The Genetics of

Bacterial Drug
Resistance
Cha pte r two

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 Introduction
It is now a point of common agreement that we have entered the “post-antibiotic era” of
medicine…a time when we will have more and more cases of multi-drug resistant
microbes for which we have few therapeutic answers in our arsenal. This is because we
have been unwise about our choices of how and when to treat, and more and more, this
is becoming a topic stressed in USMLE questions. Concern that vancomycin-resistant
Staphylococcus aureus (VRSA) and carbapenem-resistant Klebsiella pneumoniae (CRKP)
might spread globally is well-founded, and if we want to prevent the problem from
spreading, we need to understand how it began.

Forms of Drug Resistance

three general categories of drug resistance:

• Intrinsic
if a microbe lacks the target molecule for a given drug, it is said to be intrinsically
resistant. For example, Mycoplasma lacks a cell wall, the target for the penicillin-style
drugs. Therefore, Mycoplasma is intrinsically resistant to penicillin.

• Chromosomal
this means that the genes encoding resistance are found on the chromosome of the
microbe. The most important example of such a trait in medicine is
methicillin-resistant Staphylococcus aureus (MRSA), which arose shortly after the

penicillin-binding protein (PBP)

in hospitals worldwide. Most chromosomal genes encode structural proteins.

• Plasmid-mediated
this means that the resistance genes are carried on plasmids which are
extrachromosomal circles of DNA within the bacterial cell. most plasmid resistances
encode enzymes. The drug resistances encoded on plasmids include:
lactamases which inactivate penicillin and cephalosporins
for tetracycline and sulfonamides
Phosphorylases, acetylases and adenosylases which modify aminoglycosides
and chloramphenicol
methylases for macrolides, lincosamides
inactivate ribosomal binding sites for tetracycline
Ligase which creates cell wall pentapeptides that end in D-Ala-D-Lac which will
not bind vancomycin

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The problem, of course, is that microbes would simply like to remain alive. It is actually
not in their best interests to kill their hosts, but often medicine has taken a “scorched
earth” approach to kill all of them rather than foster a more healthy and realistic
discussion of management of well-being in the presence of a diverse microbiome.

spontaneously, and those microbes which possessed the resistance genes had selective
advantage in the presence of our drugs. Over many decades, the resistant populations
became the predominant clones, and so we have been forced over time to develop
more and more antimicrobics.

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 The DNA of Bacteria
Bacteria have 3 types of DNA molecules which can be transcribed and translated to
create the proteins useful for their lives.
• Chromosome
The largest molecule of DNA in the cell which contains all genes essential for life. It is
a large, closed circle.
Most chromosomal genes encode structural proteins.
Because the chromosome encodes all essential genes, mutations in chromosomal
genes have a high chance of damaging the bacterium.

• Plasmid
Circular genetic elements outside of the chromosome which encode non-essential
(but potentially useful) genes.

Most plasmid genes encode enzymes.

Today, the most common newly arising drug resistances are encoded on plasmids,
and as you will see in our discussion, bacteria have developed accelerated
techniques for trading plasmids with one another to assist their survival.

• Bacteriophage genome
The viruses of bacteria (bacteriophages or phages) may integrate their DNA into the
chromosome of the cell they infect.
This is an attribute of the lifecycle of the “temperate” or “lysogenic” phage, and for
as long as the viral DNA is stably integrated into the bacterial chromosome, its
genes are transcribed and translated just as if they belonged to the bacterium.
This type of symbiosis is referred to as lysogenic conversion, and is the source of
many important pathogenic traits in bacteria:
› Labile toxin of ETEC
› O antigen of Salmonella
› Verotoxin of EHEC
› Erythrogenic exotoxins of Streptococcus pyogenes
› Diphtheria toxin
› Cholera toxin
› Botulinum toxin
› Shiga toxin

We will go through the mechanism by which this occurs in detail, but for now, the
mnemonic LOVED CBS can help you remember these important traits. (If you are loved,
that Can Bring Sequelae…like pregnancy, since the bacteria in each of these cases, are
“pregnant” with phage.)

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 Recombination
Since prokaryotes do not have a nuclear membrane (or any internal membranes), they
do not have a mechanism to protect their DNA from enzymatic processes happening in
the cytosol. If DNA were donated from one bacterial cell to another, therefore, the newly
received DNA would not survive long. The process of recombination is designed to solve
this problem by packaging DNA into circles which cannot be cleaved open, because
their restriction endonuclease sites are protected by methylation. There are two forms
of recombination which depend on the shape of the newly arrived, donated DNA
molecule:
• Homologous recombination (for short linear pieces)
• (for circular pieces with restriction endonuclease sites
in common with the chromosome or plasmid).

Remember that recombination must happen AFTER any DNA has been donated from

since every form of genetic exchange except one will require some sort of
recombination at the end.

Homologous Recombination

3
2
1
1 2
1
3 2
3
4

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• The DNA (entering from outside) must be a linear piece with some
sequence homology with the chromosome.
• The recipient cell must have recombinase A, which lines up the homologous
sequences and does the cutting and pasting that removes the chromosomal piece
and replaces it with the exogenote DNA.
• There is a of incoming DNA for what was previously in the
chromosome.
• The newly excised DNA is destroyed by exonucleases.

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 Medical Education

is a mechanism by which small circles of DNA (plasmids,

temperate phage or transposons) can be incorporated into the chromosome.
• The incoming circle must have a restriction endonuclease site in common with the
chromosome.
• When a cut is made in both circles of DNA at the restriction endonuclease sites, the

• This effectively incorporates all of the small circle into the large circle, and no DNA is
lost in the process.

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 Horizontal Gene Transfer

showed that bacteria were able to exchange DNA when grown together in culture.

Bacterial culture with Traits A+ B+ Bacterial culture with Traits A- B-

Culture
together
Bacteria with Traits A+ B+ Bacteria with Traits A- B-

Bacteria with Traits A+B- Bacteria with Traits A-B+

Although it would take decades to elucidate the mechanisms behind these results, we
now know that horizontal genetic exchange in bacteria can occur via 3 mechanisms:
• Transformation
• Conjugation
• Transduction

If one of these mechanisms creates a new variant with improved survival ability, then
natural selection will cause that variant to become a larger proportion of the population.
Unfortunately, we provide the selection pressure to cause bacteria to continuously
adapt to the pharmacologic agents that we use.

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1. TransFormation (Free DNA)

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• Uptake and incorporation of free DNA from the environment by competent cells,
followed by incorporation using homologous recombination.

• In order to do this, cells must become “competent” or able to bind free DNA to their
membrane and import it. Some bacteria are naturally competent (natural
transformers):
Streptococcus pneumoniae
Bacillus spp.
H
Neisseria spp.
(mnemonic: Some Bacteria Have Natural competence)

• Once the DNA is inside the cell, the remainder of the process is homologous
recombination, requiring recombinase A, and a one-to-one exchange of exogenote
DNA for what was previously in the chromosome.

• If the acquisition of the new DNA allows the bacterium to have selective advantage
(e.g. become encapsulated), then it has acquired a virulence trait by this method.

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2. Conjugation
Conjugation is transfer of DNA using cell-to-cell contact. It is the analogy to sexual
exchange in eukaryotes, and as such, requires a donor (male) and recipient (female).
The gender role of the bacterium is determined by the presence or absence of a fertility
factor, which is a plasmid with the genetic coding for the process.

• Tra (transfer) operon – a group

of genes which control the
production of sex pili, the
conjugal bridge and the
enzymes for DNA metabolism
o n ori V
er
op

• oriV – the origin of vegetative

tra

replication. The site where a

replication fork will form when
the plasmid replicates.

• IS – insertion site, a restriction

endonuclease site that can
allow the plasmid to integrate
into the chromosome.
ori T IS
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• oriT – the origin of transfer. The
site of a break in one strand of
the DNA duplex that will lead the
strand across the conjugal
bridge and into the recipient.

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 Mating Types of Bacteria

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F- F+ Hfr

• There are 3 mating types of bacteria.

F- cell is the recipient in any cross. It has chromosomal DNA but does not have
a fertility factor.
F+ cell is a donor and has both chromosomal and fertility factor DNA in plasmid
form.
Hfr (high frequency recombinant) cell is a donor and has its fertility factor
integrated into the chromosome as an episome.

• There are two possible conjugal crosses. Each requires a donor and a recipient, so they
are F+ to F- and Hfr to F-.

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 The F+ by F- Cross

• The F+
sex pili to create a bridge to the
cytoplasm of the F- cell.

F+ F-

ori T
• A break in plasmid DNA is made
at oriT and a single strand is
transferred to the recipient.

• Both cells duplicate the second

strand of plasmid DNA.
• Each cell at the end has a
fertility factor, so the recipient
has become a donor in the
process.

F+ Medical Education

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 The Hfr Chromosome

Remember that in the Hfr chromosome, the fertility factor plasmid incorporated into the
chromosome by the process of The two ends of the

two types of DNA. The process is still begun with a break at oriT, but now, because the
episome is included within the chromosome, chromosomal alleles will follow oriT across
the conjugal bridge. Which chromosomal genes will be successfully transferred
depends on their , since it is a race to get across the conjugal
bridge before it breaks.

tra
ori T

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 -
Cross

• The Hfr cell creates a conjugal bridge to the

cytoplasm of the F- cell by modifying one of
its sex pili.

• A break is made in a single strand of the

episome at oriT and leads across the bridge.
Hfr F-

original fertility factor, but thereafter,

chromosomal genes are pulled in order.

• Because of the fragility of the conjugal

bridge, and the size of the entire Hfr
1 chromosome, it is not possible for the entire
3 2 molecule to cross over.

• The bridge breaks, and the DNA that has

made it into the F- cell must be
homologously recombined in order to be
saved.

2 1
3 • The only possible homology in the
chromosome of the F- recipient is that of
rec A the chromosome-origin DNA (not
episomal).

variables (speed of getting across the

bridge and speed of recombination), it
3
becomes true that the genes closest to oriT
2 are those that are most likely to be
1
successfully transferred.

• Because the tra operon is not transferred, the F-

cell remains a recipient with new
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chromosomal genes.
Hfr
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 3. Transduction

Transduction is the delivery of DNA from one bacterium to another using a phage as a
vector. Because phages have two types of lifecycles, there are two types of
transduction, both of which are accidents of the viral lifecycle.

Generalized transduction (an error of the lytic phage lifecycle)

The Lytic Phage Lifecycle:

1 Lytic (virulent) phage infects bacterial cell
2 Bacterial DNA is destroyed and phage
1 2 reproduces itself.
3.
3 Phage heads with phage DNA leave cell by
lysis to infect new cells

The accident of generalized transduction:

3A Defective phage with bacterial DNA injects
DNA into new cell
3B Homologous recombination stabilizes new
DNA

3 3A 3B

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 The Temperate Phage Lifecycle

Bacteriophages that undergo a stage of stable association of their genome within the
bacterial cell are referred to as temperate or lysogenic phages. The association
remains stable as long as a repressor gene in the viral genome prevents disassociation

bacterial cell. If the repressor gene is inactivated, the phage enters induction, removing
its genome from the chromosome and beginning a phase of rapid lytic replication.

Integrates phage DNA

Lysogenic phage into chromosome by
infects bacterium
recombination

If the repressor gene is

inactivated, the viral
The virus moves
into a lytic phase
of its lifecycle of the chromosome
and progeny
phage are made Medical Education

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 Specialized Transduction

If an fails to excise all the viral DNA and instead takes

bacterial DNA along, then this defective DNA is capable of causing specialized
transduction.

1• If during excision, the two restriction

1 endonuclease sites fail to realign, the
2 two molecules of DNA are pinched
apart from one another in an
1
erroneous fashion. This leaves a portion
of the viral genome in the
chromosome and puts a portion of the
chromosome within the viral shells that
are formed. Neither microbe can live
without the DNA it needs.

2• A generation of defective tranducing

phages are created, which are
3 capable of injecting the defective DNA,
but are not able to complete the
lysogenic life cycle.

1 1
3• If rec A is available in the next infected
cell, however, the genes captured from
rec A

cell can be transferred to the other via

homologous recombination.
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 Conjugation Transduction
Transformation
F+ - -
Generalized Specialized

Free DNA from Cell-to-cell transfer Error of Error of

Mechanism through sex pilus
environment packaging excision

Chrom. Chrom.
Fertility genes next to
DNA transferred Any factor closest to Any integration
plasmid oriT of site of
episome phage

Recombination Homologous No Homologous

Requires competency, Error of lytic Error of

Sex change No sex
Comments sensitive to virus LC temperate
in F- change
extracellular DNAses phage LC

Streptococcus
Most gram negatives,
pneumoniae PBP MRSA
all resistances, VRSA*
mutations

*Neisseria gonorrhoeae is a special case of transfer via conjugation: it takes two

plasmids to accomplish the job of one, because the plasmid with the DR genes has lost
its tra operon. As long as there is another fertility factor in the cell that can build the
conjugal bridge, the DR plasmid can sneak across in a process known as mobilization.
So, if you ever see reference to “conjugation using non-conjugative plasmids” this is
what they are referring to.

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 Transposition and the Development of
Multi-Drug Resistance Plasmids

“mobile genetic elements” in a plant related to corn. Since then, we have learned that it
is similar elements capable of moving themselves from place to place inside a bacterial
cell that have been the origin of an explosion of multiple drug resistance plasmids.

Transposition can be replicative (make multiple copies of a section of DNA), or

conservative in which case it simply moves the single copy from one place to another.

remember that not all molecules of DNA within a bacterium are equally easily
transmitted, so if the movement causes insertion into a fertility factor, the mechanism of
transfer to other cells is assured.

Transposable elements come in multiple sizes (cassettes, integrons, insertion

sequences, transposons) depending on the amount of genetic material contained
within. Insertion sequences are often considered the smallest mobile element,
encoding simply the genes which cause transposition. Transposons
additional genes, so our interest is in those that encode drug resistances.

indirect repeats
segment. An indirect repeat reads identically right to left on the bottom strand and left
to right on the top strand. This allows protection as a circle when it moves.

The transposase enzyme carried with the transposon makes a staggered cut in a new
location of DNA inside the cell and pastes the transposon into that location. This creates
gaps on either side of the location where the transposon landed, and those gaps are
repaired to create direct repeats (directly superimposable nucleotide sequences) on
either side of the mobile element. When transpositional events collect multiple
resistance genes in a fertility factor plasmid, the result is a resistance transfer factor, of
which the plasmid of vancomycin-resistant Enterococcus is an example.

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 Screening for Antibiotic Susceptibility

1. Kirby-Bauer Agar Disc Diffusion

The Kirby-Bauer is a rapid screening assay to test the isolate from a patient against a
variety of antibiotics at a variety of concentrations.

• Filter paper discs soaked in different antibiotics are dropped onto the surface of the
dish. The antibiotics diffuse out from the disc, creating a concentration gradient that
will either inhibit the microbe or not.
• The dish is incubated and examined for zones of inhibited growth in the proximity to

• This yields a quick, qualitative result that the isolate is resistant, intermediate or
sensitive to the drug.

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2. Minimal Inhibitory Concentration (MIC)
The follow up to the Kirby-Bauer screening is to test for the minimum concentration of
the drug which will inhibit growth of the isolate (MIC).
• Make a serial dilution of the drug in buffer and include a no drug control.
• Put a known number of bacteria from the patient’s isolate into each tube, incubate
and examine for turbidity as a measure of increased cell count.
• The MIC is the concentration of drug in the last tube in the dilution series with no

MIC

8.0 4.0 2.0 1.0 No drug 100 µl

control
µg/ml
Serial dilution

40 ml
agar
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MBC

3. Minimal Bactericidal Concentration (MBC)

The MBC is a sub-plating step following the MIC and is not done routinely in all hospital
labs.

• This dilution effectively removes the drug from the presence of the bacteria.
• If colonies grow after culture, the organisms were not dead. Thus, the MBC shown

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 Techniques of Infection Control

• Sterilization
complete removal or killing of any viable agent.
• Disinfection
removal of pathogens from an area.
• Antiseptics
disinfectants for the skin.
• Pasteurization
rapid heating and cooling of a liquid to remove pathogens.

Techniques of Physical Control:

• Heat
o

• Radiation
creates thymine dimers in DNA
• Filtration

forms like Mycoplasma

Techniques of Chemical Control:

Membrane-targeting agents
alcohol, phenol, detergents. Enveloped viruses can be inactivated with these agents.

Protein denaturing agents

chlorine, ethylene oxide, formaldehyde, glutaraldehyde, heavy metals, hydrogen
peroxide, iodine. Naked viruses can be inactivated with these chemicals.

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 Before you leave, can you…

bacteriophage genome.

bacteria (conjugation, transformation, transduction).

3. Explain the role of lysogenic conversion and the bacterial pathogenicity factors which
result from it.

recipient cell.

plasmids.

6. Identify the means by which the major bacterial pathogens acquire and trade
resistance genes.

and sterilize to prevent disease transmission.

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