β -Lactamases: which ones are clinically important?

Rice and Bonomo

β-Lactamases: which ones are clinically important?

DEFINING CLINICAL IMPORTANCE

Louis B. Rice, Robert A. Bonomo Medical Service, Department of Veterans Affairs Medical Center and Departments of Medicine and Pharmacology,. Case Western Reserve University, Cleveland, Ohio, USA

Abstract The introduction of a large array of β-lactam antibiotics has spawned the emergence of an even larger variety of β-lactamases designed to confer resistance to these agents. β-lactamases are produced by both gram-positive and gram-negative bacteria, but their clinical importance is far greater among the gram-negatives.The virtual explosion in our knowledge about the variety of these enzymes can often create confusion and frustration among those not well versed in the field. In this paper, we attempt to focus the discussion of βlactamases on those enzymes that are of the greatest clinical importance, the Ambler Class A and C enzymes.We also discuss the growing importance of the Ambler Class B metallo β-lactamases, which hydrolyze carbapenems and are increasing in prevalence in areas of significant carbapenem usage. © 2000 Harcourt Publishers Ltd

INTRODUCTION

β

-Lactamases are widely distributed in bacteria.1–3 The role played by these efficient molecules in β-lactam resistance has fueled considerable pharmaceutical industry investment into developing new antimicrobial agents over the past three decades. In some instances, pharmaceutical research has been directed toward identifying and developing non-β-lactam antimicrobial classes, such as the aminoglycosides and the fluoroquinolones. However, the safety and efficacy of β-lactams as a class has provided powerful motivation for developing newer versions of older molecules (extended-spectrum cephalosporins, ureidopenicillins), as well as newer classes of β-lactam agents resistant to hydrolysis by commonly encountered β-lactamases (carbapenems, monobactams). β-Lactamases represent the most common mechanism of β-lactam resistance in gram-negative bacilli. The number of different β-lactamases that have been discovered and characterized over the past decade is truly impressive. In a comprehensive 1995 review Bush, Jacoby and Medeiros listed no fewer than 126 named enzymes.2 Of these, enough genetic information was available for a dendrogram of relatedness for 88 different β-lactamases. A recent review of a web site devoted solely to the variants of the TEM and SHV-type β-lactamases (www.lahey.org/lcinternet/studies/webt.htm) revealed 75 variants of TEM and 24 variants of SHV.With such an explosion of information, the need to focus attention on those of significant clinical importance becomes urgent. In this paper, we will offer our view of those enzymes that are of the greatest clinical importance. It can be persuasively argued that all β-lactamases are of potential clinical 178

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importance. However, a discussion of the relatively few types commonly encountered in the clinical setting will be the goal of this article.

 2000 Harcourt Publishers Ltd

doi: 10.1054/drup.2000.0144, available online at http://www.idealibrary.com on

Different definitions of clinical importance are conceivable. A structural chemist may prefer one in which the most importance is placed on enzymes that offer insight into important drug-enzyme interactions. A microbiologist may prefer to emphasize those enzymes present in unusual pathogens, or that confer unusual or prototypic antimicrobial resistance profiles. A geneticist, on the other hand, may prefer to emphasize enzymes that inform us regarding the evolution of β-lactamases, or those enzymes with the most interesting regulation. We will choose a definition that clinically important β-lactamases are those commonly expressed by frequently isolated pathogens, or that are beginning to appear in clinically important, and formerly susceptible pathogens. When data are available, we also favor enzymes whose expression results in therapeutic failure when susceptible antibiotics are used. When considering nosocomial pathogens, it is reasonable to use the National Nosocomial Infection Surveillance (NNIS) data generated by the Centers for Disease Control and Prevention as a starting point (http:// www.cdc.gov/ncidod/hip/Surveill/nnis.htm). According to NNIS, bacterial pathogens constituted eight of the top 10 causes of nosocomial infection in coronary care units for the period between 1992 and 1997 (Table 1). Of these, seven produce β-lactamases that can significantly alter therapeutic choices. There are no reliable national data on the relative percentages of community acquired infections caused by specific bacterial species.We have compiled our own list of 11 common community-acquired pathogens and their β-lactamase production (Table 1). Of these, β-lactamase-mediated resistance is an important clinical consideration for eight. As Table 1 indicates, the β-lactamases of greatest clinical importance fall into Ambler molecular classes A and C.4 These β-lactamases fall into the Bush-Jacoby-Medeiros functional group 1 and 2, respectively.2 Class A enzymes are primarily penicillinases (with little activity against other β-lactam classes), plasmid-mediated (although not always), constitutively expressed (although not always) and susceptible to inhibition by the clinically available β-lactamase inhibitors clavulanic acid, sulbactam and tazobactam. Class C enzymes, in contrast, are primarily cephalosporinases (with activity against penicillins as well), chromosomally encoded (although not always), inducible by exposure to some β-lactam agents (in most cases) and resistant to inhibition by β-lactamase inhibitors. In our discussion, we will address specifically the Ambler Class A and Class C β-lactamases, emphasizing their common characteristics and important differences. We will distinguish the importance of different enzyme classes for individual genera and species as well, since the relative impact these enzymes have on the resistance phenotype varies between species. Finally, we will review some Class B β-lactamases of growing clinical significance.

Clinically important β -lactamases Table 1 Common nosocomial and community-acquired pathogens and their β-lactamase production Nosocomial Pathogen

β-lactamase (Ambler class)

CoNS* S. aureus Enterococcus E. coli Enterobacter spp. C. albicans K. pneumoniae S. marcescens P. aeruginosa Other Candida spp.

+ (A) + (A) –

Community-acquired Pathogen

β-lactamase (Ambler class)

+ (A, rare C) + (C, rare A)

S. pneumoniae S. pyogenes Viridans streptococci S. aureus E. coli

– – – + (A) + (A)

– + (A, rare C) + (C, some A) + (primarily C) –

K. pneumoniae H. influenzae M. catarrhalis N. gonorrhoeae P. mirabilis

+ (A) + (A) + (A) + (A) + (A)

B. fragilis

+ (mostly A)

* CoNS – coagulase negative staphylococci.

AMBLER CLASS A Β-LACTAMASES Ambler Class A enzymes are primarily penicillinases. Their existence has been recognized for decades, and their prevalence in clinically important species such as Staphylococcus aureus and Escherichia coli has prompted the development of several new classes of β-lactam antibiotics designed to avoid inactivation.Among these antimicrobial classes are the cephalosporins (beyond the first generation), the carbapenems, the monobactams and the β-lactam-β-lactamase inhibitor combinations. These new antimicrobial classes have proved exceptionally effective in the treatment of infections caused by β-lactamase-producing bacteria, although the enzymes in some cases have evolved to address the new antibiotic challenges. Gram-negative bacilli The two most important class A enzymes found in gram-negative bacilli are TEM-1 and SHV-1.1 TEM-1 is the most common β-lactamase found in E. coli, but has also been described in Klebsiella pneumoniae, Enterobacter spp., Haemophilus influenzae and Neisseria gonorrhoeae, among others.TEM1 is most commonly plasmid-encoded and is often transferable in vitro to laboratory recipient strains. TEM-1 may also be chromosomally encoded, a finding that has been attributed to its presence on mobile elements (transposons).1 The transferability of many TEM-related plasmids has certainly contributed to its prevalence in clinical E. coli isolates. Although numbers vary depending upon the patient population under study, currently 40% or more of nosocomial E. coli isolates are resistant to ampicillin, most through the production of TEM-1.5 TEM-1 is expressed constitutively, although the level of expression can vary between strains depending on the number of plasmid copies present within the cells, or on the nature of the promoter found upstream of the blaTEM-1

gene.6,7 A single base pair change in the blaTEM promoter (identified initially associated with the closely related blaTEM) results in a 10-fold increase in enzyme expression.6 A more 2 common mechanism for increased TEM-1 production by clinical strains is an increase in the gene copy number, either by incorporation into high copy-number plasmids, or by a duplication of the blaTEM genes themselves.8,9 The levels of ampicillin resistance conferred by TEM-type enzyme production range from roughly 1028 µg/ml to >32 000 µg/ml. In virtually all cases, the levels of resistance exceed concentrations achievable in human tissues after normal dosing, so the resistance conferred should be considered absolute. SHV-1 was originally described as a plasmid-mediated penicillinase in E. coli, although in clinical isolates it is found more commonly in K. pneumoniae.1,10,11 Its host range appears to be somewhat more restricted than TEM-type enzymes, a difference that is difficult to explain, since both types of β-lactamases are found on transferable and often broad host-range plasmids. It has been known for years that virtually all K. pneumoniae strains produce low levels of a Class A β-lactamase. The original chromosomal β-lactamase described in K. pneumoniae was designated LEN-1, which is more than 90% identical to SHV-1.12 More recent data suggest that SHV-1 may be the most common of the chromosomal βlactamases found in K. pneumoniae.13 Expression of SHV-1, when the blaSHV-1 is chromosomal, is generally modest, conferring ampicillin MICs of 64–128 µg/ml.14 A single point mutation in the -10 region of the promoter may, however result in very high levels of blaSHV-1 transcription.14 When combined with outer-membrane-protein changes, such high-level production can result in clinically significant levels of resistance not only to ampicillin, but to broad-spectrum agents like ceftazidime, suggesting phenotypically that an extended-spectrum β-lactamase is present.14 Plasmid-mediated versions of blaSHV-1 whose nucleotide sequence has been determined have generally possessed the

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Rice and Bonomo high-production promoter, and have therefore been associated with very high levels of ampicillin resistance.15 The SHV-1 enzyme differs from the TEM-1 enzyme in that, when produced in large amounts, it can exhibit clinically significant activity against third generation cephalosporins. In addition, it tends to be less susceptible than TEM-1 to inhibition by clinically available β-lactamase inhibitors, particularly sulbactam and tazobactam.Therefore, increased production of SHV-1 can be associated with clinically significant levels of resistance to some β-lactam-β-lactamase inhibitor combinations.16 In one strain of K. pneumoniae, increased chromosomal expression of blaSHV-1 combined with reduction in an outer membrane porin to result in resistance to both ceftazidime and to β-lactam-β-lactamase inhibitor combinations.14 Structural considerations for Class A β-lactamases The tertiary structures of several Class A β-lactamases have been determined at the atomic level.At present,there are seven primary Class A β-lactamase structures deposited in the protein data bank: S. aureus PC1, Bacillus licheniformis 749/C, Streptomyces albus G, E. coli TEM-1, NmcA from Enterobacter cloacae, E.coliToho-1 and the chromosomal SHV-1 β-lactamase from K. pneumoniae. Despite divergence of nucleotide and amino acids sequences, the Class A enzyme structures are similar in overall appearance and topology (Fig. 1). Particularly similar are TEM-1 and SHV-1. The critical amino acids used for catalysis by the SHV-1 enzyme (Ser70, Lys73, Ser130, Asn132, Glu166, and Lys234) deviate only .23 Å from the corresponding amino acids in TEM-1. The width of the substrate binding cavity is 0.7 to 1.2 Å wider in SHV-1 than in TEM-1.17 The increased width may explain some of the known differences in substrate binding profiles, such as the greater activity of SHV-1 against some extended spectrum cephalosporins, and the greater resistance to inhibition by mechanism-based inactivators characteristic of SHV-1. Differences are also demonstrable in the binding of the β-lactamase inhibitory protein (BLIP). It has been hypothesized that the differences at positions 104 (Glu in TEM-1 vs.Asp in SHV-1) and 215 (Lys in TEM-1 vs. Arg in SHV-1) may explain the differences in BLIP binding.17 Extended-spectrum variants of TEM and SHV The clinical significance of the TEM and SHV β-lactamases has been enhanced by the rapid emergence of mutants that

a Fig. 1

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confer high levels of resistance to many extended-spectrum cephalosporins.2,18 These mutants, the extended-spectrum βlactamases (ESBLs) appear in settings where significant quantities of cephalosporins are used.19–22 As alluded to above, there are now nearly 100 variants of the TEM and SHV enzymes that confer an extended spectrum of activity. The extended spectrum results from point mutations in the TEM or SHV genes. The sites for these mutations are most commonly in or immediately around the active site of the enzymes, and in many cases serve to ‘open up’ the active site to allow binding and hydrolysis of the bulky extended-spectrum cephalosporins.2 The extended spectrum comes at a price, however, resulting in a diminished ability of the enzyme to hydrolyze penicillins and an increased susceptibility to inhibition by β-lactamase inhibitors.18 There are no data available to help determine whether the reduced activity against the penicillins or the enhanced susceptibility to the inhibitors are important for the treatment of clinical infections, but inhibitor combinations have been found to be highly effective in treating ESBL-producing K. pneumoniae in some animal models of infection.23–25 TEM and SHV variants have also been reported that possess mutations making them less susceptible to inhibition by clavulanic acid.26–28 These mutations almost always result in increased susceptibility to extended-spectrum cephalosporins. Enzymes with potent activity against extended-spectrum cephalosporins and high-level resistance to inhibition have not been reported, and may well be chemically unachievable. Nevertheless, plasmids carrying both a TEMderived ESBL and a highly expressed SHV-1 have been reported to confer resistance to both classes,16 so the mere presence of an ESBL should not be taken to imply susceptibility to β-lactam-β-lactamase inhibitor combinations. Inoculum effects Among the more important side effects of β-lactamase-mediated resistance is the phenomenon of the inoculum effect. Phenotypically, this effect in manifest by a higher MIC when the inoculum of organisms is 107 CFU/ml that when it is 105 CFU/ml.24 This phenomenon reflects the fact that the hydrolysis of a β-lactam antibiotic represents a one-to-one interaction between β-lactam and β-lactamase. In simple terms, if the β-lactamases outnumber the β-lactam molecule, then the organism will be resistant. If they do not, the organism will

b

Ribbon diagrams of three Class A β-lactamases. (A) TEM-1; (B) SHV-1; (C) PC-1.

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c

Clinically important β -lactamases be inhibited.Whether a strain will be susceptible or resistant will therefore depend upon the relative quantities of β-lactam and β-lactamase in the periplasmic space. Circumstances that increase the quantity of β-lactam molecule in the periplasmic space, such as high concentrations of extracellular antibiotic or rapid passage of the antibiotic through the outer membrane porins, will make the strain more susceptible. On the other hand, circumstances that serve to reduce the quantity of β-lactam antibiotic in the periplasmic space, such as low external concentrations of antibiotics (relative to the number of bacteria present) or porin changes that restrict antibiotic entry into the periplasmic space, will make the strain more resistant.29,30 Inoculum effects have been particularly important for ESBLs. Many ESBLs confer high level resistance to ceftazidime, but appear susceptible to other extendedspectrum cephalosporins such as ceftriaxone, cefotaxime and cefepime.18,24 Despite this apparent susceptibility, inoculum effects are frequently demonstrable. In some animal models, these apparently active extended-spectrum cephalosporins have been ineffective, and clinical failures in the treatment of human infections have been reported.24 As a result, most experts agree that any ESBL-producing strain should be considered resistant to all extended-spectrum cephalosporins regardless of the results of standard in vitro susceptibility tests, especially for the treatment of infections involving sites other than the urinary tract. Gram-positive bacteria The only clinically important β-lactamase produced by grampositive bacteria is PC1, the β-lactamase almost universally produced by S. aureus and coagulase-negative staphylococci.31 This is a molecular Class A β-lactamase and shares several characteristics with the TEM and SHV enzymes (Fig. 1). It is a penicillinase that has little if any activity against most cephalosporins. It is most commonly plasmid-mediated.32 It is located on a transposon,33 and it is susceptible to inhibition by β-lactamase inhibitors.34 There are also several important differences between this β-lactamase and those described above. Unlike the TEM and SHV β-lactamases, expression of PC1 is inducible by exposure to β-lactam agents.35,36 Regulation of PC1 production occurs by a two-component system in which a putative trans-membrane sensor component (BlaR1) detects the presence of an inducer in the environment, then interacts with a putative repressor (BlaI) in a manner that prevents BlaI binding to the β-lactamase operator, resulting in increased transcription of the β-lactamase (blaZ) gene.37,38 This regulation system is highly analogous to that used to regulate the expression of penicillin binding protein PBP2a, the low affinity PBP responsible for methicillin resistance in staphylococci. In fact, in some strains the pbp2a regulatory sequences have been inactivated, and expression of methicillin resistance is regulated in trans by the β-lactamase regulatory machinery.35 This cross-regulation may explain in part the virtually universal production of β-lactamase by methicillin-resistant strains of S. aureus. Another important difference between the TEM-1 (and SHV-1) and PC1 is the fact that PC1 is released into the surrounding medium, rather than sequestered in the periplas-

mic space (since gram-positive bacteria lack outer membranes, they have no periplasmic space).This ‘loss’ of β-lactamase from the individual bacterium reduces the potential protection against β-lactam antibiotics, particularly in lowinoculum infections. In this light, it is curious that mutants of PC1 similar to the ESBLs have not been described. It is possible that the protection offered by the outer membrane is an important factor in the clinical emergence of ESBL-producing bacteria. By their ability to concentrate large amounts of enzyme in the peri-plasmic space, and restrict entry of the antibiotic, the gram-negative organisms can create a ‘single cell inoculum effect’ that amplifies the rather modest levels of resistance afforded by single amino acid changes in Class A enzymes, thereby increasing the chance that they will withstand the antibiotic challenge that selects for their emergence. Although no extended-spectrum variants of PC1 have been described, four serotypes of this β-lactamase (A, B, C, D) have been recognized.31 These enzymes all have roughly the same amount of β-lactamase activity. However, compared to the others, serotype A has been shown to inactivate cefazolin relatively efficiently.31 Several characteristics of β-lactamase production by S. aureus are interesting microbiologically and clinically. The first is the phenomenon of borderline oxacillin resistant S. aureus (BORSA) strains. These strains exhibit oxacillin MICs that are near the breakpoint for susceptibility (ca. 4 µg/ml) but do not express PBP2a. Oxacillin MICs for BORSA strains are increased substantially with increased inoculum (to ca. 64 µg/ml at 5 × 107 CFU/ml) and decline with the addition of β-lactamase inhibitors, suggesting the presence of a β-lactamase-mediated resistance mechanism.39 In fact, these isolates produce a large amount of the serotype A β-lactamase, and are almost exclusively within the staphylococcal phage group 5.39 Animal studies suggest that this mild elevation in MICs will not be an important impediment for the treatment of S. aureus infections with semi-synthetic penicillins,34 and to date there are no data from human infections that suggest this mechanism of resistance is important. One area where excessive production of serotype A β-lactamase may well be important is in the use of cefazolin for prophylaxis of clean surgical procedures. Data from animal models suggests that Type A β-lactamase producing strains are more likely to produce infection when cefazolin is used for prophylaxis.40 Retrospective analyses of human perioperative wound infections also suggests that a higher percentage of patients that receive cefazolin prophylaxis develop infections due to Type A β-lactamase-producing S. aureus than do those subjected to other prophylactic regimens.39 Although the data are not strong enough to recommend the movement away from cefazolin as the mainstay of surgical prophylaxis, they do suggest that patterns of S. aureus wound infections within institutions should be monitored closely, and adjustments made in prophylactic regimens if an increase in infections caused by BORSA strains is observed. In addition, it is not unreasonable to consider regimens other than cefazolin for the antibiotic treatment of deep-seated staphylococcal infections, especially if the isolate exhibits a borderline susceptibility to oxacillin.  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 178–189

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Rice and Bonomo Other Class A β-lactamases of clinical importance Haemophilus influenzae and Moraxella catarrhalis are important pathogens for infections of the upper and lower respiratory tracts. In most recent surveys, 30–40% of H. influenzae strains are penicillin-resistant by virtue of β-lactamase production. In most cases, this β-lactamase is TEM-1.41 However, in the minority of cases H. influenzae produce another class A β-lactamase designated ROB-1.41 ROB-1 exhibits characteristics very similar to TEM-1 and therefore the distinction between these two enzymes is not of clinical importance. M. catarrhalis strains are widely resistant to penicillin, also because of β-lactamase production. In most cases, M. catarrhalis (80–90%) produce a Class A β-lactamase designated BRO-1.42 As with ROB-1, this enzyme is not active against cephalosporins and is readily inhibited by β-lactamase inhibitors, so the distinction between it and other enzymes is not clinically important. Among the other Class A-type β-lactamases of clinical importance are the chromosomal β-lactamases of Bacteroides fragilis and other Bacteroides species.43 These β-lactamases are considered as molecular class A β-lactamases and are active primarily against earlier penicillins such as penicillin G and ampicillin. However, in high inocula they also exhibit activity against a variety of cephalosporins. In addition, there has been a class A β-lactamase described from Bacteriodes vulgatus that is active against cefoxitin.44 Hence, the only cephalosporins that should be considered reliably active against B. fragilis group organisms are the cefamycins (cefoxitin and cefotetan).43 Piperacillin is considerably less susceptible to hydrolysis by these enzymes than is ampicillin.43 Addition of β-lactamase inhibitors restores virtually complete activity against these organisms to ampicillin and piperacillin, making the β-lactam-β-lactamase inhibitor combinations attractive alternatives for the treatment of intra-abdominal infections.43 The common β-lactamases found in Bacteroides have very poor activity against the carbapenems imipenem and meropenem, explaining the excellent anti-anaerobic activity of this class of antibiotics.43 AMBLER CLASS C Β-LACTAMASES The other major group of β-lactamases of clinical importance are the AmpC β-lactamases (Ambler Class C, Bush-JacobyMedeiros Group 1). They represent a group of β-lactamases produced to some degree by virtually all gram-negative bacteria (Salmonella and Klebsiella being the only known exceptions). Chromosomally encoded versions of these enzymes are particularly important in clinical isolates of Citrobacter freundii, Enterobacter aerogenes and cloacae, Morganella morganii, Pseudomonas aeruginosa and Serratia marcescens. Although the catalytic efficiencies (kcat/Km ratios) of Class C β-lactamases for penicillins and cephalosporins are very similar, the turnover numbers vary dramatically. Class C β-lactamases hydrolyze first generation cephalosporins such as cephaloridine and nitrocefin with kcat values as high as 5000 s-1. Although cephalosporin substrates such as cefoxitin, cefuroxime, and cefotaxime demonstrate lower turnover numbers (lower kcat values), the Km values are also low. In contrast, the kcat values for penicillins such as benzylpenicillin and ampicillin are low for Class C 182

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Fig. 2 Ribbon diagram of Class C β-lactamaseGC1 from Enterobacter cloacae.

enzymes.The favorable kcat/Km ratio (catalytic efficiency) is a result of the low Km values.45,46 Class C β-lactamases are slightly larger than Class A enzymes, and have alkaline isoelectric points (pI > 7.0).They are distinguishable from the common Class A enzymes by the phenotypic resistance to cephalosporins and β-lactam-β-lactamase inhibitor combinations they confer. It is not as easy to distinguish these enzymes from extended-spectrum variants of TEM-1 or SHV-1. However, the activity of the Class C enzymes against the cephamycins (and to a lesser extent their resistance to inhibition by β-lactamase inhibitors) can generally be used to distinguish between the two classes in the clinical setting. Structural considerations The atomic structures of four Class C β-lactamases are currently known: C. freundii 1203, E. cloacae P99, E. cloacae GC1 and E. coli AmpC47–50 (Fig. 2). In comparison to Class A enzymes, Class C β-lactamases have larger active site cavities which may allow them to bind the bulky extended-spectrum cephalosporins (oxyimino β-lactams). That the size of the binding cavity is important is suggested by the fact that the GC-1 enzyme possesses a unique tandem repeat (Ala211Val212-Arg213) not present in P99 β-lactamase. As a result, GC1 has a wider binding cavity (by 0.6–1.4 Å). It is hypothesized that this conformational flexibility and its influence on adjacent structures facilitates hydrolysis of oxyimino β-lactams by making the acyl-enzyme intermediate more open to attack by water. Resistance to inhibition by clavulanate, however, has been more often attributed to the lack of a functional analogue of Arg244 found in Class A enzymes.47 The interaction of Class C enzymes with β-lactamase inhibitors is an area of active study. Regulation of expression Under normal circumstances in clinically important Class C enzyme-producing gram-negative bacilli (most prominently C. freundii, Enterobacter spp., P. aeruginosa and S. marcescens), β-lactamase production is repressed. The details of the repression are worked out in the most detail for Enterobacter spp. (See Table 2 and Fig. 3A and B).51–53 The

Clinically important β -lactamases

a

b Fig. 3 (A) Summary of ampC expression.AmpR serves as either a repressor or and activator of ampC transcription, depending upon whether it is associated with UDP-MurNac-pentapeptide (repressor) or anhydromuramyl-tripeptide (activator). It binds upstream of the ampC gene to serve its function. Under normal (non-induced) circumstances,AmpR is associated with UDPMurNac-pentapeptide and acts as a repressor. In settings where anhydromuramyl-tripeptide is in excess of the pentapeptide, the former compound associates with AmpR, forming an activator of ampC transcription.Without AmpR present, ampC is transcribed constitutively, but at low levels. (B) The functions of AmpD, an amidase that breaks down anhydro-MurNac-tripeptide and AmpG, a permease that allows peptidoglycan breakdown products to re-enter the cytoplasm.

repression or activation is closely tied to the processes of cell wall synthesis and breakdown. The molecule that serves as both the repressor and the activator of ampC transcription is AmpR, a transcriptional regulator of the LysR family. Under normal circumstances, AmpR is present as a repressor by virtue of its interaction with UDP MurNac-pentapeptide, a

peptidoglycan precursor molecule. In this form AmpR is incapable of activating ampC and in fact serves as a repressor of ampR expression. In the setting of high concentrations of cell-wall-breakdown product anhydro-MurNActripeptide (or perhaps anhydroMurNac-pentapeptide), however, UDP-MurNAc-pentapeptide is displaced from its site in  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 178–189

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Rice and Bonomo Table 2 Components in the regulation of AmpC β-lactamase production Gene

Protein

Function

ampC

AmpC

ampR

AmpR

ampD

AmpD

ampG

AmpG

Hydrolyzes β-lactam antibiotics DNA binding protein; Transcriptional regulator of AmpC. Activity as a repressor or activator depends upon the molecule with which it is associated Amidase; cleaves the amide bond between amino acids of muropeptides Permease involved in the recycling of cell wall peptides

Table 3 Plasmid-encoded Amp C β-lactamases β-Lactamase

Organism

MIR-1 CMY-1 CMY-2 CMY-2b CMY-3 CMY-4 CMY-5 MOX-1 FOX-1 FOX-2 FOX-3 LAT-1 LAT-2 BIL-1 ACT-1 ACC-1 DHA-1

K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E.coli K. oxytoca K. pneumoniae K. pneumoniae E. coli K. oxytoca K. pneumoniae K. pneumoniae E. coli K. pneumoniae K. pneumoniae K. pneumoniae

Adapted from reference 50.

AmpR, resulting in the conversion of AmpR to an activator of ampC transcription.51,54 This increase in anhydro-MurNActripeptide or pentapeptide concentration in the cytoplasm of the cell occurs most commonly by one of two mechanisms.The first is through the action of β-lactam antibiotics, certain of which cause the release of significant quantities of anhydro-MurNAc-tripeptide and/or pentapeptide from the peptidoglycan.55 This anhydro-UDP-MurNAc-tripeptide enters the cell through a channel (AmpG) and overwhelms the recycling ability of the cytosolic amidase (AmpD) specific for recycling of muropeptides.56 Under these circumstances, β-lactamase is produced only as long as the antibiotic is present in the medium. The mechanism for constitutive high-level production of β-lactamase most commonly involves a mutation of the ampD gene, reducing the quantity of or eliminating AmpD from the cytoplasm.51,56 Under these circumstances, a constant high level of anhydro-MurNAc-tripeptide is present in the cytoplasm, and AmpR serves as a constitutive activator of ampC transcription. Constitutive production can also result from deletion of ampR, but in this circumstance β-lactamase production is generally at a low level. Clinical importance As Table 1 indicates, Class C β-lactamase-producing bacterial species, particularly Enterobacter spp. and P. aeruginosa, are important nosocomial pathogens.The extent to which Class C enzyme production is important for these organisms can be determined by looking at the ceftazidime-susceptibilities of the species themselves. Data from the Centers for Disease Control and Prevention for the late 1980s indicate that between 30 and 40% of nosocomial Enterobacter isolates and roughly 10% of nosocomial P. aeruginosa isolates express resistance to ceftazidime.57 In virtually all of these 184

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instances, it can be safely assumed that the expression of a Class C enzyme plays a role in resistance. A clinically important development has been the recent increase in plasmid-encoded Class C β-lactamases. The phenotypic expression of a plasmid-encoded β-lactamase is identical to that of a derepressed chromosomally-encoded cephalosporinase. MIR-1, found on a mobile plasmid in K. pneumoniae, was the sentinel enzyme for the subsequent recognition of these β- lactamases.58,59 Plasmid-mediated class C β-lactamases have been described in many gram-negative bacteria from all parts of the world. Host strains harboring these enzymes include K. pneumoniae, E. aerogenes, Salmonella spp. (seftenberg and enteritidis), E. coli, Proteus mirabilis, Morganella morganii, and Klebsiella oxytoca60–68, with the majority being described in K. pneumoniae and E. coli. It is worrisome that the majority of these enzymes have been described in K. pneumonia and E. coli (Table 3).These are among the most frequently recovered isolates from patients in the hospital and in the community.The potential for further dissemination of AmpC β-lactamases by mobilization on plasmids is quite disturbing. The decrease expression of certain outer-membrane proteins (porins) in organisms harboring chromosomal or plasmidic Class C β-lactamases leads to resistance to carbapenems (imipenem). Bradford et al. recently reported an isolate of K. pneumoniae resistant to carbapenems.69 This strain possessed a new plasmidic cephalosporinase called ACT-1 and was missing an outer membrane protein. Stapleton et al. also described a similar scenario with the report of CMY4 in E. coli.70 These findings with plasmid-mediated AmpC enzymes are reminiscent of the mechanism by which P. aeruginosa most commonly expresses resistance to imipenem, in which AmpC production and down-regulation of the OMPD2 porin combine to result in resistance.71

Clinically important β -lactamases Plasmid-encoded AmpC cephalosporinases can be grouped into four general categories. Group 1 consists of those who originated from the chromosomal AmpC of C. freundii (BIL-1, CMY-2, LAT-1, LAT-2). The Group 2 plasmid borne cephalosporinases are related to the chromosomal cephalosporinase of E. cloacae (MIR-1 and ACT-1). Group 3 are related to the AmpC of P. aeruginosa (CMY-1, FOX-1 and MOX-1). Group 4 are related to the CMY-1 β-lactamase (CMY1 cluster).60,61,68 CLASS B β-LACTAMASES Unlike the serine-dependent β-lactamases (Class A, C, and D), class B β-lactamases are metallo enzymes.As such, Class B βlactamases require zinc or another heavy metal for catalysis and are inhibited by chelating agents. Except for a few notable exceptions (see below), Class B β-lactamases are able to confer resistance to a wide range of β-lactam compounds, including cephamycins and carbapenems. Class B β-lactamases are resistant to inactivation by clavulanate, sulbactam, and tazobactam. Aztreonam, a monobactam, is neither hydrolyzed nor acts as an inhibitor.72 This highly resistant phenotype coupled with the increasing prevalence of these enzymes in a growing number of clinical isolates makes them a major concern for the medical community. Class B β-lactamases can be grouped into three different subclasses; B1, B2, and B3.72,73 B1 (Bacillus spp, B. fragilis and the broad host range IMP-1 metallo β-lactamase) and B3 Class B β-lactamases (Stenotrophomonas maltophilia) demonstrate a broad substrate profile, i.e. the β-lactamase from B. fragilis is able to hydrolyze penicillins, cephalosporins and carbapenems. The B2 class B β-lactamases (i.e. from Aeromonas) exhibit a narrow substrate profile. The Aeromonas metallo β-lactamase can only hydrolyze imipinem and ampicillin and is inhibited by cefoxitin. Structure and regulation The atomic structures of four Class B β -lactamases (Bc-II from B. cereus 569/H/9, CcrA from B. fragilis, L1 from S. maltophilia, and IMP-1 from P. aeruginosa) have been solved.74–77 Although the genes encoding these β-lactamases show very little primarily structure sequence identity (17–37%), the three-dimensional structures of the three known metallo β-lactamases are similar (see Fig. 4 for an example of the crystal structure of a Class B β-lactamase). Because of the metal ion, the catalytic pathway of metallo β-lactamases does not involve an acyl enzyme intermediate as it does in Class A and C.78–80 The catalytic pathway in Class B involves a hydrolytic water molecule (the ‘bridging’ water molecule) that possesses enhanced nucleophilicity due to the proximity to the metal ion. The addition of the hydroxide to the carbonyl carbon of the β-lactam leads to the formation of a transient, non-covalent reaction intermediate. Metallo β-lactamase expression is both constitutive and inducible. The majority of metallo β-lactamases are chromosomally encoded.The metallo β-lactamase of B. cereus, S. maltophilia, A. hydrophilia, and A. jandaei are inducible.72 In A. jandaei regulation of the metallo β-lactamase appears to involve a 2 component-signal transduction systems.81

Fig. 4 Ribbon diagram of the binuclear zinc β-lactamase from Bacteroides fragilis.

Increasing prevalence First discovered in a non-pathogenic strain nearly 40 years ago (Bacillus cereus) this Class of β-lactamases are now recovered from a wide variety of bacteria. Genes encoding these β-lactamases have been found on plasmids, integrons (see below), and in the bacterial chromosome.72 Plasmid transfer accounts for the rapid spread to other species – a particular problem in Japan where carbapenems are used in abundance.At present, the most widespread metallo β-lactamases are IMP-182–86 and CcrA (found in B. fragilis).72,75,87 IMP-1 is encoded within the variable region of class-1 integrons.82,83,88,89 Clinical Importance The metallo β-lactamase most commonly encountered in the clinical setting is the one characteristically expressed by S. maltophilia, which makes this species predictably resistant to the carbapenems. Of increasing concern, however, are the plasmid-mediated metallo enzymes found in species other than S. maltophilia. IMP-1 is the other metallo β-lactamase with the greatest clinical importance. This enzyme is encoded on a mobile gene located in an integron-like element.This integron has been found in Serratia marcescens, Klebsiella pneumoniae, P. aeruginosa, Pseudomona putida, Burkholderia cepacia, and Alcaligenes xylosoxidanes.84–86 It is generally accepted that imipenem resistance in P. aeruginosa is most commonly caused by a combination of high-level AmpC production and down-regulation of outer-membrane protein OMPD2. However, a growing number of strains are expressing IMP-1 and the closely related and recently described VIM-1 and VIM-2.88,89  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 178–189

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Rice and Bonomo Carbapenem therapy of serious intra-abdominal infections may be limited by the metallo β-lactamase from B. fragilis.This β-lactamase gene is present in only a limited number of isolates, and expressed in large quantities in even fewer. Evidence suggesting that the gene encoding this β-lactamase resides on a transposable element is cause for concern. Therapeutic directions for Class B enzyme-producing pathogens A number of β-methyl-carbapenem derivatives were screened as inhibitors of the IMP-1 metallo β-lactamases. A report by Nagano et al.90 describes a novel compound, J-110, 441, as a potent inhibitor of the IMP-1 metallo β-lactamases. J-110,441 is a potent inhibitor of Ccr A from B. fragilis, L1 from S. maltophilia and type II β-lactamase from B. cereus. In addition J110, 441 shows activity against TEM β-lactamases and the chromosomal C β-lactamase of E. cloacae.The combination of imipenem or ceftazidime with J-110,441 had a synergistic effect on the antimicrobial activity of these β-lactamase producing bacteria. J-110,441 was very effective against IMP-1 but had decreased activity against L1 and type -2 enzymes. CONCLUSIONS AND FUTURE CONCERNS A wide variety of β-lactamases are present in clinically important bacteria. The most prevalent enzymes are unable to hydrolyze the newer and more potent β-lactam antibiotics. However, the emergence of extended-spectrum variants of older enzymes and the appearance of previously undescribed broad-spectrum enzymes argue for continuous vigilance in monitoring prevalence and characteristics of β-lactamases, for judicious use of currently available antibiotics and for continued efforts to develop novel antimicrobial compounds. While the available studies provide us with reasonable estimates of which enzymes pose the greatest clinical challenge at the present time, predicting enzymes that will emerge, evolve or disseminate in the future is more difficult. The past 50 years have taught us that the evolution of β-lactamases in human pathogens will be related to the antimicrobial challenges faced in the clinical setting.The past two decades have been associated with widespread use of cephalosporins and the emergence of many β-lactamases that hydrolyze this highly effective and safe class of antibiotics. Insofar as emerging β-lactamase-mediated resistance to cephalosporins limits the appeal of this class, it is likely that the carbapenems will be used more frequently for the treatment of clinical infection. If increased carbapenem use does occur, it can be anticipated that enzymes capable of conferring resistance to these agents, including the Class B enzymes, the Class C enzymes (along with porin mutations) and the rare carbapenem-hydrolyzing Class A enzymes will continue to proliferate in the clinical setting. Note added in proof Recently, the x-ray structure of the OXA-10 β-lactamase from P. aeruginosa has been solved at 2.4 resolution.Although the overall structure of OXA-10 is similar to Class C and Class A enzymes, several important differences exist. Firstly, there is a three-residue-long strand that provides an extension of the substrate-binding cavity. Secondly, there is also the presence of a completely new fold in the Ω loop. This area of the 186

enzyme bears no acidic amino acids that would act as a general base in producing the hydrolytic water for deacylation of the acyl enzyme intermediate as in Class A.91

Received 28 April, 2000; Revised 11 May, 2000; Accepted 15 May, 2000 Correspondence to: Louis B. Rice MD, Medical Service 111(W),VA Medical Center, 10701 East Blvd., Cleveland OH 44106, USA.Tel: +1 216 791 3800, ×4801; Fax: +1 216 231 3289; E-mail: [email protected]

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