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J Antimicrob Chemother 2010; 65: 1842 – 1852 doi:10.1093/jac/dkq217 Advance Access publication 23 June 2010 The 2009 Garrod Lecture: The evolution of antimicrobial resistance: a Darwinian perspective Sir Richard Sykes * Department of Biosurgery and Surgical Technology, Imperial College, Hammersmith Campus, Du Cane Road, London W12 0NN, UK *Tel: +44-20-8383-2273; E-mail: [email protected] Microbes have evolved over 3.5 billion years and are arguably the most adaptable organisms on earth. Restricted genetically by their inability to reproduce sexually, bacteria have acquired several additional mechanisms by which to exchange genetic material horizontally. Such mechanisms have allowed bacteria to inhabit some of the most inhospitable environments on earth. It is thus hardly surprising that when faced with a barrage of inimical chemicals (antibiotics) they have responded with an equal and opposite force. This article compares and contrasts the evolution of antimicrobial resistance to b-lactam antibiotics over the last 70 years in two bacterial species, namely Staphylococcus aureus, a highly evolved human pathogen, and Pseudomonas aeruginosa, an opportunistic nosocomial pathogen. Keywords: microbes, b-lactam antibiotics, Staphylococcus aureus, Pseudomonas aeruginosa Introduction ‘Infection 2009’ is a very significant event in this year’s calendar and I feel very privileged having been invited to present the Garrod Lecture at this prestigious meeting. This is also a very special year as we are celebrating two famous anniversaries, the 200th anniversary of Darwin’s birth and the 150th anniversary of On the Origin of Species, a publication that enunciated the greatest general principles in biology. These principles relate to the concept of evolution through a process of natural selection: adapt or perish. Darwin had eventually come to this view following his voyage on HMS Beagle, and almost 30 years of painstaking research, leaving no stone unturned. Darwin worked in a hostile environment, during a period when the general belief was that the world had been created in 6 days, and that this had been accomplished 6000 years in the past. It was a period when nothing was known about genetics or the microbial basis for infectious diseases. The concept of spontaneous generation was still regarded as the route through which new life forms appeared. However, as with the concept of evolution, things were changing rapidly. In 1866 Gregor Mendel working in a monastery garden had published his work on experiments with plant hybrids and the basic principles of heredity. However, they remained largely undiscovered in the monastery library at Brunn for many years and were never available to Darwin. At around the same time Pasteur was attacking the concept of spontaneous generation with his experiments on fermentation. This work, as we all know, led to the understanding of the role of microorganisms as agents of infectious disease. The next 30 years or so was a period of intense activity in the identification and characterization of the agents of disease in man, animals and plants. It soon became clear that these microorganisms were remarkable creatures as they could be found in all ecological niches examined. A perfect example of survival: adapt or perish. We can only imagine what impact this work on microorganisms would have had on Darwin had it been carried out in the early nineteenth century as it would have provided a perfect example for Darwin’s theory. Darwin was driven to his conclusions through fossil evidence and the changes that he observed through both natural and artificial selection. He had to befriend the pigeon fanciers and the dog breeders who were using artificial selection techniques to create new breeds. However, his work was dedicated to creatures that reproduce sexually where the overriding factor for genetic variation is during the process of meiosis (Figure 1a) and the mixing of genes between partners. This is a slow process due to long gestation periods. However, bacteria multiply asexually by binary fission (Figure 1b) with generation times of minutes rather than days, months or years. Microbes, which have evolved over 3.5 billion years, are arguably the most adaptable organisms on earth, inhabiting nearly every corner of the globe. Some invade the cavities of a wide variety of insects and invertebrates while others colonize the skin, blood, eyes and internal organs of animals and man. They thrive in the most inhospitable places such as hydrothermal vents on the ocean floor and at the Poles. As already described, they divide by binary fission and thus produce clones of each other. Any genetic variability under these circumstances requires random point mutations within the genome that probably occur at a rate of 1×1025 – 1×1028. Although this is a very low frequency, it is important to remember the # The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] 1842 JAC Garrod Lecture (a) Daughter nuclei II Daughter nuclei Interphase Meiosis I Meiosis II Homologous chromosomes Prokaryotic chromosome Plasma membrane Cell wall (b) Duplication of chromosome Continued growth of the cell Division into two cells Figure 1. Comparison of cell division in eukaryotic cells (meiosis; a) and bacteria (binary fission; b). very high concentrations of organisms in the gut, for example, and so point mutations do play a role in genetic variability. However, if this were the only mechanism open to bacteria to adapt their genetic structure then it is inconceivable they could have been so successful. Because they are unable to reproduce sexually, bacterial species have acquired several additional mechanisms by which to exchange genetic material horizontally, giving them tremendous genetic flexibility. The horizontal transfer of genetic material between the same and different species can be accomplished through three main mechanisms (Table 1). Each of these methods of genetic exchange can introduce sequences of DNA that share little or no homology with the DNA of the recipient cell. The inherited DNA can remain outside the chromosome (extrachromosomal) or it can be integrated. All three mechanisms are capable of introducing antibiotic resistance genes associated with a number of different genetic entities, the three major ones being: (i) plasmids,1 which are circular double-stranded DNA molecules separate from chromosomal DNA and capable of replicating independently of chromosomal DNA (host-to-host transfer requires direct mechanical transfer by conjugation or uptake by transduction); (ii) transposons (or jumping genes), which are mobile sequences of DNA that can move to different positions within the genome of a single cell (transposition)2 (in the process, such activity can produce mutations; transposons in bacteria often carry 1843 Garrod Lecture Table 1. Mechanisms of genetic transfer between bacteria Mechanism Transformation Transduction Conjugation Mode of DNA uptake uptake of naked DNA transfer of DNA by bacteriophage transfer of DNA by cell-to-cell contact and plasmid transfer genes for antibiotic resistance and can jump from chromosome DNA to plasmid DNA and back, a good mechanism for developing multiresistant strains and also for causing mutations); and (iii) integrons,3 which are gene-capture systems found in plasmids, chromosomes and transposons, through which pieces of DNA called gene cassettes can be incorporated, expressed and disseminated. The integron contains an integrase to allow its insertion into the genome, a promoter that drives expression and a gene that allows integration of cassettes. Horizontal gene transfer is believed to be the essential mechanism for the attainment of genetic plasticity in many species of bacteria. Thus during bacterial evolution the ability of bacteria to adapt to new environments most often results from the acquisition of new genes through horizontal transfer rather than by alteration of gene functions through numerous point mutations and vertical transmission: a good example of early gene therapy. In this article I want to review the development of antimicrobial resistance over the last 70 years and discuss how bacteria have adapted to the wide range of inimical environments that we have created. To illustrate how bacteria have adapted to the onslaught of antibiotics I have chosen two organisms, Staphylococcus aureus and Pseudomonas aeruginosa, the former being a highly evolved human pathogen and the latter an opportunistic nosocomial pathogen. I will restrict most of the discussion to the interaction of these organisms with b-lactam antibiotics. My intention is not to blind you with science but to tell a story, and at times relate my own involvement in the process. S. aureus Interaction with b-lactam antibiotics S. aureus is a well-known major human pathogen commonly found on the mucous membranes and skin of around one-third of the population. At the time of the isolation of penicillin in the late 1930s, S. aureus, along with Streptococcus pyogenes, was a major cause of morbidity and mortality, hence penicillin, which specifically targeted these organisms, was seen as a wonder drug. However, the euphoria did not last for long as penicillinresistant strains of S. aureus soon started to appear. By 1955, 80% of all S. aureus strains in London hospitals were resistant to penicillin.4 At this point we need to review the mode of action of penicillin and the mechanisms through which resistance to penicillin can occur. The mechanism of antibiotic action in general can be expressed as illustrated in Figure 2. Ideally, a given antibiotic reaches its target site(s) unimpeded and intact, binds to and 1844 Antibiotic Target(s) Cell response Amount of antibiotic reaching target Nature of target Nature of response Figure 2. Mode of action of antibiotics and factors that influence their activity. inactivates the essential target(s) and, as a result, the cell rapidly loses viability and dies. In the 1940s it was found that penicillin had an effect on cell wall structure and morphology and, by the 1950s, penicillin was shown to be a specific inhibitor of cell wall synthesis. In the 1960s the structure of bacterial cell walls and the mechanisms of their synthesis were elucidated in detail. In Gram-positive bacteria such as staphylococci, the cell wall is a strongly linked peptidoglycan structure that is responsible for the shape and integrity of the cell wall (Figure 3a). Early studies recognized two major enzyme groups located on the cytoplasmic membrane that were involved in peptidoglycan formation and that are susceptible to penicillin action, namely transpeptidases and carboxypeptidases (Figure 4).5 It is now recognized that there are multiple targets in the cell membrane, referred to as penicillin-binding proteins (PBPs) that are all involved in cell wall synthesis.6 Penicillin acts as a steric analogue of the D-Ala-D-Ala peptide (which is a building block mimetic) and thus binds to the cell-wall-synthesizing enzymes. As a result, the enzymes are inactivated resulting in a weakened cell wall structure causing the cell wall to rupture, resulting in cell death. Penicillin has a high affinity for these enzymes and thus is extremely effective in binding to this target site. Resistance to b-lactam antibiotics A major factor in bacterial resistance to antibiotics is the failure of the antibiotic to reach its target (Figure 2). This may be due to poor membrane permeability and/or antibiotic inactivation. For b-lactam antibiotics to reach their target on the bacterial cytoplasmic membrane they must pass through the cell wall envelope. In staphylococci and other Gram-positive bacteria this consists of one or more layers of peptidoglycan linked to an anionic polymer (Figure 3a). Although the cell wall acts as a molecular sieve with an exclusion limit of 100000 Da, it is not an effective barrier against penetration by b-lactam antibiotics (300 Da). Thus permeability plays little or no role in resistance to penicillin in staphylococci. One of the major mechanisms of resistance to b-lactam antibiotics is enzymatic inactivation (Table 2). Even before penicillin had been introduced, Abraham and Chain7 reported on the identification of an enzyme that could inactivate the molecule, which they termed penicillinase. This and related enzymes, now referred to as b-lactamases, hydrolyse the cyclic amide bond (the b-lactam ring) of susceptible b-lactams to produce inactive products (Figure 5). These open ring molecules are no longer recognized by the cell-wall-synthesizing enzymes. In 1955 all resistance to penicillin among staphylococci was due to enzymatic inactivation by b-lactamase, which is a single protein coded for by a single gene: these genes were JAC Garrod Lecture (a) LTA Transpeptidase Carboxypeptidase Peptidoglycan Protein Cytoplasmic membrane Phospholipids Cytoplasm (b) O-antigen repeat n n n n n n Outer core Porin Outer membrane LPS Inner core Lipid A Efflux pump Lipoproteins Peptidoglycan Periplasm MDO (periplasmic b-glucans) Undecaprenyl phosphate sugars Inner membrane Phospholipids Cytoplasm Protein Figure 3. Structure of the cell walls of S. aureus (a) and P. aeruginosa (b). LTA, lipoteichoic acid; LPS, lipopolysaccharide. shown to be plasmid mediated with cell-to-cell transfer occurring by transduction (that is by bacteriophage). Most staphylococci are lysogenic, which means they carry phages, and staphylococcal R-plasmids are transmitted from cell to cell by these lysogenic phages. The b-lactamase produced by staphylococci is an inducible extracellular enzyme, thus production occurs only when necessary in the presence of b-lactam antibiotics.8 The number of plasmid copies can be quite high and, along with the population effect, enzyme levels can represent up to 1% of the dry weight of the cell. Such large quantities of enzyme pass into the external milieu to inactivate penicillin before it has an opportunity to reach the target site, producing high levels of resistance. It is important to recognize here that there are two opposing enzymatic mechanisms at work that both have an affinity for the antibiotic: the transpeptidase or cell-wall-synthesizing enzymes and the b-lactamase. Therefore, in order to be effective, the b-lactamase must have an affinity for the b-lactam antibiotic at or around the MIC for the 1845 Garrod Lecture GlcNAc GlcNAc MurNAc GlcNAc MurNAc MurNAc L-Ala L-Ala L-Ala D-Glu D-Glu D-Glu meso-DAP meso-DAP-?-NH2 meso-DAP D-Ala D-Ala D-Ala D-Ala LD-Carboxypeptidase D-Ala DD-Carboxypeptidase D-Ala meso-DAP meso-DAP D-Glu D-Glu L-Ala MurNAc Transpeptidase L-Ala GlcNAc MurNAc GlcNAc Figure 4. Penicillin-sensitive enzymes involved in cell wall biosynthesis. GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; DAP, diaminopimelic acid. Table 2. Mechanisms of antibiotic resistance in S. aureus and P. aeruginosa Organism Resistance mechanism Antibiotics affected S. aureus enzymatic inactivation/modification alteration of target antibiotic trapping efflux pumps b-lactams, aminoglycosides methicillin, macrolides, vancomycin vancomycin, daptomycin fluoroquinolones, tetracyclines P. aeruginosa enzymatic inactivation/modification alteration of target efflux pumps porin loss b-lactams, aminoglycosides aminoglycosides, fluoroquinolones most antibiotics carbapenems organism. In the case of staphylococci the MIC of penicillin is 1027 M, similar to the affinity of the b-lactam for the b-lactamase. Thus the enzyme is an extremely potent weapon against penicillin particularly when produced in high concentrations. Obviously the plasmids carrying genes for b-lactamase production did not just appear. Plasmids are common in bacteria and there is little doubt that horizontal gene transfer through bacteriophages was a common event. Only on the introduction of penicillin was such a b-lactamase-producing gene useful to the bacterium. already developed mechanisms and adapting them to cope with a new environment. It is interesting to compare this adaptive process with that seen in methicillin-resistant organisms, which we shall come to shortly. Soil samples from the British Museum collected in the seventeenth century were shown to contain strains of Bacillus spp. producing b-lactamase. Once penicillin appeared on the scene those organisms carrying the b-lactamase gene would have a significant advantage and could transfer that advantage quickly to other organisms by the horizontal route thus quickly producing resistant strains. All of this happened in a short period of time and is a great example of adaptive survival. Evolution of b-lactamase Where did this enzyme come from in the first place? It now turns out that many soil microbes (including bacteria, streptomycetes and fungi) are capable of producing b-lactam antibiotics, and they in turn have developed defence mechanisms to protect themselves against the antibiotics produced. The staphylococcal b-lactamase enzyme has been shown to have close homology with that from the soil organism Bacillus licheniformis.9 In turn, this enzyme is closely related to the transpeptidase involved in cell wall biosynthesis,10 a clear process of evolution using 1846 Semi-synthetic penicillins This situation put enormous pressure on the pharmaceutical industry to develop more agents that could resist the action of b-lactamases. The isolation of the penicillin nucleus [6-amino-penicillanic acid (6-APA)] in 1959 led to the development of b-lactamase-stable semi-synthetic penicillins such as cloxacillin and methicillin (Figure 6).11 These compounds showed a high degree of resistance to hydrolysis by staphylococcal b-lactamase. However, following the introduction of JAC Garrod Lecture R Ser NH S CH3 N OH R Ser S CH3 CH3 N O O O O O HO R NH CH3 HO R NH S HO O NH CH3 H2O Ser NH S O CH3 O CH3 NH O HO CH3 O HO acyl intermediate Figure 5. Inactivation of penicillin by b-lactamase. methicillin into clinical practice in 1961, methicillin-resistant strains [methicillin-resistant S. aureus (MRSA)] soon started to appear. Over the past 50 years these organisms have acquired multidrug-resistant traits and have spread worldwide to become one of the most important causative agents of hospital-acquired infections. Then in the late 1990s these organisms began to show up in the community. This is a great example of a dangerous human pathogen continually evolving to deal with a changing environment. These globally spread epidemic clones of MRSA are the product of the convergence of several unusual evolutionary processes. Methicillin resistance The development of resistance to methicillin is completely different from that seen with the early penicillins. First, resistance to penicillins was rapid whereas resistance to methicillin was slow to develop. Staphylococcal b-lactamase has a poor affinity for methicillin, which therefore reaches the target site intact and inactivates the organism. Thus methicillin resistance is not due to permeability or enzymatic inactivation but is primarily due to production of a modified target PBP to which the antibiotic cannot bind. However, as was the case with the early penicillins, resistance emerged following the acquisition of foreign DNA, in this case through the mecA gene (Figure 7).12 This gene is the determinant of a unique PBP (PBP2A) that has a low affinity for b-lactam antibiotics but can still function as a surrogate for the native staphylococcal PBPs, the cell-wall-synthesizing enzymes. The mecA resistance gene appears to have evolved from a domestic gene of the fully b-lactam-susceptible Staphylococcus sciuri, a frequent colonizer of the skin of both wild and domestic animals and one of the most abundant staphylococcal species on this planet.13 However, in order for the mecA gene to establish itself in S. aureus, it had to be incorporated into a unique molecular vector called the staphylococcal chromosome cassette (SCC), which has the ability to deliver a variety of different resistance or virulence mechanisms to S. aureus. The successful grafting of the SCCmec gene into the methicillin-susceptible S. aureus (MSSA) cell is not a straightforward process and requires additional factors, particularly a unique and apparently rare genetic background that allows maintenance and expression of the mecA gene. Hence, the relatively few successful SCCmec acquisition events that give rise to an MRSA strain appear to require the absence of a genetic barrier that is very common in many S. aureus lineages.14 This explains why we see only a handful of lineages around the world. As with the gene for b-lactamase production, the SCC cassette is transferred by transduction. The origin of the mecA gene is a fascinating event and could not possibly have been a result of the appearance of methicillin. Penicillin was used extensively in animals from 1949 and S. sciuri strains have remained susceptible to penicillin as they carry no plasmids carrying the b-lactamase gene. The gene homologue of mecA is a PBP transpeptidase and so the gene codes for a cell-wall-synthesizing enzyme in S. sciuri.15 However, if this gene is up-regulated then the organism shows resistance to penicillin. Therefore it is reasonable to assume that the extensive use of antibiotics in animals led to the up-regulation of the gene, which was subsequently transferred to MSSA strains thus converting them to methicillin resistance. Once again we see that a cell-wall-synthesizing enzyme has been hijacked for resistance purposes. In the late 1990s MRSA-infected patients from the community who lacked any of the usual risk factors for MRSA infections began to make their appearance. These organisms were rapidly transmitted from person to person causing skin structure 1847 Garrod Lecture H H N OMe H S H H N Me H S Me N Me OMe O N O Methicillin Me O CO2H CO2H Cl 6-Aminopenicillanic acid N O NH H3C S CH3 N O CH3 Cloxacillin O HO H2N H N HOOC S S N O NH2 O N CH3 O CH3 O O O COOH O Cephalosporin C H O COOH 7-Aminocephalosporanic acid COOH O H N NH OH CH2OH O H HN H H Clavulanic acid S N O + OH NH3 H H OH O S Imipenem N O O S – NH2 O N Thiapenem N HN O CH3 O HN O CH3 OH N O Monobactam Figure 6. Structures of a range of b-lactams. 1848 N SO3H O Aztreonam SO3H JAC Garrod Lecture MSSA orfX b-Lactamase inhibitors Excision Integration mecA SCCmec MRSA mecA SCCmec MSSA orfX mecA SCCmec Figure 7. Excision and integration of SCCmec containing the mecA gene. 16 infections, osteomyelitis and pneumonia. They were also responsible for fatal diseases such as severe sepsis and necrotizing fasciitis. In contrast to the healthcare-associated MRSA, these community-associated MRSA showed diverse genetic backgrounds indicating the loss of a genetic barrier to the acquisition of the mecA gene. Subsequently, a few highly epidemic clones have emerged and spread in the community. Many of these strains are associated with the Panton –Valentine leucocidin toxin gene (pvl), which seems to correlate with the high frequency of skin and soft tissue infections caused by these organisms.17 In the mid-1990s ,0.1% of individuals in the community carried MRSA strains whereas .50% of S. aureus hospital isolates were resistant to methicillin in the same period.18 Now we see high carriage rates of 6%– 8% in underserved groups in the community.19 Once b-lactamase had been overcome with the development of b-lactamase-resistant penicillins, if the organisms were to survive they had to find other defence mechanisms. Modification of the target site is an obvious mechanism of defence but is much more complicated than antibiotic inactivation. This extended genetic reservoir accessible to staphylococci (mobile genetic elements plus DNA transfer mechanisms afforded by horizontal gene flux) is fundamental to the acquisition, maintenance and dissemination of resistance in these organisms. Resistance to cephalosporins In 1954 the isolation of cephalosporin C from a sewage outlet in Sardinia by Brotzu, and the subsequent identification of the cephalosporins as b-lactam antibiotics by Abraham and Newton in Oxford, caused a great deal of excitement, as they were shown to be resistant to staphylococcal b-lactamase. The cephalosporins were another example of naturally occurring b-lactam antibiotics produced by a soil microorganism. The isolation of 7-aminocephalosporanic acid (7ACA) (Figure 6) in the early 1960s allowed the development of semi-synthetic cephalosporins, the first of which were cefalotin and cefaloridine. Although these early semi-synthetic cephalosporins were active against staphylococci, they were considered to be of more value against Gram-negative organisms, which were beginning to become a serious problem in the hospital environment. Staphylococcal b-lactamase inactivated cefaloridine but not cefalotin so cefalotin was used widely at the time. For many years there had been an ongoing search in the industry for naturally occurring inhibitors of b-lactamase in the hope that they could be used in combination with the early penicillins. In 1976 the b-lactamase inhibitor clavulanic acid (Figure 6) was isolated from Streptomyces clavuligerus and this was shown to be a potent inhibitor of staphylococcal b-lactamases.20 This compound was combined initially with amoxicillin to produce co-amoxiclav (Augmentin), which is active against non-methicillin-resistant staphylococci. To date, no staphylococcal enzymes have been seen that show resistance to clavulanic acid. Resistance of staphylococci to other antibiotics Over the last few years, the widespread development of resistance to methicillin has led to the wider use of glycopeptide antibiotics such as vancomycin. In 2000, MRSA strains carrying the transposon Tn1546-based enterococcal vancomycin resistance mechanism were identified.21 This resistance appears to be due to modification of the target site, with the D-alanine-D-alanine moiety of the peptidoglycan precursor (to which glycopeptides bind) being replaced with D-alanine-D-lactate. These organisms are also resistant to clindamycin, aminoglycosides, trimethoprim/sulfamethoxazole, rifampicin and the fluoroquinolones. New compounds introduced over the last few years such as linezolid and daptomycin have also selected out resistant strains.22 It is now clear that in some instances staphylococcal resistance is due to a multiplicity of factors including altered PBPs, large amounts of b-lactamase production, antibiotic trapping and efflux pumps (Table 2). These can also be associated with virulence factors that encourage the maintenance of plasmids carrying antibiotic resistance genes. P. aeruginosa P. aeruginosa is a non-fermenting Gram-negative organism commonly found in the natural environment. However, it is also a nosocomial pathogen, an opportunistic organism that has a propensity for infecting critically ill and immunocompromised patients. It can also colonize medical equipment such as catheters and prostheses through its ability to form biofilms. It represents a phenomenon of bacterial resistance since practically all known mechanisms of antimicrobial resistance can be seen in it. P. aeruginosa has a large genome with 6.26 Mb equivalent to 5567 genes, whereas S. aureus has 2.81 Mb equivalent to 2594 genes. Such a large genome provides P. aeruginosa with much greater adaptability allowing it to survive in most hostile environments and even dissolve oil slicks. Interaction with b-lactam antibiotics Like most of the Enterobacteriaceae, P. aeruginosa showed little susceptibility to the early penicillins due to the inability of the drug to reach its target site, the PBPs on the cytoplasmic membrane. The cell wall of Pseudomonas is very different from that of S. aureus (Figure 3b). On the outside of the cell there is often a layer of alginate polysaccharide that is important in biofilm formation. The outer membrane is the main barrier to entry of 1849 Garrod Lecture b-lactam antibiotics, which can only cross this barrier through aqueous channels called porins. P. aeruginosa possesses several different porins including OprF, which is present in all strains, and OprD, which is a specialized porin involved in the uptake of amino acids and which has become adapted for the specific uptake of the carbapenems.23 These porins are also closely associated with efflux systems that are responsible for rejecting molecules from the bacterial cell. In evolutionary terms these systems have developed to allow the organism to survive in inimical environments and have now adapted themselves to deal with antibiotics. There are four genetically different efflux systems in P. aeruginosa, all of which are composed of three components, namely an energy-dependent pump, an outer membrane porin and a linker protein that couples these two membrane proteins together.24 All classes of antibiotics are susceptible to extrusion by these systems. Hence, there are significant permeability barriers to the entry of b-lactam antibiotics into P. aeruginosa, unlike the situation in staphylococci. Molecules such as b-lactams have great difficulty penetrating the outer membrane and even if they do there is a high probability that they will be extruded. Even compared with Escherichia coli, P. aeruginosa is 100 times more impermeable to b-lactams. However, in common with S. aureus, these organisms produce b-lactamase and over the last 45 years there has been a constant battle between the production of new and more potent antibiotics and the microorganisms’ ability to destroy them. This is an incredible example of the eternal battle that Huxley described as ‘Nature, red in tooth and claw’ (Huxley was himself quoting from Tennyson). b-Lactamases All wild-type strains of P. aeruginosa produce an inducible chromosomally encoded b-lactamase designated AmpC, which is produced constitutively at low levels and is capable of hydrolysing cephalosporins.25 However, in the presence of b-lactam antibiotics the level of enzyme can increase up to 1000-fold. This enzyme, along with the permeability barrier, provides high-level resistance to the early penicillins and cephalosporins. Unlike the situation in staphylococci, the enzyme in P. aeruginosa is located strategically in the periplasmic space. The introduction of carbenicillin in 1965 provided the first penicillin with activity against P. aeruginosa. Carbenicillin is able to enter the cell through the porin system, shows poor affinity for the AmpC cephalosporinase and is thus able to inactivate the cell-wall-synthesizing enzymes. Carbenicillin was used widely for the treatment of P. aeruginosa infections particularly in burns patients. In 1969 a strain of P. aeruginosa was isolated in the Burns Unit at the Birmingham Accident Hospital with MICs of carbenicillin of .250 mg/L—very different from the low-level resistance that had been seen previously. At the time I was working in Bristol with Mark Richmond doing my PhD thesis on the b-lactamases of P. aeruginosa. We asked Dr Edward Lowbury for a culture of his organism. On examination the strain produced two b-lactamases, one the normal AmpC cephalosporinase but the other was an enzyme shown to be indistinguishable from the TEM b-lactamase that at the time was commonly found in strains of E. coli and known as RP-1.26 This was the first report of such an enzyme in P. aeruginosa. It was shown subsequently that the gene was carried on a 1850 transposon with the capability of moving between the chromosome and a non-chromosomal location. Within a few months we were contacted by Bill Newsome at Papworth Hospital, Cambridge who also had a strain of P. aeruginosa highly resistant to carbenicillin. We showed that it was a different enzyme from RP-1 even though it was transposon mediated.27 It is now known as PSE-1 (where PSE stands for Pseudomonas-specific enzyme). These were both rare occurrences at that time. There are now four carbenicillin-hydrolysing enzymes that are Pseudomonas specific and all closely related, with the exception of PSE-4, which appears to have been acquired from another species.28 The next development came with the introduction of thirdgeneration cephalosporins with activity against Pseudomonas including cefoperazone and ceftazidime. Ceftazidime came out of a programme at Glaxo based on the oxyimino cephalosporins with which I was closely associated in the 1970s. These compounds initially showed resistance to both the chromosomal enzyme and the transposon-mediated enzymes and were used extensively in Pseudomonas infections. Thienamycin, a naturally occurring carbapenem antibiotic isolated from Streptomyces cattleya, led to the synthesis of imipenem29 (Figure 6), a novel b-lactam introduced in 1985, again showing high levels of activity against P. aeruginosa. Subsequently, while working at the Squibb Institute for Medical Research in the late 1970s, we isolated a novel group of molecules called monobactams (Figure 6), which led to the development of aztreonam, another compound active against Pseudomonas.30 This continuous attack on P. aeruginosa with new and powerful drugs has resulted in a powerful response, most of which has been concentrated on drug inactivation. One feat of adaptability occurred in cystic fibrosis patients who at the time were being bombarded with ceftazidime because of its resistance to the AmpC and TEM-type b-lactamases. This resulted in a spontaneous mutation in the regulatory gene AmpR leading to the derepression of enzyme production. Now P. aeruginosa began behaving more like staphylococci, producing large quantities of enzyme bleeding out into the environment and producing a population effect and high resistance. The gene for this enzyme is still restricted to a chromosomal location in P. aeruginosa but it will only be a matter of time before it gets incorporated into a mobile genetic element similar to the situation seen in the Enterobacteriaceae. Organisms carrying this derepressed gene are highly resistant to third-generation cephalosporins. Since 1990, the constant exposure of P. aeruginosa to antibiotics in the hospital environment has resulted in a plethora of b-lactamase types that exhibit differing substrate profiles. The number of discrete enzymes in 1972 was 13, in 1999 it was 282 and by 2004, 532. These enzymes, capable of inactivating b-lactam antibiotics, can be divided into four types based upon nucleotide and amino acid sequences (Table 3). The class A enzymes include the carbenicillinases (Table 3), which are transposon mediated and are represented by the PSE enzymes.31 Also in class A are the enzymes referred to as extended-spectrum b-lactamases (ESBLs), which made their appearance from 1990. They include the new TEM-type enzymes, SHV enzymes, PER and VEB (South-East Asia) and GES (Guinea). These enzymes are all closely related but with differing substrate profiles that have obviously evolved due to JAC Garrod Lecture Table 3. b-Lactamases found in P. aeruginosa Class Type A carbenicillinases B C D metallo-b-lactamases cephalosporinases oxacillinases Examples Comments PSE ESBLs including TEM, SHV, PER, VEB and GES VIM, IMP, GIM and SIM AmpC five groups increased selection pressure. Most of the variations take place by amino acid substitutions at the active site. Dissemination of these class A ESBL-encoding genes by plasmids and integrons provides a very important role in the resistance and dissemination of antibiotic resistance. Class B enzymes are the metallo-b-lactamases (Table 3), which are dependent upon zinc ions for activity and are referred to as carbapenemases,25 because of their resistance to the carbapenems such as imipenem but sensitivity to the monobactam aztreonam. These enzymes contain the VIM, IMP, SPM, GIM and SIM enzymes all named on the basis of their initial geographical location. Many of these enzymes are closely related but again differ by changes at the active site. Many of the genes for these enzymes are carried on mobile elements called gene cassettes that are inserted into class 1 integrons. Such integronlocated resistance genes provide increased potential for expression and dissemination. Several of these class 1 integrons have been found in transposons, allowing integrons to be transposed, which increases the threat of genes being disseminated among diverse groups of bacteria. Resistance to imipenem is also characterized by a deficiency of the OprD porin. Loss of this porin in the presence of derepressed AmpC provides a high level of resistance. The class C enzymes (Table 3) are the cephalosporinases of the AmpC type, which are naturally occurring inducible enzymes in P. aeruginosa.25 The Class D enzymes (Table 3) are the oxacillinases of which there are five different groups in P. aeruginosa.26 Initially these enzymes were active against the early penicillins and cephalosporins but now, like many of the other enzyme types, they have broadened their profile to include third-generation cephalosporins such as ceftazidime. These enzymes are all closely related as a result of amino acid substitutions. They can be chromosome-, plasmid- or integronlocated genes and so spread easily. These enzymes are not susceptible to b-lactamase inhibitors. Alteration of the target site is a rare occurrence in resistance to antibiotics in P. aeruginosa as compared with staphylococci. There are examples of low-affinity PBPs for certain antibiotics but they are not a major cause of resistance. From an evolutionary point of view, the ability of Pseudomonas to produce a plethora of b-lactam-hydrolysing enzymes located behind a lipid barrier has been so successful in overcoming the onslaught of b-lactam antibiotics that the necessity and complexity of an altered target site has not proved necessary. This is in complete contrast to the situation seen with the staphylococci. The mechanisms of resistance seen in P. aeruginosa are illustrated in Table 2. PSE-1 to PSE-4, transposon mediated ESBLs, disseminated by plasmids and integrons carbapenemases may be encoded by chromosome, plasmids or integrons Summary Following the publication of On the Origin of Species in 1859 and the subsequent debate at Oxford in 1860, the concept of evolution through natural selection and consequently the ability to adapt slowly started to be accepted. However, today there are still significant numbers of people around the world who continue to question the theory of evolution. Because of their ability to reproduce rapidly and their enormous genetic flexibility, bacteria have provided a perfect example of Darwin’s principles when faced with the onslaught of a plethora of antibiotics. The increasing availability of antibiotics in the 1950s and 1960s led many in authority to predict the ‘beginning of the end’ for infection. Nothing could be further from the truth! Our quests to counter the inevitable emergence of microbial resistance are puny compared with the power of Nature to defeat our feeble attempts! Resistance is increasing remorselessly and the output of novel antimicrobial compounds is decreasing. However hard we try and however clever we are, there is no question that organisms that have been around for 3 billion years, and have adapted to survive under the most extreme conditions, will always overcome whatever we decide to throw at them. Transparency declarations None to declare. References 1 Lipps G, ed. 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