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Chapter 33

Molecular Epidemiology of Antibiotic


Resistance in Humans and Animals
Sebastian G.B. Amyes1 and Benjamin A. Evans2
1
University of Edinburgh, Edinburgh, UK, 2Anglia Ruskin University, Cambridge, UK

DEFINITIONS for Mycobacterium tuberculosis this is an uncommon


mechanism for resistance as the resultant resistant bacte-
Any discussion of epidemiology of antibiotic-resistant ria, which proliferate in the presence of the antibiotic, are
bacteria has inherent flaws. Epidemiology implies a rigor- not sufficiently competitive when the antibiotic is
ous study where a population of bacterial isolates are col- removed and they simply got diluted out when the patient
lected prospectively, in particular that all patients with a finished treatment and the bacteria with the susceptible
specific infection have specimens taken and all of these target gene proliferated. However, some bacteria have
are examined in the laboratory. Of course, this almost ameliorated the fitness cost imposed by resistance through
never happens, especially with community isolates. generating a compensatory mutation which cancels out
Specimens are often only taken in more difficult cases or the cost of resistance, allowing the resistant bacteria to
if the patient has not responded to previous antibiotic proliferate in the absence of the antibiotic. In terms of
treatment. This can bias the data so that the proportion of epidemiology, while the same mutation may be found in
resistance reported is considerably higher than it actually several different bacteria, the mutations will have arisen
is. This is particularly true in community respiratory independently and in isolation from one another, rather
infections, where specimens are virtually never taken on than have been transferred between bacteria, with the pos-
first consultation (Fig. 33.1). sible exception of naturally transformable bacteria such as
Virtually all resistance studies are based on surveil- Streptococcus pneumoniae [1].
lance, which determines the proportion and type of resis-
tance in strains submitted to the laboratory. It does not
take account of the procedures that selected the isolates PLASMIDS
studied. This may have a profound effect not only on the
In 1963, it was reported that some Shigella strains in
proportion of resistance but also the proportion of individ-
Japan could quickly become resistant to four unrelated
ual resistance genes observed within the resistant popula-
antibiotics. This is an event that would occur with a fre-
tions. As epidemiology is the popular term, it will be used
quency of one in 1028 by spontaneous mutation, so
but with the caveat that it has serious limitations.
another mechanism was responsible. The bacteria were
acquiring four resistance genes which were being intro-
duced into the bacteria by plasmids [2].
MUTATIONS
Plasmids were soon found in almost all clinical bacte-
The ability to become resistant to an antibiotic is initially ria except M. tuberculosis and their ability to transfer
manifested by mutation, often at the binding site of the between related strains suggested that they spread promis-
antibiotic target. Mutations occur spontaneously in an cuously through the clinical population. Their characteri-
individual gene at approximately 1 in 107 cell divisions zation was initially by their antibiogram and later by their
and the mutated bacteria are then selected by the presence incompatibility group, a complicated genetic study exam-
of the antibiotic. The selection of spontaneous mutations ining the ability of an unknown plasmid to co-exist in the
that cause resistance is a common response of bacteria same cell as a standard plasmid of known grouping. The
when initially challenged with a new antibiotic. Except principle is based on the understanding that plasmids with
Molecular Medical Microbiology. DOI: http://dx.doi.org/10.1016/B978-0-12-397169-2.00033-0
© 2015 Elsevier Ltd. All rights reserved. 599
600 PART | 5 Antibacterial Agents

similar maintenance genes cannot co-exist and one will GENOTYPING


be excluded.
Clinical plasmid DNA usually ranges from 50 300 When bacterial chromosomal DNA is extracted, it tends
kilobases and became relatively easy to extract and iso- to disintegrate. However, if it is mixed with random PCR
late. The ability to cut this DNA at specific sites with primers, there is amplification of the DNA and when
restriction endonucleases meant that plasmids produced a these amplicons are separated in an agarose gel they are
very distinctive pattern or fingerprint when the fragments able to produce a banding pattern that can be used as a
were separated according to size in an agarose gel. The ‘fingerprint’. This method, known as RAPD PCR, is unre-
presence of the same plasmid in many different clinical liable because it is very dependent on the amplification
isolates led to the, often erroneous, belief that plasmids conditions and results are often not transferable from one
were promiscuous and were rapidly transferring from one laboratory to another. A modification of the method is to
clinical strain to another. The reason for this assumption design primers from known repetitive regions within the
was that the methods for typing the bacteria were more DNA (REP PCR); although more consistent than RAPD
primitive and relied upon phenotypic tests rather than PCR, it still suffers from the inherent variations in the
examination of the DNA of the bacterium. amplification procedure.
It was the ability to isolate chromosomal DNA virtu-
ally intact that provided the first major consistent geno-
typing system. If the cells are lysed within an agarose
matrix and the released DNA is treated with a restriction
endonuclease, the resultant fragments can be fingerprinted
in a similar manner to plasmids. In this case, the restric-
tion endonuclease needs to have a large recognition site,
so that it cuts the genomic DNA relatively rarely. Most
of the fragments are of similar size and, if separated by
conventional electrophoresis, would bunch together.
Therefore the gel is ‘pulsed’, that is the direction of the
current is applied at an angle of 60 to the direction of
migration and then switched to 60 on the opposite side.
The net result is that the DNA will migrate down the gel
but the switching turns the large fragments of DNA,
which start to separate as the slightly larger fragments
take longer to turn. This process, known as pulsed-field
gel electrophoresis or PFGE, allowed very accurate typing
FIGURE 33.1 Biasing of resistance data. If the specimens sent to the
diagnostic laboratory are not a true reflection of the rate of a particular
of bacteria, especially if all the studies are performed in
infection, particularly if the sampling is biased, the resistance rate the same laboratory, but is less accurate when results
reported is often meaningless. from different laboratories are compared (Fig. 33.2).

FIGURE 33.2 Pulsed-field gel electrophoresis, This


diagram shows how, by altering the direction of the
electrical field, DNA fragments of similar size are
separated from each other in an agarose gel.
Chapter | 33 Molecular Epidemiology of Antibiotic Resistance in Humans and Animals 601

As DNA sequencing became more common, it was bacteria that shared the same plasmid(s) were actually the
possible to identify genes that were essential for the main- same strain and that the plasmids were not promiscuously
tenance of the bacterium; this is known as multi-locus moving from one strain to another. It seems that once the
sequence typing or MLST. These genes cannot tolerate plasmid has found a suitable host it remains there and that
many mutations that alter the amino acids (non-synony- what is actually being observed is the dissemination of an
mous) without losing the integrity of the encoded product; individual bacterial clone with its resident plasmid(s).
however, mutations that do not change the translated Clonal spread became even more evident with the
amino acid (synonymous) can be used to identify differ- Salmonella typhi outbreak in South Asia in the 1990s.
ences and similarities. Therefore up to seven of these Isolates from all over India were found to have the same
‘housekeeping’ genes are identified; they are often chosen plasmid conferring ampicillin, chloramphenicol and tri-
randomly and without an understanding of the need to methoprim resistance; however, when the strains were
have a low non-synonymous:synonymous ratio. In other genotyped by PFGE, most were found to be identical [5].
words, there should be no selection pressure to alter the Subsequently, strains from Pakistan [6] were genotyped
proteins produced by these strains [3]. The identified and they were also found to be identical (Fig. 33.3) as
genes are amplified by internal PCR primers and the were strains from Vietnam [7]. Therefore, it was the
amplicon is sequenced. The number and type of changes spread of a single bacterial strain that was resistant
are scored and a ‘sequence type’ is allocated. The infor- because of its resident plasmid. The plasmid itself was
mation can be digitalized and is therefore easily transfer- not migrating significantly between strains. This was
able and compared between laboratories. MLST is now remarkable because the bacterium was a community-
the preferred genotyping method for many laboratories; acquired organism with an incredible capacity to spread.
when compared mathematically MLST is not as sensitive Thus cross-infection was the crucial feature in the dissem-
as PFGE but it does allow more international comparison ination of resistance in this organism and it is now fairly
and analyses of longer-term evolution, whereas PFGE clear that cross-infection of bacteria, that have become
will more readily show variations appearing within iso- resistant, is responsible for much of the observed ‘spread’
lates over a shorter evolutionary timescale, such as from of resistance.
an individual hospital. The most obvious example of clonal spread of multi-
With the advent of cheaper next-generation sequenc- resistant bacteria within the hospital environment is
ing technologies, it is becoming cost-effective to sequence meticillin-resistant Staphylococcus aureus or MRSA. The
entire bacterial genomes. This has the huge benefit that meticillin resistance is encoded by the mec gene; this has
all of the genetic information about the bacterial strain been acquired by Staphylococcus aureus, from an
will be obtained and can be compared with sequence data unknown source, on relatively few occasions to form a
from any other source. Using these data, many different genomic island (SCCmec). Once established, the
analyses can be performed, from virtual PFGE, where all Staphylococcus aureus underwent some alterations as
sites recognized by a restriction endonuclease are identi-
fied and the sizes of the virtual bands calculated, through
to whole-genome sequence comparisons. Whole-genome
sequencing is still too expensive for large-scale routine
use. However, it has been used effectively in studying
bacterial epidemiology. During an Escherichia coli out-
break in Germany in 2011 in which a number of people
died, the genome of the isolate responsible was sequenced
and assembled in less than 3 days, demonstrating that
such detailed real-time monitoring of outbreaks is now
possible [4].

PLASMID OR BACTERIUM, WHICH IS


REALLY RESPONSIBLE FOR THE SPREAD
OF RESISTANCE?
As genotyping techniques improved, it was possible to
FIGURE 33.3 Genotypying of multidrug-resistant Salmonella enterica
demonstrate that two isolates were, for all practical pur- serovar Typhi from India and Pakistan. Many of the strains are identical
poses, identical. As this was applied to larger hospital to one another despite the fact that they were isolated from patients who
populations, it soon became clear that many multiresistant resided more than 2000 km apart.
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they passed through the clinical population until certain This example shows the importance first of robust
resistant strains started to predominate, probably because strain identification, which can only be successfully done
of a combination of virulence factors and resistance with examination of the bacterial DNA. No phenotypic
genes. In the United Kingdom, two lineages predominated method is sufficiently robust for epidemiology. Secondly,
and were originally known as EMRSA-15 and EMRSA- it shows the importance of clear and acceptable definitions
16, the E designating ‘Epidemic’ [8]. These bacteria of resistance versus intermediate or sensitive and finally it
spread rapidly between patients and were almost solely demonstrates that typing methods have to be based on
responsible for the increase in MRSA levels to nearly DNA rather than phenotypical characteristics, which are
40% of all Staphylococcus aureus isolations in English inherently unreliable.
hospitals. Unfortunately, the nomenclature for identification of
Other multiresistant bacteria followed the same clonal multiresistance is often misleading. In the case of
dissemination. Carbapenem-resistant Acinetobacter bau- Streptococcus pneumoniae, penicillin resistance is usually
mannii spread rapidly between patients in 60 different a synonym for multiresistance. In the Hungarian example
hospitals, predominantly in the south of England [9]. It although only 2% were truly penicillin-resistant, nearly
was found that two clones of this Gram-negative bacte- 40% were macrolide-resistant and in most areas macrolide
rium were responsible. When the spread of resistance is resistance is far greater than penicillin resistance. In gen-
being measured, the emergence and evolution of the resis- eral, these strains should be called multiresistant. The
tance gene has taken place largely undetected as resis- manifestation of macrolide resistance in Europe is fairly
tance levels remain low. When a favourable arrangement consistent with the spread of the ermB gene, which
of plasmid and host is formed, then the resistant strain is encodes a protein that alters the target site. This contrasts
likely to spread, through a combination of positive selec- sharply with strains isolated in North America where the
tion and cross-infection, to become predominant within predominant gene is mefA, encoding an efflux pump [11].
that particular infection niche. The new techniques have allowed the monitoring of
the spread of multiresistant Streptococcus pneumoniae
around the world; in particular the outbreak in a day care
STREPTOCOCCUS PNEUMONIAE centre in Reykjavı́k, Iceland, where the prime case was a
The clonal dissemination of resistant bacteria can often be child who had been on holiday in northern Spain and
harder to determine in the community. Good epidemiol- returned with a Spanish strain (serotype 6A) and then
ogy requires not only robust data but also a reliable typing passed it on to his playmates [12]. Indeed, the spread of
technique. The marker of resistance in Streptococcus multiresistant Streptococcus pneumoniae is now clear,
pneumoniae has conventionally been to penicillin; if the with a few individual clones disseminating through the
organism is resistant to 0.06 mg/l but not to 2 mg/l, then populations carrying the appropriate resistance genes.
it is referred to as intermediate and above 2 mg/l as resis-
tant. Furthermore the typing of the bacteria was conven-
tionally performed by serology. In the 1990s, Hungary
ACQUISITION OF RESISTANCE GENES
reported some of the highest incidences of resistance in Plasmids had to have acquired resistance genes because the
Europe and all through the spread of a serotype, virtually Murray collection of bacteria, now nearly 100 years old,
unknown elsewhere, type 19A. The typing was performed shows the same plasmids in bacteria that we find today but
by serology and the bacteria had been identified solely by without most of the resistance genes [13]. So plasmids
α-haemolysis and optochin sensitivity. Resistance was have been acquiring these genes by genetic interactions.
defined as an MIC . 0.06 mg/l, in contrast to international The most frequent method is by transposition. Essentially
convention, resulting in a resistance level of 40%. The the transposon is a mobile element that can insert a copy of
Hungarian isolates were re-evaluated, this time confirm- itself into another section of the same DNA molecule or a
ing strain identity using not only optochin sensitivity and different one within the cell. This means that a plasmid,
bile solubility of α-haemolytic streptococci but also con- entering a cell with a transposon on another DNA element,
firming the presence of the lytA gene, responsible for can acquire that transposon. These elements are usually
autolysis. The definition of resistance was determined by identifiable by having two flanking insertion sequences and
the international definitions and the typing was performed they possess the gene for the transposition and recombina-
by PCR amplification of the genes responsible for the tion. Many transposons can insert randomly at any point in
serotype. With these criteria, the isolates from Hungary the DNA and their presence is usually the result of antibi-
were actually very similar to those in the surrounding otic selective pressure stabilizing the insertion of a transpo-
countries, penicillin resistance was 2% and the serotypes son. Often a plasmid comprises a build-up of different
were virtually identical to those elsewhere with only the transposons that have integrated, one into another. The
rarest identification of serotype 19A [10]. resistance genes often become permanent because the
Chapter | 33 Molecular Epidemiology of Antibiotic Resistance in Humans and Animals 603

insertion of a subsequent transposon has disabled a previ- bacteria. The aminoglycoside-modifying enzymes (acetyl-
ously established transposon. transferase, adenyltransferase, phospho-transferase) all
Although transposons evolved long before the anti- came from either the aminoglycoside-producing organ-
biotic era, they also had to acquire the resistance genes. isms or those in their immediate vicinity. So resistance
Within the DNA of many transposons, there are hotspots was widespread long before the antibiotic era, all that was
that can acquire gene cassettes, which often comprise required was that the genes had to be transported into
resistance genes. These integrons have an integrase to clinical bacteria.
achieve this and they often acquire a single resistance When the synthetic antibacterial trimethoprim was
gene without the promoter and insert them downstream of introduced, resistance occurred quickly. Bacteria had
a suitable promoter. The ability to ‘pick up’ different never previously been in contact with a compound such
resistance genes provides a high degree of flexibility as this, so there were no detoxifying enzymes that were
for the host DNA and, if the event takes place regularly, effective. However, there were plasmid-encoded resistance
then there will be individual bacteria in a colony with the genes passing from one bacterium to another. The target of
ability to resist almost any antibiotic challenge. trimethoprim is bacterial chromosomal dihydrofolate
It is probably the result of transposon insertion into reductase and the plasmid produces an additional dihydro-
plasmids that resistance genes tend to cluster together. folate reductase that is also less susceptible than the bacte-
The ‘foreign’ origin of their DNA, with a different GC rial enzyme, so although the chromosomal enzyme
ratio from the host, is often easy to identify and these is inhibited, the plasmid dihydrofolate reductase is still
clusters have been termed resistance islands. Some bacte- able to convert dihydrofolate to tetrahydrofolate and thus
ria, such as Acinetobacter baumannii, have been able to effectively ‘bypasses’ the inhibited target site [16].
capture not just individual resistance genes into resistance The source of these plasmid enzymes has not been
islands but whole resistance islands from other bacteria. immediately clear as they were presumably not located on
The best example of this is the 89-kilobase resistance mobile elements before trimethoprim was introduced. A
island in strain AYE, which carries 45 resistance genes plasmid-mediated enzyme was identified in India, it only
[14]. From DNA analysis, this resistance island is com- conferred a degree of insusceptibility about ten-fold
posed of resistance genes from Escherichia coli, and greater than that of the chromosomal enzyme. Analysis of
whole resistance islands from Pseudomonas aeruginosa the sequence of this type IV plasmid dihydrofolate reduc-
and Salmonella species. Within the resistance islands are tase revealed that it was similar to the chromosomal dihy-
the remnants of the transposons and integrons that congre- drofolate reductase of Escherichia coli and presumably
gated in their previous bacterial hosts. It is also interesting had resulted from mutations within the gene and then
to note that this resistance island has complete operons the gene migrated onto a plasmid. Further evidence of
conferring heavy metal resistance and four antiseptic the bacterial source of these genes came from the type
resistance genes, indicating the possible selective environ- S1 plasmid-encoded dihydrofolate reductase in
ments that have applied pressure to form these resistance Staphylococcus aureus. This enzyme confers a 1000-fold
islands. decrease in susceptibility to trimethoprim. The type S1
enzyme is identical to the chromosomal enzyme of
Staphylococcus epidermidis except for four non-
ORIGINS OF RESISTANCE GENES synonymous mutations, which together are responsible for
The resistance mechanisms for the ‘mobile’ resistance decreased binding of trimethoprim [17]. The source of
genes had to be different from the mutated target site, these dihydrofolate reductases appears to be the chromo-
usually manifested by a single gene it had to be dominant somal enzymes of bacteria in the immediate location and,
within the organism. The most common mechanism was since the introduction of trimethoprim, they have mutated
to produce an enzyme that detoxified the antibiotic. and these mutations have been selected with drug usage.
These enzymes did not evolve since the antibiotic era Sulphonamide resistance appears to have resulted in the
but were present in non-clinical bacteria before the use same manner with the mutation of the chromosomal dihy-
of antibiotics. Most of them were in soil organisms and dropteroate synthetase.
were used as secondary metabolites; this was to defend A modification of the bypass mechanism has been
the microorganisms against competing bacteria. observed with plasmid-encoded glycopeptide resistance in
Sometimes the bacterium that produced the antibiotic Enterococcus spp. Vancomycin is an antibiotic produced
had to produce a detoxification process so that it was not by a soil organism. It binds to the D-alanyl-D-alanine resi-
destroyed by the compound that it was producing to due at the end of the pentapeptide, which is the precursor
improve its competitiveness in the soil [15]. Through a for the cross-linking of peptidoglycan. The mechanism of
series of mobilization events these genes have then been resistance is to prevent the formation of the D-alanyl-
transported onto the plasmids that we find in clinical D-alanine residue. In most cases, the plasmid encodes an
604 PART | 5 Antibacterial Agents

enzyme that reverses the production of D-alanyl-D-alanine residue in the active site which is the point of catalysis.
to leave D-alanine residues. A second enzyme converts Despite the catalytic serine, the amino acid sequences of
pyruvate to D-lactate and a third enzyme ligates the classes A, C and D are completely different. These four
D-alanine residues to D-lactate to form D-alanyl-D-lactate. classes actually represent a leading example of convergent
D-alanyl-D-lactate now forms the end of the pentapeptide evolution for there is considerable pressure for bacteria,
chain and vancomycin does not bind to it, thus conferring especially those in the environment, to protect
resistance. In the final stage of cross-linking, the end resi- themselves.
due is lost (D-lactate in this case) so the peptidoglycan The class A β-lactamases are the largest group and
manufactured using D-alanyl-D-lactate is the same as that have had the greatest impact clinically. They have been
using D-alanine [18]. considered to be the earliest β-lactamases to evolve and
The presence of an operon of three genes to encode a were the chromosomal enzymes of Gram-positive bacte-
single resistance mechanism is unusual. Operons com- ria. They have migrated from their original hosts on
prising very similar genes have been found in transposons and are now commonly found carried by
Streptomyces toyacaensis and Amycolatopsis orientalis, plasmids in Gram-negative bacteria. Their natural sub-
the latter being the species that produces vancomycin strates were the penicillins. They became predominant
(Fig. 33.4). These three genes are the defence mechan- with the clinical launch of ampicillin, especially with the
isms that the vancomycin-producing bacterium and its emergence of the TEM-1 enzyme, a β-lactamase that has
close neighbours have evolved to prevent their inhibition an active site ideally suited to hydrolysing this antibiotic.
by this glycopeptide. The complete operons have This is the most common resistance mechanism to any
migrated to the plasmids in clinical bacteria that have antibiotic and, in some areas, 25% of the population
become vancomycin resistant [19]. carry the gene in their commensal bacteria. In 1982,
there was an outbreak of Klebsiella oxytoca in a neonatal
unit in Liverpool. The bacteria became cephalosporin-
CHANGES IN β-LACTAMASE GENES resistant and this was because the gene encoding the
TEM-1 β-lactamase had first duplicated and then, in one
DURING THE ANTIBIOTIC ERA copy, there was a mutation in codon 164, changing argi-
By far the largest group of resistance genes are the nine to serine. Arginine is a basic amino acid forming
β-lactamases, over 1000 have been identified in clinical ionic bonds with two acidic residues, glutamic acid 171
bacteria. Most precursor β-lactamases of the clinically and aspartic acid 179 (Fig. 33.5). These bonds ensure
significant enzymes were carried to clinical bacteria from that the entrance to the active site is too small to allow
environmental bacteria. There were, however, relatively the entry of the cephalosporins; removal of the bonds
few of these events and most of the β-lactamases that we opens the entrance and allows them to bind. The degree
now find are modifications to those ancestral enzymes of resistance is relatively low and did not confer clini-
that have occurred through selection of mutations arising cally significant levels of resistance. The entrance to the
during the antibiotic era. active site was increased even further by mutations on
The simplest β-lactamase classification method is that the β-3 sheet at the side of the active site. In particular a
of Ambler which categorized them by their amino acid mutation of glutamic acid 240 to lysine pulled the β-3
sequence. With this method, there are four classes, A D. sheet away from the active site, increasing the binding of
Class B enzymes have one or two zinc ions at the point of the larger cephalosporins and providing clinical levels of
catalysis whereas the other three classes have a serine resistance, especially to slow-penetrating cephalosporins

FIGURE 33.4 Origins of the van genes.


The likely source of the van resistance
genes can be identified when their structure
is compared to operons in Streptomyces
toyacaensis and Amycolatopsis orientalis.
Data taken from [19].
Chapter | 33 Molecular Epidemiology of Antibiotic Resistance in Humans and Animals 605

mutations that also affect the ability to bind and hydrolyse


cephalosporins. The most common worldwide is known
as CTX-M-15, from cluster CTX-M-1. It is certainly the
most prevalent because of the spectrum of cephalosporins
to which it is able to confer resistance. It appears to have
derived from a mutation at codon 240 in the enzyme
CTX-M-3 converting aspartic acid to glycine [21]. This
has the same effect as the codon 240 mutation in SHV
above, in that it increases the spectrum to confer resis-
tance to ceftazidime. CTX-M β-lactamases are now uni-
versal, severely restricting both the use and development
of cephalosporins.
The class C enzymes appear to have come under
selective pressure later with the emergence of Gram-
negative bacteria. Most natural penicillins are not effec-
tive against the majority of Gram-negative bacteria, which
is possibly the reason for their initial success, but the
cephalosporins do inhibit and the predominant chro-
FIGURE 33.5 Active site of the TEM-1 β-lactamase. Diagram shows
mosomally encoded β-lactamases in Gram-negative bacte-
the position of the active site serine 70 residue and the residues around ria are the class C enzymes whose preferential substrates
the active site responsible for ESBL activity. are the cephalosporins. These chromosomal β-lactamases
have been effective against clinical cephalosporins, partic-
ularly in Enterobacter spp., where the gene repression
such as ceftazidime. In the last 30 years, over 100 com- machinery has been disabled and the β-lactamase is con-
binations of mutations have been found, between them stitutively produced. In some areas, nearly 100% of the
providing resistance to most cephalosporins. These are strains have had mutations in the repressor gene.
amongst the earliest examples of extended-spectrum The crucial step in the epidemiology of these genes
β-lactamases (ESBLs) [20]. was their capture onto transposons and subsequent car-
A less common plasmid-encoded class A β-lactamase riage by plasmids. This has occurred with a number of
is SHV-1. This enzyme shares about 60% identity with species, but most commonly with those of the genus
TEM-1 and has a similar substrate profile. It also has Citrobacter. The first was the BIL-1 β-lactamase (later
been a precursor of ESBLs and started to mutate. The piv- referred to as CMY-2), which shows 90% homology with
otal mutation was an alteration at codon 238 converting the chromosomal β-lactamase of Citrobacter freundii
glycine to serine. This mutation increased the ability to [22]. Examination of the genetic environment shows that
bind fast-penetrating cephalosporins, particularly cefotax- this transfer from the bacterial chromosome has occurred
ime. A subsequent mutation at codon 240 converting glu- independently on numerous occasions. The resultant
tamic acid to lysine increased the ability to bind and plasmid-encoded enzyme not only gives widespread resis-
hydrolyse slow-penetrating cephalosporins such as cefta- tance to cephalosporins but also to β-lactamase inhibitors.
zidime. There are also about 100 combinations of SHV It would be considered that these resistance genes were
mutations found, also providing resistance to most cepha- even more effective than the class A ESBLs, but they are
losporins [20]. actually much rarer. In a recent study in Glasgow, they
The TEM and SHV ESBLs, though still present, are only represented 5% of the cephalosporin-resistant strains
not currently the cause of most cephalosporin resistance. and they were never found in strains with ESBLs. It
The chromosomal β-lactamases of Kluyvera ascorbata seems that the class A ESBLs are the more advantageous
and Kluyvera georgiana, species closely related to for the cell.
Escherichia coli, have been transported onto plasmids to
clinical members of the Enterobacteriaceae. They are
known as CTX-M β-lactamases because of their ability to EPIDEMIOLOGY OF CARBAPENEM
hydrolyse the fast-penetrating cephalosporin cefotaxime.
However, they did not emerge until the clinical introduc-
RESISTANCE
tion of ceftriaxone, closely related to cefotaxime with a The β-lactams seen as the final defence against Gram-
much longer half-life. There are five genetic clusters of negative infection are the carbapenems and the epidemiol-
these enzymes, presumably originating from five different ogy of resistance to them has recently been extensively
chromosomes. Within each cluster, there are a number of studied. Although the majority of β-lactamases able to
606 PART | 5 Antibacterial Agents

confer resistance largely fall into molecular classes A, B The mechanism of catalysis of these enzymes has not yet
and D, there are some examples from class C. Patients been fully elucidated. Another enzyme, OXA-51, shared
infected with bacteria carrying the CTX-M-15 β-lacta- nearly 70% homology with OXA-23 and 60% homology
mase and treated with carbapenems of limited activity, with OXA-58 [27] (Fig. 33.6); however, this enzyme is
often become resistant to those drugs during therapy. The encoded by the chromosome. This was the forerunner of
CTX-M-15 β-lactamase provides some basic ability to nearly 100 closely related chromosomally encoded class D
bind the drug but the resistance is augmented by the acti- β-lactamases collectively known as OXA-51-like [28].
vation of chromosomally encoded efflux pumps. This also Indeed every strain of Acinetobacter baumannii possesses
reduces the susceptibility to more active carbapenems; if one of these OXA-51-like β-lactamase genes. It is not
the patient is subsequently treated with the more active known how many of these can confer carbapenem resis-
drugs resistance to them is likely to occur rapidly [23]. tance but, when purified, the original OXA-51 β-lactamase
The class B enzymes, found encoded by the chromo- could also hydrolyse carbapenems. However, most strains
somes of both Gram-positive and Gram-negative bacteria, carrying OXA-51-like β-lactamase genes do not confer car-
are also effective against penicillins and also thienamycin, bapenem resistance unless there is an insertion sequence
a naturally occurring carbapenem from Streptomyces cat- carrying a promoter sequence upstream of the gene. The
tleya. These enzymes were considered to be the major prevalence of OXA-51-like β-lactamase genes, gives a clue
mechanism of resistance against carbapenems. The zinc to the origin of OXA-23 β-lactamase, which is derived
ion at the catalytic point of class B enzymes is usually from the chromosomal β-lactamase of a related species,
able to hydrolyse carbapenems as well as other β-lactams. Acinetobacter radioresistens.
Many species carry the genes for these enzymes on their
chromosome; however, a few genes have migrated onto
plasmids. The two main groups are those of the IMP and EPIDEMIOLOGY OF ANTIBIOTIC
VIM clusters. The origins of these genes are not clear but
the IMP enzymes emerged soon after the introduction of
RESISTANCE IN ANIMAL BACTERIA
imipenem and the VIM appeared later after the introduc- This is a highly contentious issue as it is presuming that
tion of meropenem. The NDM group of enzymes, which the use of antibiotics in animals is a direct precursor to
have been found in Asia, North America and Europe [24], the development of resistance in humans. Without much
is becoming the predominant carbapenemase in the evidence to suggest it does, the addition of growth promo-
Enterobacteriaceae. ters (low-dose antibacterials) to animal feedstuffs has
The Sme and NMC-A class A β-lactamases conferring been banned in the European Union. In particular, the
carbapenem resistance have been known for about 20 addition of avoparcin was particularly questioned because
years, but have been found only on a few occasions. The resistance to this growth promoter also gave resistance to
KPC (Klebsiella pneumoniae carbapenemase) β-lactamase the glycopeptide vancomycin, and there was some resis-
shares about 45% homology with these earlier enzymes tance in bacteria in animals treated with avoparcin.
and has emerged in Klebsiella species particularly in the Interestingly glycopeptide resistance in clinical bacteria
USA. These enzymes have been much more successful in Europe has been considerably lower than that found in
than their class A predecessors and they have spread, car- similar bacteria in patients in the USA, where avoparcin
ried by plasmids, to many bacteria worldwide. Klebsiella has never been used.
pneumoniae is a true pathogen and the widespread There have been cases of direct transmission of resis-
distribution of this enzyme in this species is a serious tant bacteria from food-producing animals to man. The
hindrance to treatment [21]. two best examples are Salmonella enteritica serovar
The species Acinetobacter baumannii has become a Typhimurium phage type DT104 and ciprofloxacin-
successful pathogen within hospitals purely from its abil- resistant Campylobacter jejuni. In the former case, there
ity to become multidrug-resistant and, in particular, to the were a number of human infections that matched the ani-
carbapenems. Even before the introduction of the first mal bacteria. There were exactly the same resistance
carbapenem, a strain isolated in Edinburgh was found to genes. Similarly, Campylobacter jejuni treated with enro-
carry a plasmid-encoded class D β-lactamase able to con- floxacin become resistant by alterations to the DNA gyr-
fer high-level carbapenem resistance [25]. This enzyme, ase, which cross-resist to the human fluoroquinolone
OXA-23, has now been found in clinical strains world- ciprofloxacin and there have been sporadic cases of direct
wide and is still the most prevalent cause of carbapenem transmission. Both these bacteria are faecally transmitted
resistance in Acinetobacter baumannii. Another plasmid- and cause infections in the community in poor hygiene
encoded class D β-lactamase, OXA-58, was found to con- conditions.
fer high-level carbapenem resistance [26]. It shared nearly There are also examples of resistance genes that have
60% sequence homology with the OXA-23 enzyme. emerged in animal bacteria and that have transmitted to
Chapter | 33 Molecular Epidemiology of Antibiotic Resistance in Humans and Animals 607

Pairwise (OG:100%, UG:0%) (FAST:2,10)Gapcost:30%

100
60

65

70

75

80

85

90

95
OXA-23
OXA-27
OXA-49
OXA-64
OXA-71
OXA-51
OXA-78
OXA-66
OXA-76
OXA-65
OXA-69
OXA-68
OXA-77
OXA-70
OXA-75
OXA-40
OXA-26
OXA-72
OXA-25
OXA-58

FIGURE 33.6 Dendrogram of OXA carbapenemases in Acinetobacter baumannii. Dendrogram shows the four major groups of OXA β-lactamases;
the enzyme from which each group is named is shown in bold and the two major plasmid-mediated enzymes, responsible for the majority of carbape-
nem resistance in Acinetobacter baumannii, are shown by the grey arrows.

clinical bacteria but are no longer in their original host bacteria was CTX-M-14, whereas in humans it has been
bacteria. Apramycin is an aminoglycoside used to treat the CTX-M-15 β-lactamase. In molecular terms, they are
animal infections. Resistance genes do emerge in treated very distinct from one another and it is not possible for
bacteria conferring resistance to apramycin with cross- one to evolve into the other. The dissimilar predominance
resistance to the clinical antibiotic gentamicin. The resis- probably reflects the different cephalosporins used to treat
tance mechanism is a very specific aminoglycoside human and animal infections. More latterly more animal
acetyltransferase type 3-IV [29]. Although this gene has bacteria have emerged with the CTX-M-15 ESBL, but
been found in clinical bacteria it is very rare and most this is many years after CTX-M-15 ESBLs have become
gentamicin resistance genes are manifested by aminogly- prominent in human bacteria. In most cases, the emer-
coside resistance genes that have never been found in gence of resistance in human bacteria has probably been
animal bacteria. within the clinical bacterial population itself [31]. As we
A more directed method to study the potential trans- have seen, the spread is by cross-infection and this will
mission of resistance genes from animals to man, is to have occurred virtually entirely within the clinical popula-
examine the particular problems in clinical bacteria and tion. A further supporting view is in India, which has the
look for their precursors in animal bacteria. It was found highest incidences of resistance in community bacteria
that the MRSA strains found in animals were of different ever recorded and, at that time, there was virtually no
genotypes from those found in humans, suggesting no administration of antibiotics to animals because of the
direct link. Furthermore, a study that examined bacteria cost [32]. It appears that humans are capable of spreading
isolated from animals and animal products through resistance genes without the help of animal bacteria [33].
slaughter, food production, food preparation, hospital Antibiotic resistance is, however, prevalent in animal
kitchen preparation and the food given directly to patients bacteria regardless of whether it then enters human bacte-
showed none of the serious pathogens currently found in ria. Animals have successively been treated with growth
hospitals [30]. promoters (and still are in the USA) and also prophylacti-
There are, however, reports of resistant bacteria in cally when a single member of a herd becomes infected.
food-producing animals, in particular the class A CTX-M This barrage of antibiotics accounts for 50% of all anti-
ESBLs. In the UK, the most common enzyme in resistant biotics used in this country. There are strict rules that
608 PART | 5 Antibacterial Agents

state the number of days after an animal finishes treat- [7] Wain J, Nga LTD, Kidgell C, James K, Fortune S, Diep TS, et al.
ment before slaughter so that no residual antibiotic can Molecular analysis of incHI1 antimicrobial resistance plasmids
enter the food chain and select resistance. Clearly, though from Salmonella serovar Typhi strains associated with typhoid
animals are often infected with resistant bacteria and they fever. Antimicrob Agents Chemother 2003;47:2732 9.
[8] Moore PCL, Lindsay JA. Molecular characterisation of the domi-
pass these bacteria on to others in the herd. It has been
nant UK methicillin-resistant Staphylococcus aureus strains,
shown that individual resistant bacteria can spread rapidly
EMRSA-15 and EMRSA-16. J Med Microbiol 2002:51:516 21.
through a herd and this is often clonal dissemination as it [9] Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME,
is in humans. Brown S, et al. Multiplex PCR for genes encoding prevalent OXA
carbapenemases in Acinetobacter spp. Int J Antimicrob Agents
2006;27:351 3.
CONCLUSIONS [10] Dobay O, Rozgonyi F, Hajdú E, Nagy E, Knausz M, Amyes SGB.
Antibiotic susceptibility and serotypes of Streptococcus pneumo-
The increase of antibiotic resistance is difficult to monitor niae isolates from Hungary. J Antimicrob Chemother 2003;51:
and requires rigid protocols. The vast majority of the 887 93.
increase in number of resistant bacteria is caused by the [11] Dobay O, Rozgonyi F, Amyes SGB. Molecular characterisation of
spread of strains that have already become resistant. This Hungarian macrolide-resistant Streptococcus pneumoniae isolates,
is witnessed by the predominance of both multiresistant including three highly resistant strains with the mef gene. Int J
clones and of a few individual resistance genes. The Antimicrob Agents 2005;25:488 95.
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relatively rare event when compared with the vast num- the introduction of a multiresistant clone of serotype-6b
Streptococcus pneumoniae from Spain to Iceland in the late
bers of identical resistance genes and multiresistant bacte-
1980s. J Infect. Dis 1993;168:158 63.
ria. Once really successful resistance genes have
[13] Datta N, Hughes WM. Plasmids of the same Inc groups in entero-
established themselves in clinical bacteria, the selective bacteria before and after the medical use of antibiotics. Nature
pressure to select new genes is considerably reduced, par- 1983;306:616 7.
ticularly in the absence of new clinical antibiotics. [14] Fournier PE, Vallenet D, Barbe V, Audic S, Ogata H, Poirel L, et
Therefore, in order to restrict the spread of resistance, the al. Comparative genomics of multidrug resistance in
containment of multiresistant bacteria through infection Acinetobacter baumannii. PLoS Genet 2006;2:62 72.
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stop new resistance genes emerging. self-resistance before antibiotic biosynthesis incapacitates them?.
Mol Microbiol 2007;63:937 40.
[16] Amyes SGB, Smith JT. R-factor trimethoprim resistance mecha-

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