay for detection of gentamicin in serum samples in a dose-
gentamicin biosynthetic pathway from M. echinospora was per- dependent manner was developed based on the estimation of
formed using HPLC-electrospray ionization mass spectrometry extracellular ATP levels in bacterial cultures exposed to stan-
(Park et al., 2007). Further, Cascaval et al. (2007) developed a dard gentamicin concentration and to serum specimens (Nilsson,
reactive extraction method for selective separation of gentamicin 1978). However, the gentamicin measurements in serum samples
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 189

were affected by the presence of sodium phosphate, glucose, and analysis. Gentamicin may be analyzed either by evaporative
heparin, while the presence of bilirubin had no effect (Nilsson, light scattering detection (Clarot et al., 2004) or by pulsed
1980). electrochemical detection (Adams et al., 1998; Cai and Mou,
A sensitive and selective fluorimetric method was developed 2005; Manyanga et al., 2008). These detection techniques
for the determination of gentamicin based on the coupling of enable direct detection of gentamicin without derivatization.
gentamicin with a fluorogenic reagent having a benzoxadia- Further, a precolumn derivatization allows the removal of
zole moiety, such as 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole interfering substances in the separation process (Stead, 2000).
(NBD-Cl), which in presence of 50% (v/v) methanolic phos- Since gentamicin possesses no UV or fluorescence chro-
phate buffer (pH 7.2) gave an intense fluorescent product mophores, it is not amenable for spectro- or fluorometric de-
having excitation and emission wavelengths at 465 and 530 tection. Therefore, after separation it may be derivatized with
nm, respectively. This method was applied for the estimation a suitable UV absorbent or fluorescent agent that permits
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of gentamicin in fermentation broth (Kabasakalian, Kalliney, analysis with the available spectrophotometric or fluorometric
and Magatti, 1977) and in plasma samples (Al-Majed et al., detectors. The most widely employed pre- or postcolumn
2003). A synchronous first-derivative fluorimetric method is derivatization agents for detection by fluorescence or UV-
demonstrated for the estimation of total potency of gentam- light absorption include dansyl chloride (Peng, Gadalla, and
icin which is based on a Hantzsch reaction, i.e., acetylacetone Peng, 1977), 9-fluorenylmethyl chloroformate (FMOC) (An-
and formaldehyde reacted with the amino group of gentam- halt and Brown, 1978; Posyniak, Zmudzki, and Niedziel-
icin molecule to form N -gentamyl-2,6-dimethyl-3,5-diacethyl- ska, 2001; Stead and Richards, 1996), fluorescamine (Walker
1,4-dihydropyridine, which is a fluorescent compound having and Coates, 1981), o-phthaldialdehyde or 1,2-phthalic dicar-
excitation and emission wavelengths at 405 and 399 nm, re- boxyaldehyde (OPA) (Al-Amoud, Clark, and Chrystyn, 2002;
spectively. This method is simple, but has a low detection limit Anhalt, 1977; Seidl and Nerad, 1998; United States Pharma-
(Wang et al., 2000). Chemiluminescence (CL) is a very sensitive copeia, 2003), o-phthalaldehyde-thioglycolic acid (Weigand and
analytical technique that finds application for the detection of Coombes, 1983), and 1-fluoro-2,4-dinitrobenzene (FDNB) (Ar-
gentamicin, mainly in combination with flow-injection analysis celloni et al., 2001; Barends, Van der Sandt, and Hulshoff, 1980).
For personal use only.

(FIA) manifold using an immobilized tris(4,7-diphenyl-1,10- Although HPLC offers high degree of specificity, both the sam-
phenanthrolinedisulfonic acid) ruthenium(II) on the electrode ple cleanup and derivatization processes are time-consuming
surface (Blanchard et al., 1998). A peroxyoxalate chemilu- and the yields are very low from the derivatization process,
minescent reaction was developed based on the reaction of which are considered as disadvantages of the HPLC tech-
peroxyoxalate with gentamicin (previously derivatized with nique. The quantitative estimation of gentamicin using HPLC
o-phthaladehyde) in presence of imidazole (Fernandez-Ramos are reported for different biological matrices like serum (An-
et al., 2006). The main advantages of this coupling are the halt and Brown, 1978; Barends, Van der Sandt, and Hulshoff,
short analysis time, automation, high versatility, and satisfac- 1980; Maitra et al., 1977), plasma (Walker and Coates, 1981),
tory precision. milk (Kijak, Jackson, and Shaikh, 1997), urine (Al-Amoud,
Clark, and Chrystyn, 2002), tissues (Cherlet, Baere, and Backer,
2000; Lecaroz, Campanero, Gamazo, and Blanco-Prieto,
HPLC Methods 2006), etc.
Gentamicin and other aminoglycosides are polybasic
cationic in nature and are generally separated on strong cation- Radioimmunoassay
exchange columns followed by ion-pair HPLC typically on C18 Radioimmunoassay (RIA) methodology is found to be rapid,
reversed-phase columns using alkylsulphonate ion-pair reagents sensitive, and precise for quantification of serum gentamicin
(Anhalt and Brown, 1978; Claes, Busson, and Vanderhaeghe, levels (Berk, Lewis, and Nelson, 1974). Currently, most RIAs
1984). The ion-pair is generally preferred over the ion-exchange are automated with solid phase separation process assisted by
HPLC in view of its high efficiency, stability, reproducibility, antibody bound to a solid phase like polystyrene assay tubes
specificity, and sensitivity of detection (Anhalt, 1977; Walker or magnetic particles. The factors that inhibit its wide usage
and Coates, 1981). Biological samples contain many matrix include the expensive equipment involved and the problems as-
components which may possibly interfere in the HPLC anal- sociated with handling of radioactivity. Further, the shelf-life
ysis. So it is necessary to extract and concentrate the analyte is only about 2 months for the iodinated gentamicin reagent
from the matrix leaving behind the protein and other matrix that is now commercially available for use in all gentamicin
components that may otherwise interfere in the subsequent RIAs. RIA has been successfully applied for the quantitative es-
derivatization, separation, and detection steps. To minimize timation of gentamicin in different biological fluids like blood
the interference potential, the ion-exchange extraction step is (Mahon, Ezer, and Wilson, 1974) and serum (Berk, Lewis, and
followed by the reversed-phase HPLC (Stead, 2000). Further, Nelson, 1973; Broughton and Strong, 1976). This technique
the use of solid-phase extraction columns and cartridges has can detect gentamicin concentration as low as 0.01 μg l−1 in
increased the performance of cleanup of the sample for HPLC serum.
 190 C. GANESH KUMAR ET AL.

Enzyme Immunoassay of fluorescence quenching is quantified by a fluorometer (Shaw

A competitive ELISA method based on antigen-capture type et al., 1977). This method was later automated for rapid analysis
for the quantitative estimation of gentamicin in serum samples of clinical serum samples (Shaw, Watson, and Smith, 1979). A
(Mattiasson et al., 1978; Standefer and Saunders, 1978), fer- fluorescence polarization immunoassay (FPIA) for gentamicin
mentation broth (Lecaroz et al., 2007), milk (Haasnoot et al., analysis was developed which is based on the detection of the
1999; Jeong et al., 2007), micro-dialysed samples (Hanes and extent of antibody binding of fluorescein-labeled gentamicin by
Herring, 2001), and tissue samples (Haasnoot et al., 1999) and measuring the fluorescence polarization using a specialized flu-
a competitive ELISA method based on antibody-capture for de- orometer (Watson et al., 1976). An improved method of FPIA
tecting gentamicin in human serum samples (Kolosova et al., was applied for the measurement of gentamicin in blood samples
1998), milk (Ara et al., 1995; Loomans et al., 2003), and swine spotted on filter paper (Fujimoto et al., 1989). Yang et al. (2002)
tissues (Chen et al., 2008) was developed. The noncompetitive developed an immunoaffinity column based on a sol-gel-derived
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ELISA usually involves binding of the antigen in the sample mesoporous biomaterial immobilized with anti-gentamicin an-
to an excess of the immobilized antibody. The extent of bind- tibody for flow-injection fluorescence immunoassay to quanti-
ing is quantified with a second (enzyme-labeled) antibody di- tatively estimate gentamicin in serum with a detection limit of
rected against a different epitope. In this method, the so-called 200 ng ml−1 .
sandwich ELISA, the measured signal is directly proportional
to the concentration of antigen (gentamicin) in the sample. This
method was applied for the estimation of other aminoglycosides. Capillary Electrophoresis
An improved parallel affinity sensor array (PASA) system func- A capillary electrophoretic immunoassay (CEIA) coupled
tioning with an automated chip-based ELISA and a CL reaction with a detection system using laser-induced fluorescence po-
monitored by a charge-coupled device (CCD) camera was re- larization was developed for estimation of gentamicin (Wan and
cently developed for the detection of gentamicin in milk samples Le, 1999). Kaale et al. (2000) reported the analysis of gentam-
(Knecht et al., 2004). The enzyme multiplied immunoassay tech- icin using CE with UV detection at 330 nm after precapillary
nique (EMIT) assay was used for the quantitative determination derivatization with phthalic dicarboxaldehyde (OPA) and mer-
For personal use only.

of gentamicin in serum samples (Pesce and Bodourian, 1981). captoacetic acid (MAA) with a gentamicin concentration limit
The EMIT gentamicin assay (Holt et al., 1994) was found to for quantification of four gentamicin components ranging from
be rapid, precise, and reliable for detection over a concentra- 0.3 to 1.0 mg l−1 . CE-mediated microanalysis of gentamicin
tion range of 0.3–16.3 μg ml−1 . A homogeneous immunoassay with in-capillary derivatization was also demonstrated by the
based on particle-enhanced turbidimetric inhibition immunoas- same authors (Kaale et al., 2001). Further, this technique was ap-
say (PETINIA) technology was developed to quantify the con- plied for the quantification of gentamicin in human serum sam-
centration of gentamicin in serum and plasma (Wei et al., 1999). ples (Kaale et al., 2005). Further, Micellar Electrokinetic Cap-
illary Chromatography method with the use of sodium tetrabo-
rate (100 mM, pH 10.0), sodium deoxycholate (20 mM), and
Fluoroimmunoassay β-cyclodextrin (15 mM) as background electrolytes enabled
Heterogenous fluoroimmunoassay (FIA) generally uses flu- the separation of all major (C1 , C1a , C2 , and C2a ) and minor
orophore labels such as rhodamine or fluorescein. This method (C2b ) components of gentamicin (Wienen and Holzgrabe, 2003).
involves the mixing of a gentamicin/fluorescein dye conjugate The addition of cetyl trimethylammonium bromide (CTAB) and
(tracer) and the sample (gentamicin). In presence of gentamicin sodium pyrophosphate as background electrolytes allowed the
in the sample, it competitively binds with the gentamicin/dye complete separation between the major gentamicin components
conjugate for the binding sites on the antibody. The amount of (C1 , C1a , C2 , C2a ) and failed in the separation of the latter two
drug concentration in the sample is proportional to the rate of gentamicin components (Yuan et al., 2005). This method was
increase in the fluorescent intensity. This method was used for further modified by applying the same conditions but a reversed
the detection of gentamicin in serum samples (Burd et al., 1977). polarity enabled the complete separation between the major and
A solid phase immunofluorescence method (FIAX system) for minor gentamicin components with direct UV detection without
quantification of gentamicin in serum or plasma samples was de- derivatization (Curiel et al., 2007).
veloped which detected gentamicin at concentrations as low as For clinical applications, several other assay methods were
50 μg l−1 and showed very low cross reactivity with other antibi- developed. The enzymatic method employing a radioactive
otics except netilmicin and sisomicin (Tsay, Wilson, and Keefe, tracer (Holmes and Sanford, 1974) has many advantages like
1980). A quenching FIA method for estimating serum gentam- convenience, specificity, accuracy, and precision as compared
icin levels was developed which is based on the fluorescence to the microbiological assay (Smith et al., 1974). Other meth-
quenching of fluorescein-labeled gentamicin upon its binding ods reported for quantification of gentamicin in fermented cul-
with antibodies (anti-gentamicin serum). The addition of appro- ture filtrates and other biological samples include hemaggluti-
priate amount of antiserum to a mixture of labeled and unlabeled nation inhibition assay (Mahon, Feldman, and Scherr, 1977),
gentamicin competes for antibody binding sites and the extent latex agglutination inhibition card assay (Doern et al., 1981),
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 191

chemiluminescent immunoassay based on immobilized anti- Response Element (RRE) in the envelope gene of HIV-1. RRE
body and biotin–avidin system (Yang et al., 1995), histoim- is the binding site of the viral Rev protein and plays a role in the
munoblot assay (Mihelic-Rapp and Giebel, 1996), gas–liquid transport of unspliced RNA from the nucleus to the cytoplasm.
chromatography (Mayhew and Gorbach, 1978), GC/MS (Iso- Inhibition of Rev–RRE interaction by aminoglycosides blocks
herranen and Soback, 2000), capillary GC/MS (Preu, Guyot, the production of HIV-1 in tissue culture (Zapp et al., 1993). The
and Petz, 1998), LC/MS/MS (Babin and Fortier, 2007; Heller transactivation-responsive element (TAR) is another stem-loop
et al., 2005; Löffler and Ternes, 2003), HPLC/thermospray MS structure in HIV-1 RNA that is bound by aminoglycosides (Mei
(Getek, Vestal, and Alexander, 1991; Graham, Speicher, and et al., 1995). TAR is a substrate of the viral Tat protein and is
Williamson, 1997), and HPLC/ESI-MS (Cherlet, Baere, and essential for viral replication.
Backer, 2000; Heller et al., 2005; Park et al., 2007). Gentamicin C is a clinically important aminoglycoside com-
prising a mixture of three components, gentamicin C1a , C2 , and
Critical Reviews in Biotechnology Downloaded from [Link] by SUNY Health Sciences Center on 10/08/14

MODE OF ACTION C1 , that have different patterns of methylation at the 6 posi-

Aminoglycosides find clinical application as antibacterial tion of ring I (see Figure 3). Yoshizawa, Fourmy, and Puglisi
agents since they specifically inhibit the protein synthesis by (1998) elucidated the 3D-structure of gentamicin C1a bound to
rapid binding to bacterial ribosomes and also disrupt the integrity an A-site RNA complex using NMR techniques. Gentamicin
of the bacterial cell membrane (Spahn and Prescott, 1996). The C1a binds in the major groove of the A-site of the RNA. In this
aminoglycosides are positively charged at biological pH (Botto study, it was established that rings I and II of gentamicin C1a
and Coxon, 1983), which contributes to RNA binding. Different exhibits binding interaction similar to that observed in case of
classes of aminoglycosides bind specifically to different sites on the complex of paromomycin and A-site RNA template. How-
the rRNA based on the structural complementarity between the ever, ring III in gentamicin C1a interacted with the conserved
two. For example, gentamicin, paromomycin, neomycin, and base pairs U1406. U1495 and G1405. C1496 in the upper stem
kanamycin are presumed to bind the A-site on the 16S rRNA region. Based on these observations, it was proposed that all the
in E. coli in a similar fashion and were demonstrated to pro- aminoglycosides that targeted the A-site of 16S rRNA bind in a
tect bases like A1408 and G1494 as revealed from the chemi- common manner, similar to the rings I and II in paromomycin
For personal use only.

cal footprinting experiments (Moazed and Noller, 1987). They and gentamicin.
were known to disturb the decoding function of protein synthe- Most aminoglycosides are highly active against bacterial ri-
sis through a complex, multistep process. The decoding func- bosomes, but do not affect the cytoplasmic ribosomes in human
tion of the ribosome is due to the small ribosomal subunit and cells. The sensitivity of eukaryotic ribosomes to some aminogly-
takes place at the ribosomal A site, a region located near the cosides, such as gentamicin, G-418, paromomycin, hygromycin,
3P end of the 16S rRNA. The binding of the above mentioned and a few others, has been viewed as an unwanted side effect
aminoglycosides to the A-site in the decoding region interferes associated with these antibacterial drugs. But it is precisely that
with the accurate recognition of the cognate tRNA by rRNA “side” effect that opens the possibility of using these drugs for
during translation (Noller, 1991). These interactions were con- the treatment of human genetic diseases. So far, the clinical ap-
sidered to interfere with the translocation of tRNA from the plication of aminoglycosides has been limited to their use as
A-site to the peptidyl-tRNA site (P-site). antibacterials; no effort has been made to optimize their ability
Aminoglycosides and their derivatives are thought to act to cause translation errors in eukaryotic cells. This leaves hope
specifically on the ribosome, also act as inhibitors of catalytic that their capacity to “bypass” stop-codons could be improved
RNAs, such as the self-splicing group I introns (Mikkelsen by screening combinatorial chemical libraries or rational drug
et al., 1999; Von Ahsen, Davies, and Schroeder, 1991), the design especially as the structures of several aminoglycosides
hammerhead ribozyme (Stage, Hertel, and Uhlenbeck, 1995), complexed with their sites were solved (Fourmy et al., 1996;
the HDV ribozyme (Rogers et al., 1996), the hairpin ri- Mankin and Liebman, 1999).
bozyme (Earnshaw and Gait, 1998), the HIV viral RNAs
(Wang et al., 1998; Zapp, Stern, and Green, 1993) or E. coli USES OF GENTAMICIN
RNase P RNA (Mikkelsen et al., 1999), and also bind to the Aminoglycosides were available for several decades, and
mRNA (Tok, Cho, and Rando, 1999) and tRNAs (Mikkelsen therefore are widely used by most physicians. In clinical prac-
et al., 2001). They decrease the fidelity of mRNA, caus- tice, they possess many desirable properties, the most important
ing incorporation of incorrect amino acids into the growing one being the rapid bactericidal activity against a wide range
polypeptide chain. The primary intracellular target is the 30S ri- of pathogens. Some of the salient features of aminoglycosides
bosome, although some aminoglycosides appear to bind the 50S are that they are relatively low in cost, have good chemical sta-
ribosome. bility, and are rarely associated with allergic reactions. Another
Besides rRNA, aminoglycosides can bind in a saturable fash- very important characteristic is their synergistic antimicrobial
ion and with similar micromolar affinities to a variety of other activity when used in combination with β-lactam agents in the
RNA structures containing nonduplex elements, such as bulges treatment of Enterococci, S. aureus, and certain Gram-negative
or interior loops. Aminoglycosides can bind to the Rev protein bacilli such as P. aeruginosa and Klebsiella spp. interestingly,
 192 C. GANESH KUMAR ET AL.

O
OPO32-
gntB (gtmA)
O OH HO
HO OH
intramolecular carbocyclization, HO
HO oxidative dephosphorylation
OH
OH
2-Deoxy-scyllo-inosose
D-Glucose-6-phosphate

gntA (gtmB) transamination

H2N
H2N
HO gntC (gtmC)
NH2
HO
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HO OH
dehydrogenation
HO
OH
OH
Keto-2-deoxy-scyllo-inosamine
2-Deoxy-scyllo-inosamine
gntF (gtmD) transamination OH
D-Glucosamine
H2N OH
H2N O
HO
HO
HO
gntD & gntZ O
HO HO
(gtmE & gtmG) NH2
O
OH H2N
Paromamine
2-Deoxystreptamine
D-Xylose
gntD & gntZ C-methylation and
(gtmE & gtmG) epimerization
For personal use only.

OH

O
HO H2N OH
HO O
HO
OH HO
gntC & gntF O
O NH2
(gtmE & gtmG)
H2N
OH
Gentamicin A2
O
HO H2N OH
H3CHN O
HO
OH HO
O
O NH2
H2N

Gentamicin A

FIG. 3. Proposed biosynthetic pathway for gentamicin C complex. Predicted roles and gene notations used by Unwin et al. (2004)
and Kharel et al. (2004b) for different biosynthetic genes are also shown. Gene notations used by Kharel and co-workers are
specifically shown in brackets. (Continued)

and more importantly the type of synergy differs for each of these reasons, the use of aminoglycoside is usually reserved for
these groups of bacteria. clinical situations in which less toxic and more easily admin-
The major limitation of these agents is a relatively narrow istered antibiotics will not suffice. These groups of antibiotics
therapeutic ratio predisposing to both nephro- and oto-toxicity. are also commonly employed for treatment of patients suffer-
In view of their cationic nature, they are not absorbed enterally, ing from cystic fibrosis. Further, these patients are plagued with
and therefore must be given parenterally by either intravenous or P. aeruginosa upper respiratory infections and receive gentam-
intramuscular route. Their biologic distribution is altered by host icin and other aminoglycosides not only parenterally but also by
factors such as age, renal function, presence of ascites, fever, and means of inhalation.
obesity, and as mentioned earlier, they are not active in an anaer- Despite the introduction of newer, less toxic antimicrobials,
obic environment (Siegenthaler, Bonetti, and Luthy, 1986). For aminoglycosides continue to serve as therapeutic agents (Begg
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 193

OH OH
O OH
HO H2N OH O
O H3C H2N OH
H3CHN gntK O
HO H3CHN
OH HO HO
O O OH O HO
NH2 C-methylation O
H2N H2N NH2

Gentamicin A Gentamicin X2
dehydroxylation
amination gntK (gtmI) C-methylation

OH
OH OH
OH O
O H3C H2N OH
H3C H2N OH H3CHN O OH
H3CHN O HO
HO OH H3C
O
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OH O H2N O
O H2N NH2
H2N NH2
G418
JI-20A dehydroxylation
amination
gntI dehydroxylation OH
OH
H3C O H2N OH
H3CHN O NH2
OH OH HO
O H3C
O
H3C O H2N H2N NH2
H3CHN O
HO
OH O H2N JI-20B
O
H2N NH2
gntI dehydroxylation
Gentamicin C1a epimerization

OH
For personal use only.

N-methylation H3C O H2N

H3CHN O NH2
OH HO
O H3C
O
H2N NH2
OH
O
H3C H2N
O Gentamicin C2
H3CHN
HO
OH O H3CHN
O N-methylation
H2N NH2

Gentamicin C2b OH
(Sagamicin) O
H3C H2N
H3CHN O NHCH3
OH HO
O H3C
O
H2N NH2

Gentamicin C1

FIG. 3. (Continued)

and Barclay, 1995; Edson and Terrell, 1999). Their clinical im- has been successfully applied for the treatment of glandular
portance is being reassessed in the light of increasing resis- tularemia in two reported case studies (Hassoun, Spera, and
tance of pathogenic organisms to other antimicrobials (Zem- Dunkel, 2006). Intratympanic injections of gentamicin were ef-
bower et al., 1998). Gentamicin is currently the first choice fectively demonstrated for the treatment of Meniere’s disease or
aminoglycoside for the treatment of serious infections with al- vertigo (Chung et al., 2007; De Beer, Stokroos, and Kingma,
ternatives being amikacin, netilmicin, and tobramycin (Stead, 2007).
2000). Gentamicin preparations are commercially available in
three forms, namely otic, ophthalmic, and topical based on the Combination Therapy
respective function to treat infections of ear canal, eye, and The aminoglycosides have a very broad antibacterial spec-
skin. Oral and injectable forms of gentamicin were found to trum and find use in the treatment of infections caused due to
exhibit effective antibacterial activity against Yersinia pestis as aerobic and facultative Gram-negative bacilli and those suscep-
demonstrated in a mouse infection model, which suggests that tible strains of Gram-negative microorganisms that are resistant
gentamicin can be used for the clinical treatment of plaque to less toxic antibiotics. Organisms commonly susceptible to
(Goto et al., 1998). Gentamicin (at 5 mg kg−1 body weight) these drugs include Klebsiella sp., Enterobacter sp., Serratia
 194 C. GANESH KUMAR ET AL.

sp., Citrobacter sp., E. coli, Proteus sp., Acinetobacter sp., including stop codons. Aminoglycosides suppresses the transla-
and P. aeruginosa (Belknap, 1997; Davies, 1986; Kaduruga- tional termination by insertion of a near-cognate amino acid at
muwa, Clarke, and Beveridge, 1993; Lortholary et al., 1995). the stop codon (Fearon et al., 1994). The translation continues in
Monotherapy is usually practiced for the treatment of less the correct open reading frame once an amino acid is introduced
serious infections, for example, uncomplicated urinary tract at the stop codon resulting in a full-length protein synthesis.
infections, and only when other antibiotics are not appropri- Several studies are in progress to evaluate this approach to sup-
ate. Further, treatment of infections caused by P. aeruginosa press stop mutations to examine whether aminoglycosides can
is frequently complicated by intrinsic resistance to antimicro- find application for the suppression of disease-causing prema-
bial agents and by frequent development of resistance during ture stop mutations in human mRNAs. Studies were carried out
single-agent chemotherapy. Combination therapy demonstrates to utilize this approach in cell-based models of cystic fibrosis,
a synergy effect and rapid killing activity (McGrath et al., 1992) Hurler syndrome, and Duchenne muscular dystrophy (Barton-
Critical Reviews in Biotechnology Downloaded from [Link] by SUNY Health Sciences Center on 10/08/14

which contributes to its clinical efficacy. It was observed that Davis et al., 1999; Bedwell et al., 1997; Howard, Frizzell, and
the overall bacterial killing was greater with successive rather Bedwell, 1996; Keeling et al., 2001).
than simultaneous administration (Barclay et al., 1995). Com- Cystic fibrosis (CF) is the most prevalent lethal inherited au-
bination therapy with a β-lactam antibiotic or a quinolone may tosomal recessive disorder observed among Caucasian popula-
be adopted for the treatment of more serious Gram-negative tion worldwide affecting 1 in every 2,500 newborns (Consortium
infections. Combination therapy with a β-lactam permits the use CFGA, 1994). People suffering from CF secrete excess mucus in
of aminoglycosides for the treatment of infections caused by par- the pancreas, lungs, and other organs. These secretions may also
ticular Gram-positive organisms (Belknap, 1997; Davies, 1986; block the digestive tract causing poor absorption of food. The
Lortholary et al., 1995) such as S. aureus, Enterococci, or S. lungs are prone to congestion, leading to pneumonia and other
viridans endocarditis. In view of the facts like incidence of toxi- infections. CF is caused by mutations in the gene encoding the
city, the relationship between toxicity and serum concentration, cystic fibrosis transmembrane conductance regulator (CFTR)
and the dosing protocols utilized, monitoring of these drugs can protein. CFTR is a member of the ATP-binding cassette family
be useful. The combined use of gentamicin and metronidazole of transmembrane transporter proteins. It is composed of 1480-
For personal use only.

for prophylaxis against serious postoperative septic complica- amino acids, a glycosylated membrane glycoprotein that has
tions was found to be effective in the patients who underwent homology with the transport ATPases, and functions as a cAMP-
surgery for elective colorectal carcinoma (Lau et al., 1988). Fur- regulated chloride ion channel in exocrine glands and secretory
ther, a combination of doxycycline for 45 days and gentamicin epithelial cells (Frizzell, 1995). Many mutation types reported
for 7 days (each at 5 mg kg−1 of body weight) was used as an to cause cystic fibrosis include stop codon mutations (18%),
effective therapy for the treatment of human brucellosis (Sol- missense mutations (40%), frameshifts (22%), splicing muta-
era et al., 1997). A combination of ampicillin (6 μg ml−1 ) and tions (18%), and others (promoter, in-frame deletions, etc., 2%)
gentamicin (0.5 μg ml−1 ) was applied for the treatment of liste- (Hamilton, 2001). The most frequent CF mutation accounting to
riosis caused by L. monocytogenes (Temple and Nahata, 2000) ∼70% of all the CF mutations is CFTR-F508 (a deletion of the
and enterococcal endocarditis (Gavalda et al., 2003). Gentam- phenylalanine residue at position 508), while the CFTR-G452X
icin in combination with tetracycline (Boulanger et al., 2004) premature stop mutation represents the second most common
or doxycycline (Mwengee et al., 2006) exhibited antibacterial mutation accounting to ∼3.4% of all CF mutations. All these
activity against Y. pestis, suggesting its use for treatment of hu- mutations prevent the CFTR function and thereby cause abnor-
man plague. mal transport of both chloride and sodium ions across many
types of epithelial tissues as well as disrupt other membrane as-
Gene Therapy sociated functions, such as pH and ion regulation (Al-Awqati,
Aminoglycoside antibiotics were found to disrupt the trans- 1995; Stutts et al., 1995; Zeitlin, 1999). Further, Bedwell and his
lational fidelity in both prokaryotic and eukaryotic species. A colleagues reported that gentamicin can overcome the effects of
highly conserved rRNA sequence is involved in the mainte- stop mutation in the CFTR in cultured respiratory epithelial cells
nance of translational fidelity, which formed the interaction (Bedwell et al., 1997; Howard, Frizzell, and Bedwell, 1996).
site between the codon and aminoacyl-tRNA anticodon (A-site) Currently, gentamicin is the only clinically applied aminogly-
(Yoshizawa, Fourmy, and Puglisi, 1999). The aminoglycosides coside for treatment of cystic fibrosis due to its ability to suppress
bind to this region of rRNA and alter its conformation which nonsense mutations within the CFTR gene in patients and the
decreases the tRNA dissociating rates causing translational mis- results were found to be quite promising (Clancy et al., 2001).
reading resulting in protein synthesis inhibition (Karimi and Moreover, topical administration of gentamicin as nasal drops
Ehrenberg, 1994; Purohit and Stern, 1994). Recent studies have to the nasal epithelium of CF patients with W1282X nonsense
demonstrated that aminoglycosides like gentamicin play an im- mutations restored the CFTR protein function and increased the
portant role in the suppression of premature stop mutations in CFTR membrane immunostaining (Wilschanski et al., 2003).
mammalian transcript in both in vitro and in vivo conditions Similarly, the intravenous delivery of gentamicin in patients car-
which allows normal tRNAs to recognize “incorrect” codons rying the Y122X mutation resulted in the delivery of the CFTR
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 195

protein at the membrane and restored the CFTR-dependent chlo- likely for those patients having other nonsense mutations, TAA
ride transport in nasal epithelial cells (Sermet-Gaudelus et al., and TAG (Kimura et al., 2005).
2007). Nonsense-mediated mRNA decay (NMD) pathway is Hurler syndrome (mucopolysaccharidosis type I (MPS I) or
a posttranscriptional translation-dependent surveillance mecha- McKusick 25280) is a lysosomal storage disease caused due
nism functioning to prevent the synthesis of proteins carrying to the deficiency of α-L-iduronidase, a glycosyl hydrolase in-
premature termination codons (PTC) (Maquat, 2004). It was volved in the sequential degradation of the glucosaminoglycans,
very recently observed that downregulation of NMD in epithe- heparan sulphate, and dermatan sulphate. The deficiency of this
lial cells carrying W1282X mutation enhanced both the level enzyme results in the lysosomal accumulation and urinary secre-
of CFTR nonsense transcripts and the CFTR chloride channel tion of partially degraded glycosaminoglycans. The premature
activity in response to the administered gentamicin (Linde et al., stop codons, Q70X and W402X, are two of the most common
2007). α-L-iduronidase gene (IDUA) mutations accounting for up to
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Duchenne/Becker muscular dystrophy (DMD/BMD) is 70% of MPS I disease alleles in Caucasian populations. It was
caused due to defective expression of the human dystrophin re- earlier demonstrated that the Hurler syndrome fibroblast cell
sulting in the absence of the dystrophin protein in muscle fibers line heterozygous to the α-L-iduronidase stop mutations, Q70X
(Hoffman, Brown, and Kunkel, 1987). It is reported that approx- and W402X, exhibits a significant increase in α-L-iduronidase
imately 60% of the DMD/BMD patients have deletions in the activity when cultured in presence of gentamicin, resulting in
dystrophin gene itself (Forrest et al., 1988; Gillard et al., 1989; the restoration of 2.8% of the normal α-L-iduronidase activity
Lindlöf et al., 1989), while it is believed that the remaining 40% (Keeling et al., 2001). In another study, treatment of CHO-K1
of patients have small deletions or point mutations in the re- cell lines with gentamicin resulted in a 4-fold increase in the
gion that encodes the gene. Moreover, the presence of nonsense levels of α-L-iduronidase activity for some of the Q70X and
mutations within the gene account for approximately 5–15% of W402X constructs (Hein et al., 2004).
muscular dystrophies (Mendell et al., 2001; Prior, Bartolo, and
Pearl, 1995). In this condition, the patients in the age group of Drug Delivery Systems
early to mid-twenties exhibit progressive muscular degenera- Osteomyelitis is a bone disease caused by bacterial infection
For personal use only.

tion and mortality occurs due to respiratory or cardiac failure. of the bone medullar cavity, cortex, and/or periosteum. One of
Barton-Davis et al. (1999) demonstrated the possibility of treat- the most common causes is postoperative sepsis following or-
ing DMD patients with nonsense mutations using gentamicin thopedic surgery. The treatment of this bone infection remains
treatment. They used gentamicin to successfully suppress the difficult due to problems of local penetration of the system-
nonsense mutations and consequently restored the dystrophin atically administered antibiotic. Systematic administration can
expression levels to 10–20% of the normal in the skeletal mus- also pose problems of toxicity to bystander tissues (Rutledge
cle of mdx mice (animal models for DMD which possess a et al., 2003). Moreover, the bacteria adhere to the bone matrix
nonsense mutation in the dystrophin gene) both in vitro and and orthopedic implants avoiding the host defenses and antibi-
in vivo. otic action by developing a slimy film (biofilm) or acquiring
However, very few patients are applicable to this therapy a very slow metabolic rate (Ciampolini and Harding, 2000).
because of three reasons, namely (1) only 5–15% of DMD pa- Hence, a suitable drug delivery system with controlled antibi-
tients have nonsense mutations in the dystrophin gene, (2) it otic release is needed for the treatment of chronic osteomyelitis
is challenging to find nonsense mutations in the gene since which can potentially minimize bone loss and multiple surgical
dystrophin cDNA is very long (14 kb), and (3) the efficiency procedures. Gentamicin-impregnated polymethyl-methacrylate
of aminoglycoside-induced read-through is dependent on the (PMMA) beads have played a major clinical role for the treat-
type of nonsense mutation (Howard et al., 2000). Considering ment of chronic posttraumatic osteomyelitis for many years
these limitations, a novel approach was developed to identify (Armynot du Chatelet et al., 1989; Bunetel et al., 1990; Giu-
DMD patients that qualify for aminoglycoside therapy. The sys- liano, Verpooten, and de Broe, 1986). The use of gentamicin-
tem consists of monitoring dystrophin expression of myotubes impregnated PMMA beads has proved highly effective in the
differentiated from fibroblasts of DMD patients that were trans- treatment of patients with impaired renal function (Walenkamp
fected with AdMyoD and cultured in gentamicin. The dystrophin and Vree, 1981), management of localized bone and soft tissue
expression in gentamicin-exposed myotubes was monitored by infections in humans (Henry and Galloway, 1995), and in treat-
in vitro dystrophin staining and Western blotting analysis. The ing septic arthritis in dogs (Brown and Bennett, 1988). These
results indicated that gentamicin was able to induce dystrophin beads have the potential of releasing more gentamicin by diffu-
expression in the differentiated myotubes by the read-through sion process than the solid bone cement plugs due to the much
of the nonsense mutation TGA in the gene; a read-through of increased surface area of the many small beads used for the
the nonsense mutations TAA and TAG did not occur and conse- purpose. However, the drawback of PMMA beads usage is that
quently did not lead to dystrophin expression. Therefore, it was these beads are not resorbable and need a second surgery after
suggested that aminoglycoside therapy was far more effective a few months to remove the implanted beads after the release
for DMD patients who have nonsense mutation TGA and un- of gentamicin and be replaced either by new beads to prolong
 196 C. GANESH KUMAR ET AL.

the antibiotherapy or by a bone substitute to accelerate bone croporous PCL matrices (Chang, Perrie, and Coombes, 2006;
reconstruction (Neut et al., 2003). Coombes et al., 2004). Further, a controlled delivery system
Subsequent research efforts focused on bioceramics such as of gentamicin-impregnated polycaprolactone (PCL) fibers was
self-setting tricalcium phosphate (β-TCP), sintered hydroxya- prepared by gravity spinning the particulate suspensions of
patite (HAP) cements, and bioglass which were used as bone gentamicin in PCL solution. These fibers find potential appli-
implants and acted as gentamicin-delivery systems for local cation for controlled release of gentamicin to combat infec-
antibiotic treatment of bone infections because of their struc- tion associated with periodontal disease, musculoskeletal in-
tural similarity to the mineral phase of bone and their osteo- juries, and implantation of fiber-based tissue substitutes, such
conductive and bone-binding properties (Bohner et al., 2000; as vascular prostheses (Chang et al., 2008), despite their es-
Joosten et al., 2004; Shinto et al., 1992; Thoma, Alex, and tablished applications in soft tissue engineering (Williamson
Randzio, 1992; Yang et al., 1996). This approach also offsets and Coombes, 2003; Williamson, Chang, and Coombes,
Critical Reviews in Biotechnology Downloaded from [Link] by SUNY Health Sciences Center on 10/08/14

the problems of brittleness and the difficulty of shaping hard 2004).

ceramic materials to fit bone defects. Further, composite micro- Nasal drug delivery has been investigated as an alternative to
spheres containing poly(3-hydroxybutyrate-3-hydroxyvalerate) the parenteral routes for systemic drug delivery that shows poor
(PHBV) and wollastonite were developed as controlled re- oral bioavailability (Behl et al., 1998). Some limiting factors ob-
lease gentamicin-delivery systems for treatment of chronic os- served for nasal drug delivery are the inadequate drug adsorption
teomyelitis (Li and Chang, 2005). Mesoporous bioactive glasses and the nasal mucocilary clearance. Nasal mucocilary clearance
(MBG) find application as a promising controlled gentamicin- severely limits the time allowed for drug absorption to occur
delivery system (Xia and Chang, 2006). Later developments and does not permit sustained nasal drug administration. In this
in this area included the use of copolymer blends of polyan- context, mucoadhesive preparations were developed to increase
hydride matrix (erucic acid and sebacic acid dimer beads, a the contact time between the dosage form and mucosal layers of
bioerodable composite impregnated with gentamicin) (Domb nasal cavities, which enhance drug absorption (Ugwoke et al.,
and Maniar, 1993; Nelson, Hickmon, and Skinner, 1997), cal- 2000; Vidgren et al., 1991). Different semi-synthetic polymers
cium phosphate/polylactide (Schnieders et al., 2006; Soriano based on cellulose derivatives, such as hydroxypropyl methyl-
For personal use only.

and Evora, 2000), coralline hydroxyapatite-gelatin composite cellulose (HPMC), hydroxypropyl cellulose (HPC), and car-
microspheres (Sivakumar and Rao, 2002), collagen/poly(lactic- boxymethylcellulose sodium (CMC Na) were used previously
co-glycolic acid) (PLGA) (Schlaap and Friess, 2003), Eudragit in the preparation of mucoadhesive microspheres for nasal drug
RS100 microspheres (Singh et al., 2008) for sustained release delivery (Nakamura et al., 1996; Vidgren et al., 1992; Zhou
of gentamicin for effective cure of osteomyelitis. Different bone and Donovan, 1996). Duchateau et al. (1986) studied the ef-
cements produced by various manufacturers vary in composition fect of bile salts on the nasal absorption of gentamicin. Sodium
and differ in their mechanical properties and antibiotic elu- cholate and sodium taurodeoxycholate were the most active ab-
tion characteristics. Some of the bone cements/implants avail- sorption promoters and the absorption rates were 41 and 34%,
able commercially as gentamicin-delivery systems are shown respectively. They found that without using these compounds,
in Table 3. The added advantage is that no secondary surgery gentamicin was not absorbed. Illum et al. (1988) reported in-
is required for the implant removal. Further, polylactide micro- creased nasal absorption of gentamicin by using microsphere
spheres containing gentamicin find application for the treatment formulations (Illum et al., 1988). The absorption was further
of Brucella melitensis infections (Blanco-Prieto et al., 2002; enhanced by the use of lysophosphatidylcholine (lysolecithin)
Gamazo et al., 2004; Lecaroz et al., 2007; Prior et al., 2000; as an enhancer. The bioavailability of gentamicin increased to
Prior et al., 2004). Nanoparticles made of PLGA 502H contain- about 50% of the intravenous dose as compared to less than 1%
ing gentamicin were found to be most suitable nanocarriers due bioavailability observed with a simple formulation. In addition,
to their highest entrapment (6.2 μg mg−1 ) and sustained release mucoadhesive microsphere formulations based on hyaluronic
of gentamicin for treatment of brucellosis (Lecaroz, Gamazo, acid (HA), chitosan glutamate (CH), or a combination of the
and Blanco-Prieto, 2006b) and other intracellular gentamicin- two were prepared loaded with gentamicin sulphate as a model
susceptible pathogens (Lecaroz, Gamazo, and Blanco-Prieto, drug using solvent evaporation method for nasal drug deliv-
2006a). Gentamicin-impregnated collagen sponges (GICS) ery. The bioavailability of gentamicin from HA and HA/CH
have gained renewed interest for the management of sep- formulation was 50% longer as compared to a formulation pre-
tic arthritis in cattle (Hirsbrunner and Steiner, 1998), horses pared with CH alone (Lim et al., 2000). Further, hydroxypropyl
(Holcombe et al., 1997), and dogs (Owen, Moores, and Coe, methylcellulose-based microsphere formulations were prepared
2004). loaded with gentamicin sulphate for nasal drug delivery using
Gentamicin-loaded poly (ε-caprolactone) (PCL) based- spray-drying technique. Sodium cholate was used as the active
microporous matrices were prepared by a precipitation casting absorption promoters for increasing the absorption of gentam-
technique for delivery of gentamicin to treat musculoskeletal icin through the nasal mucosa (Hascicek, Gonul, and Erk, 2003).
and ocular infections and bone substitutions. This approach en- Most pathogens exhibit a high intrinsic resistance to
ables improved rate and duration of gentamicin release from mi- most available antibiotics. Hence the development of an
 MICROBIAL BIOSYNTHESIS AND APPLICATIONS OF GENTAMICIN 197

TABLE 3
Bone cements/implants available commercially as gentamicin-delivery systems

Trade name/Company Type of material Reference


R
Sulmycin Implant (Essex Pharma, Munich, collagenous sponge (2 mg gentamicin per 2.8 mg Leyh et al., 1999
Germany) collagen)
Collatamp G (Schering-Plough Animal Health Collagen sponge (2 mg gentamicin-impregnated) Owen et al., 2004
Corporation, UK)
OsteofoamTM (Birmingham Polymers, Inc. 3-D poly(lactide-co-glycolide) PLGA 75/25 Holy et al., 1997; Holy
Birmingham, AL, USA) scaffolds et al., 2001
Critical Reviews in Biotechnology Downloaded from [Link] by SUNY Health Sciences Center on 10/08/14


R
Boneloc bone cement (Polymers Gentamicin-loaded Butylmethacrylate (BMA) Nilsson and Dalen,
Reconstructive A/S, Farum, Denmark) 1998
R
Resomer (Boehringer Ingelheim KG, PLGA spheres Poly(lactic-co-glycolic acid) Baro et al., 2002
Germany) (PLGA)
SeptacinTM (Abbott Laboratories, North polyanhydride matrix (copolymer of erucic acid Stephens et al., 2000
Chicago, IL, USA) dimer (EAD) and sebacic acid in a 1:1 weight
ratio)

R
Biobon (Biomet Merck, Germany) powdered HAC Joosten et al., 2004
R
CMW-1 (De Puy Iberica SA, Madrid, Spain) Copolymer of poly(methyl methacrylate) (PMMA) Frutos et al., 2002
and 2-hydroxyethyl methacrylate (HEMA)

R
Septopal (Merck, Darmstadt, Germany) Gentamicin-loaded PMMA beads Neut et al., 2003

R
Palacos R+G (Heraeus Kulzer GmbH, Hanau, Gentamicin-loaded PMMA; Carrier: refined Dall et al., 2007
Germany) peanut oil; Filler: 10% BaSO4 ; containing 0.84 g
For personal use only.

gentamicin sulphate (0.5 g active gentamicin)

Refobacin Bone Cement R (RBC; AAP Gentamicin-loaded PMMA; Carrier: Sunflower Dall et al., 2007
Biomaterials GmbH & Co., KG, Dieburg, oil; Filler: 10% BaSO4 ; containing 0.84 g
Germany) gentamicin sulphate (0.5 g active gentamicin)

R
Cemex ISOPLASTIC (Tecres Spa, Verona, Gentamicin-loaded PMMA; Filler: 10% BaSO4 Cenni et al., 2002
Italy)

R
Simplex P (Stryker-Howmedica-Osteonics, Gentamicin-loaded PMMA-styrene copolymer DiCicco et al., 2003
Inc., East Rutherford, NJ, USA) cement
R
Osteopal V (Heraeus Kulzer GmbH, Hanau, PMMA bone cement; Zirconium dioxide as X-ray Gheduzzi et al., 2006
Germany) contrast medium

R
Palamed G (Merck KGaA, Darmstadt, PMMA bone cement containing 0.86% (w/w) Kienapfel et al., 2004
Germany) gentamicin base
Copal (Biomet Merck, Darmstadt, Germany) PMMA bone cement containing 1.62% (w/w) Ensing et al., 2006
gentamicin base
CMW 1 Radioopaque G (DePuy International PMMA bone cement containing 1.7% (w/w) Hendriks et al., 2003
Ltd., Leeds, UK) gentamicin base
CMW Smartset-G (DePuy International Ltd., PMMA bone cement containing 4.2% (w/w) Shi et al., 2006
Leeds, UK) gentamicin base
Smartset GHV Gentamicin (DePuy Acrylic bone cement containing 4.22% (w/w) Lewis and Janna, 2006
Orthopaedics, Warsaw, IN, USA) gentamicin base
VersaBondTM AB (AAP Biomaterials GmbH & Acrylic bone cement containing 4.2% (w/w) Lewis and Janna, 2004
Co., KG, Dieburg, Germany; Distributor: gentamicin base
Smith and Nephew, Inc., Memphis, TN, USA)

antibiotic-delivery system is of interest that could achieve ef- encapsulated gentamicin delivery system could provide the
ficient intracellular biodistribution of the antibiotic with re- above benefits. Liposomes were extensively used as antimicro-
duced antibiotic toxicity, while increasing the therapeutic index bial and antineoplastic drug carriers (Berestein, 1987). They are
against intracellular pathogens. In this regard, the liposome- commonly produced from naturally occurring, biodegradable
 198 C. GANESH KUMAR ET AL.

and nontoxic phospholipids and their derivatives. Liposomes Abou-Zeid, A. A., Eissa, A. E. I., and Salem, H. M. 1974. The fer-
were designed to release drugs into an extracellular or intracel- mentative production of gentamicins by Micromonospora purpurea.
lular compartment to reach their site of action (Fielding, 1991). J. Appl. Chem. Biotechnol. 24: 655–661.
The ability of liposomes to alter the drug distribution depends Abou-Zeid, A. A., Eissa, A. E. I., and Salem, H. M. 1976a. Influence
largely on their size and surface properties (Schiffelers, Storm, of some compounds on gentamicin formation by Micromonospora
purpurea. J. Appl. Chem. Biotechnol. 26: 318–322.
and Woudenberg, 2001). Thus, the encapsulation of aminogly-
Abou-Zeid, A. A., Eissa, A. E. I., and Salem, H. M. 1976b. Production
cosides into liposomes significantly improves their therapeutic of gentamicin antibiotics by Micromonospora purpurea. Indian J.
index by altering their pharmacokinetics and pharmacodynam- Exp. Biol. 4: 200–203.
ics, thus enhancing the accumulation of the drug at the infection Abou-Zeid, A. A., Salem, H. M., and Eissa, A. E. I. 1978. Production
site and reducing the nephro- and oto-toxicity that would attract of gentamicins by Micromonospora purpurea. Zentralbl. Bakteriol.
considerable interest from an application perspective (Lutwyche Naturwiss. 133: 261–275.
Critical Reviews in Biotechnology Downloaded from [Link] by SUNY Health Sciences Center on 10/08/14

et al., 1998; Morgan and Williams, 1980). A liposome-mediated Abrasimovskii, P. I., Valdimirov, A. V., and Bartoschevich, I. E. 1990.
gentamicin delivery system was developed with gentamicin Phosphate regulation of the processes of growth and biosynthesis of
encapsulated in liposomes comprising lipid compositions gentamicin in Micromonospora purpurea var. violaceae. Antibiot.
(1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- Khimioter. 35: 5–8.
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2- Adams, E., Roelants, W., De Paepe, R., Roets, E., and Hoogmartens, J.
1998. Analysis of gentamicin by liquid chromatography with pulsed
distearoyl-sn-glycero-3-phosphocholine (DSPC)) and choles-
electrochemical detection. J. Pharm. Biomed. Analysis 18: 689–698.
terol in the molar ratio of 2:1 by sonication for controlling Ahlert, J., Distler, J., Mansauri, K., and Pipersberg, W. 1997. Iden-
pulmonary infections caused by P. aeruginosa in cystic fibrosis tification of stsC, the gene encoding the L-glutamine: scyllo-
patients (Mugabe, Azghani, and Omri, 2005). inosose aminotransferase from streptomycin-producing Strepto-
mycetes. Arch. Microbiol. 168: 102–113.
Al-Amoud, A. I., Clark, B. J., and Chrystyn, H. 2002. Determination of
Other Uses gentamicin in urine samples after inhalation by reversed-phase high-
Gentamicin has been identified as a promising candidate for performance liquid chromatography using pre-column derivatisation
use in cell and tissue culture (at 50 μl ml−1 ) due to its bacte-
For personal use only.

with o-phthalaldehyde. J. Chromatogr. B 769: 89–95.

ricidal role. The cell or tissue culture samples, clinical samples Al-Awqati, Q. 1995. Regulation of ion channels by ABC transporters
containing viruses, or specimens need gentamicin supplementa- that secrete ATP. Science. 269: 805–806.
tion to prevent bacterial growth during transportation (Schafer Albracht, J. H., and De Wit, M. S. 1987. Separation of gentamicin in raw
et al., 1972). Bacteria are the causative agents of spoilage in pro- materials and pharmaceuticals by HPLC. Chromatogram. 8: 7–8.
ducing mycorrhizae in axenic systems. To counteract these prob- Alcid, D. V., and Seligman, S. J. 1973. Simplified assay for gentamicin
in the presence of other antibiotics. Antimicrob. Agents Chemother.
lems, gentamicin has been applied for the selective inhibition of
3: 559–561.
contaminating bacteria in axenic culturing of ectomycorrhizal Al-Majed, A. A., Belal, F. B., Abounassif, M. A., and Khalil, N. Y.
plants (Kidd et al., 1982). Gentamicin sulfate (at 50 μl ml−1 ) 2003. Fluorimetric determination of gentamicin in dosage forms and
also finds use in the prevention of contaminating bacteria in the biological fluids through derivatization with 4-chloro-7-nitrobenzo-
HeLa cell plaque forming test by Shigella sonnei (Jeong et al., 2-oxa-1,3-diazole (NBD-Cl). Microchim. Acta 141: 1–6.
2007). Gentamicin in combination with ultrasound treatment Anhalt, J. P. 1977. Assay of gentamicin in serum by high-pressure liquid
has been successfully applied for killing of biofilm bacteria like chromatography. Antimicrob. Agents Chemother. 11: 651–655.
E. coli and P. aeruginosa, which are commonly encountered on Anhalt, J. P., and Brown, S. D. 1978. High-performance liquid-
implant devices such as catheters, dialysis tubing, drug-delivery chromatographic assay of aminoglycoside antibiotics in serum. Clin.
tubing, heart pacemakers, artificial vascular grafts, heart values, Chem. 24: 1940–1947.
etc. (Carmen, et al., 2004; Pitt et al., 1994). Anhalt, J. P., Sancilio, F. D., and McCorkle, T. 1978. Gentamicin C-
component ratio determination by high-pressure liquid chromatog-
raphy. J. Chromatogr. 153: 489–493.
Ara, J., Gans, Z., Sweeney, R., and Wolf, B. 1995. Dot-ELISA for the
ACKNOWLEDGMENTS rapid detection of gentamicin in milk. J. Clin. Lab Anal. 9: 320–
The authors are grateful to the Director, Indian Institute of 324.
Chemical Technology, Hyderabad, for providing the facilities for Arcelloni, C., Comuzzi, B., Vaiani, R., and Paroni, R. 2001. Quantifi-
the research work on gentamicin. The authors are also thankful cation of gentamicin in Mueller–Hinton agar by high performance
to the three anonymous reviewers for their helpful comments. liquid chromatography. J. Chromatogr. B 753: 151–156.
Armynot du Chatelet, A. M., Fourcade, S., Atisso, M. A., Simeon
de Bouchberg, M., Yakoun, M., Varet, A., and Piedra C. 1989. In
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