b-lactamases: a focus on current...

b-Lactamases: A Focus on CurrentChallenges

Robert A. Bonomo1,2

1Department of Medicine, Case Western Reserve University School of Medicine, Louis Stokes ClevelandDepartment of Veterans Affairs Medical Center, Cleveland, Ohio 44120

2Departments of Pharmacology, Molecular Biology and Microbiology, Biochemistry, andProteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland,Ohio 44120

Correspondence: [email protected]

b-Lactamases, the enzymes that hydrolyze b-lactam antibiotics, remain the greatest threat tothe usage of these agents. In this review, the mechanism of hydrolysis is discussed for boththose enzymes that use serine at the active site and those that require divalent zinc ions forhydrolysis. The b-lactamases now include .2000 unique, naturally occurring amino acidsequences. Some of the clinically most important of these are the class A penicillinases, theextended-spectrum b-lactamases (ESBLs), the AmpC cephalosporinases, and the carbape-nem-hydrolyzing enzymes in both the serine and metalloenzyme groups. Because of theversatility of these enzymes to evolve as new b-lactams are used therapeutically, new ap-proaches to antimicrobial therapy may be required.

Despite the tremendous advancements inbiomedicine, the production of b-lactam

hydrolyzing enzymes, b-lactamases, by Gram-negative and -positive bacteria still remainsone of the most significant threats to humanhealth (Hauck et al. 2016). With the introduc-tion of every new class of antibiotics, bacteriahave continued to evolve resistance, as they areamazingly capable of responding to environ-mental pressure via selection of existing muta-tions and acquisition of new genes. The mostsignificant threat has been faced by b-lactamantibiotics. The rapid evolution of b-lactamas-es, especially carbapenem hydrolyzing enzymes,makes each new drug obsolete in a very short

period of time (Bush 2010a,b, 2014; Drawz andBonomo 2010).

MECHANISM OF b-LACTAM ACTION

To properly appreciate the mechanisms bywhich b-lactamases have changed the status ofb-lactams in our therapeutic armamentarium,it is important to briefly review how b-lactamskill bacteria. b-Lactam antibiotics show theirbactericidal effects by inhibiting enzymes in-volved in cell-wall synthesis, that is, penicillin-binding proteins (PBPs). The integrity of thebacterial cell wall is essential to maintainingcell shape in a hypertonic and hostile environ-

Editors: Lynn L. Silver and Karen Bush

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ment such as serum, urine, lung mucus, or gas-trointestinal tract. Osmotic stability is preservedby a rigid cell wall comprised of alternating N-acetylmuramic acid (NAM) and N-acetylglu-cosamine (NAG) units. These glycosidic unitsare linked by a transglycosidases. A pentapeptideis attached to each NAM unit; the PBPs act astranspeptidases to catalyze the cross-linking oftwo D-alanine-D-alanine NAM pentapeptides.This cross-linking of adjacent glycan strandsconfers the rigidity of the cell wall. In the1960s, Strominger realized that the b-lactamring is sterically similar to the D-alanine-D-ala-nine of the NAM pentapeptide (Drawz and Bo-nomo 2010; Fisher and Mobashery 2014; Fisho-vitz et al. 2015). As a result, PBPs “mistakenly”use the b-lactam as a substrate “building block”during cell-wall synthesis. This “error” results inacylation of the PBP, which renders the enzymeunable to catalyze further carry out transpepti-dation reactions. As cell-wall synthesis slows,constitutive peptidoglycan autolysis continuesas a result of amidases, bacterial autolytic en-zymes. The breakdown of the murein sacculus,the peptidoglycan net that surrounds the bacte-rium, leads to cell-wall compromise and in-creased permeability. In this way, the b-lac-tam-mediated inhibition of transpeptidationcauses cell lysis.

MECHANISMS OF RESISTANCETO b-LACTAMS

There are four primary mechanisms by whichbacteria can overcome b-lactam antibiotics(Drawz and Bonomo 2010; Papp-Wallace et al.2011). First, changes in the active site of PBPscan lower the affinity for b-lactam antibioticsand subsequently increase resistance to theseagents, such as in PBP2x of Streptococcus pneu-moniae. In a similar manner, penicillin resis-tance in Streptococcus sanguis, Streptococcusoralis, and Streptococcus mitis developed fromhorizontal transfer of a PBP2b gene fromS. pneumoniae.

Methicillin resistance in Staphylococcus spp.is another example of an altered PBP. Althoughthe cause for this resistance is heterogeneous, itis often conferred by acquisition of the mec el-

ement, the mecA gene, which encodes PBP2a(also denoted PBP20). This low-affinity trans-peptidase can assemble new cell wall in the pres-ence of high concentration of penicillins (i.e.,methicillin) and cephalosporins.

Second, to access PBPs on the surface of theinner membrane, b-lactams must either diffusethrough or directly traverse porin channels inthe outer membrane of Gram-negative bacterialcell walls. Resistance to b-lactams can occurwhen these porin proteins are modified suchthat they are not produced in a fully activeform. Some Gram-negative bacteria show resis-tance to carbapenems based on loss and or re-duction of these outer membrane proteins, suchas the loss of OprD, which is associated withresistance to imipenem and reduced suscepti-bility to meropenem in Pseudomonas aerugi-nosa (Papp-Wallace et al. 2011).

Third, multicomponent drug efflux pumpsystems (mex), as part of either an acquired orintrinsic resistance repertoire, are capable of ex-porting a wide-range of substrates from theperiplasm of Gram-negative bacteria to the sur-rounding environment (Papp-Wallace et al.2011). These pumps are an important determi-nant of multidrug resistance in many Gram-negative pathogens, particularly notable inP. aeruginosa and Acinetobacter spp. Otherpumps are found in the enteric bacteria butwill not be discussed in detail, as this is beyondthe scope of this review. As an example of therole played by efflux pumps, increased produc-tion of the MexA–MexB system, in combina-tion with the low intrinsic permeability ofP. aeruginosa, can contribute to decreased sus-ceptibility to penicillins, cephalosporins, carba-penems, as well as quinolones, tetracycline, andchloramphenicol.

Last, b-lactamases hydrolyze b-lactams.This is the most common and important mech-anism of resistance in Gram-negative bacteriaand will be the focus of this review. The descrip-tion of b-lactamases conferring resistance topenicillins and cephalosporins has been exten-sively detailed (Drawz and Bonomo 2010; Papp-Wallace et al. 2011). This work will build onthose reviews and highlight the role of carbape-nemases as clinically important b-lactamases.

R.A. Bonomo

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Emphasis will be placed on select class D oxa-cillinases as much still needs to be learned aboutthese less popular b-lactamases.

b-LACTAMASE HISTORY

The first b-lactamase was identified in Bacillus(Escherichia) coli before the clinical use of pen-icillin (Abraham and Chain 1988). Within adecade, a significant clinical problem emergedwhen S. aureus was observed to be resistant topenicillin owing to the production of the staph-ylococcal penicillinase, PC1. As more b-lactamswere developed by pharmaceutical companiesand introduced into the clinic, resistance toeach agent was observed; the growing numberof b-lactam antibiotics increased the selectivepressure on bacteria, promoting the survivalof organisms with effective b-lactamases (Mas-sova and Mobashery 1998). As a result, b-lac-tamases were discovered in a multitude ofGram-negative bacteria including the entericbacteria such as Klebsiellae spp., Enterobacterspp., and nonfermenters such as P. aeruginosa.

Presently, .2000 naturally occurring b-lac-tamases are now identified and each possesses aunique amino acid sequence and characteristichydrolysis profile (K Bush, pers. comm.). It isspeculated that the rapid replication rate andhigh mutation frequency permit bacteria toadapt to novel b-lactams by evolution of theseb-lactamases. Because we also enjoy the benefitsof rapid whole genome sequencing methods,novel variants of a particular family of b-lacta-mases are constantly being discovered, forexam-ple, Acinetobacter-derived cephalosporinases,ADCs, and species-specific families of OXAs.

NAMING OF b-LACTAMASES

Jacoby (2006) has provided a very nice sum-mary of this interesting practice of naming theb-lactamases. b-Lactamases have been namedon the basis of molecular characteristics orfunctional properties. Earlier, b-lactamaseswere initially designated by the name of the bac-teria or plasmid that produced them (e.g., PC1or P99). Since these original descriptions,b-lactamases have been named after substrates

that are hydrolyzed (FOX), discovery location(OHIO), patient’s names (TEM), or the namesof the discoverers (HMS). Notably, a few b-lac-tamases have been given more than one name(e.g., ARI-1 and OXA-23; YOU-1 and TEM-26;YOU-2 and TEM-12; PIT-2 and SHV-1). Theinterested reader should refer to Jacoby (2006).

CLASSIFICATION

Two major classification schemes exist for cat-egorizing b-lactamase enzymes (the Amblerand Bush–Jacoby systems). Ambler classes Athrough D use amino acid sequence homologyto categorize b-lactamases (Bush and Jacoby2010). The Bush–Jacoby system groups 1through 4 are based on substrate hydrolysisprofiles (penicillin, cephalosporin, extended-spectrum cephalosporin, carbapenem) and in-hibitor profile (inhibition by b-lactamase in-hibitors clavulanate and tazobactam) (shownin Table 1).

A “family portrait” reveals the structuralsimilarity of class A, C, and D serine b-lacta-mases (Fig. 1). Class B b-lactamases (“a classapart”) are metallo-b-lactamases (MBLs).These proteins possess either a single or pairof Zn2+ ions coordinated in their active sites.More details will be discussed below.

CLASS A HYDROLYTIC MECHANISM

b-Lactamases are bacterial hydrolases that bindand acylate b-lactam antibiotics, much likePBPs, and then use strategically positioned wa-ter molecules to hydrolyze and inactivate theantibiotic before it can reach its target (Drawzand Bonomo 2010). In this manner, the b-lac-tamase is regenerated and can inactivate addi-tional antibiotic molecules. This reaction maybe schematically represented by the followingequation:

Eþ S Ok1

k�1

E :S�!k2E� S�!k3

E� P: ð1Þ

In this scheme, E is a b-lactamase, S is ab-lactam substrate, E:S is the Henri–Michaelis

b-Lactamases: A Focus on Current Challenges

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complex, E–S is the acyl enzyme, and P is theproduct devoid of antibacterial activity. The rateconstants for each step are represented by: k1,k21, k2, and k3; k1 and k21 are association anddissociation rate constants for the preacylationcomplex, respectively; k2 is acylation rate con-stant; and k3 is deacylation rate constant.

Serine b-lactamases, for example, TEM-1,SHV-1, P99, and KPC-2, actually use a multistepprocess to inactivate b-lactams. First, after pen-icillin or cephalosporin binding, nucleophilicattack by the active site serine on the carbonylgroup of the b-lactam antibiotic results in ahigh-energy acylation intermediate. Next, thisintermediate “transitions” into a lower energycovalent acyl enzyme. Following this, a catalyticwater molecule attacks the covalent complexand leads to a high-energy deacylation interme-diate, with subsequent hydrolysis of the bondbetween the b-lactam carbonyl and the serineoxygen. Last, deacylation regenerates the activeenzyme and renders the b-lactam inactive.

Both acylation and deacylation require theactivation of the nucleophilic serine and hydro-

lytic water, respectively. In class A enzymes, theultra-high-resolution (0.85 A) structure ofTEM-1 in complex with an acylation transitionstate analogue revealed the protonated state ofGlu166, supporting the hypothesis that Glu166acts as the activating base for both acylation anddeacylation.

CLASS C, B, AND D HYDROLYTICMECHANISM

The mechanistic symmetry seen in the class Adiscussion above is mirrored in class C enzymes,in which Tyr150 likely behaves as the generalbase for both acylation and deacylation, increas-ing the nucleophilicity of Ser64 and the catalyticwater, respectively (Chen et al. 2009). The hy-drolysis reaction catalyzed by class C b-lacta-mases consists of two steps: acylation and de-acylation. In the acylation half of the reaction,Ser64 attacks the b-lactam ring carbon andforms a covalent acyl-enzyme complex. Currentstructural and conformational evidence sug-gests that during acylation the catalytic nucleo-

Table 1. Comparison of nomenclature systems

Bush–Jacoby Ambler

Defining

substrates

Inhibited

by EDTA

Inhibited by clavulanaic

acid or tazobactam Representatives

1 Class C Cephalosporinases (–) No P99Cephamycinases FOX-4

2 Class A (–) Yes2a Penicillins Yes PC12b Yes TEM-1, SHV-12be Cephalosporins Yes TEM-10, SHV-22br No TEM-302ber No TEM-502ce Yes RTG-42d Class D Penicillins (–) Variable OXA-12de Cephalosporins OXA-112df Carbapenems OXA-232e Yes CepA2f Carbapenems Variable KPC-23 Class B Carbapenems (+) No3a B1 NDM-1, VIM-2,

IMP-13b B2 CphA3a B3 L1

Based on data from Bush and Jacoby (2010).

EDTA, ethylenediaminetetraacetate.

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phile, Ser64, is deprotonated and a proton isalso transferred to the leaving group, the b-lac-tam ring nitrogen. In deacylation, it is believedthat a general base activates the structurally con-served deacylating water, whereas a general acidmay be needed to reprotonate Ser64. In the de-acylation step, the catalytic water reacts with thecovalent linkage between the enzyme and thesubstrate, leading to the release of the hydro-lyzed product. Both acylation and deacylationreactions proceed through a high-energy tetra-hedral transition state.

Despite this similarity, debate exists aboutthe role of Lys67 (Chen et al. 2009). It is generallyagreed that many mechanisms may contributeto hydrolysis in class Cb-lactamases, dependingon the enzyme and the substrate, explaining whydifferent variants and substrates seem to sup-port different pathways and mechanisms, that

is, substrate assisted or conjugate base. For thewild-type enzyme itself, the conjugate basemechanism may be well favored. Substrate assis-ted catalysis may also occur; in this mechanism,the proton from the catalytic water is transferredto the substrate ring nitrogen, whereas Tyr150stabilizes the water molecule. It is the opinion ofthis researcher that the two different hypothesesare not mutually exclusive.

Class B includes Zn2+-dependent enzymesthat follow a different hydrolytic mechanism.These MBLs use the OH group from a watermolecule that is coordinated by Zn2+ to hydro-lyze the scissile amide bond of a b-lactam(Fig. 2). The importance of a high-energy an-ionic intermediate is currently favored as beingessential to the reaction coordinate.

Class D enzymes hydrolyze b-lactams usinga slightly different scheme by featuring a carba-

Class B IMP-1 β-lactamaseClass A TEM-1 β-lactamase

Class C E. coli AmpC β-lactamase Class D OXA-1 β-lactamase

Figure 1. The structural similarity of class A, C, and D serine b-lactamases.



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mylated lysine. OXA b-lactamases use this car-bamylated lysine to activate the nucleophilicserine used for b-lactam hydrolysis (likeGlu166 in class A b-lactamases). The deacylat-ing water molecule approaches the acyl-enzymespecies, anchored at the nucleophilic serine us-ing the carbamylated lysine as a chemical an-chor. Carbapenem hydrolyzing class D b-lacta-mases have evolved the ability to hydrolyzeimipenem, an important carbapenem in clini-cal use, by subtle structural changes in the activesite. These changes may contribute to tighterbinding of imipenem to the active site and re-moval of steric hindrances from the path of thedeacylating water molecule as was shown inclass A (Verma et al. 2011).

CLINICALLY IMPORTANT b-LACTAMASES

Class A—Serine Penicillinases TEM, SHV,and CTX-M and the CarbapenemasesKPC, etc.

Class A b-lactamases are often plasmid-encod-ed, but can also be located on the bacterial chro-mosome. For example, blaSHV-1 is a chromo-somal gene in Klebsiella pneumoniae, but mayalso be found on plasmics; penA from Burkhol-deria pseudomallei is chromosomally encoded(Papp-Wallace et al. 2015). In general, class Aenzymes are usually susceptible to inactivationby the clinically available b-lactamase inhibi-tors: clavulanate, sulbactam, tazobactam, andavibactam. TEM, SHV, and CTX-M b-lacta-

mases are mostly found in E. coli and Klebsiellaespp. Many class A b-lactamases have substrateprofiles that include expanded-spectrum ceph-alosporins, and these extended-spectrumb-lac-tamases (ESBLs) have been discussed extensive-ly (Paterson and Bonomo 2005; Perez et al.2007). The widespread distribution of CTX-Mb-lactamases, especially CTX-M-14 and CTX-M-15, in E. coli is responsible for the large partof the global advanced generation cephalospo-rin resistance seen in many clinical isolates.Other class A enzymes are encoded on inte-grons, for example, GES-1 from K. pneumoniaeand VEB-1 in P. aeruginosa and Acinetobacterbaumannii (Poirel et al. 2012).

Few Ambler class A b-lactamases show car-bapenem-hydrolyzing activity. The major classA carbapenemases include KPC, GES, Nmc-A/IMI, and SME b-lactamases, with SME carba-penemases identified, to date, only in Serratiamarcescens. With the notable exception of KPCsand GES, the clinical distribution of the typesof carbapenemases is relatively limited (Bush2010a).

Currently, most carbapenem resistanceamong Enterobacteriaceae in the United Statesis attributed to plasmid-mediated expression ofa KPC-type (K. pneumoniae carbapenemase)carbapenemase. KPC-producing Enterobacter-iaceae are considered endemic in many places,for example, in Greece, along with other car-bapenemases, specifically VIM-type metallo-b-lactamases. KPC b-lactamases efficientlyhydrolyze carbapenems as well as penicillins,

HNN

N

HN

101His

223HisHis162

Asp103

–O O

HSZn 2

Zn 1

OH

NH

N

Cys181

99HisN

N

H

Figure 2. The active site of a metallo-b-lactamase.

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cephalosporins, and aztreonam and are notovercome in vitro by clinically available b-lactamase inhibitors; in fact, clavulanic acid,sulbactam, and tazobactam are hydrolyzed. Avi-bactam inhibits KPC enzymes, but is hydro-lyzed slowly (Nguyen et al. 2016).

Carbapenem resistance secondary to KPCproduction was first described in a K. pneumo-niae isolate recovered in North Carolina in 1996(Yigit et al. 2003). The blaKPC gene has beenmapped to a highly conserved Tn3-based trans-poson, Tn4401, and different isoforms ofTn4401 are described. Plasmids carrying blaKPC

are of various sizes and many carry additionalgenes conferring resistance to fluoroquinolonesand aminoglycosides, thus limiting the anti-biotics available to treat infections with KPC-producing pathogens; blaKPC has rarely beenmapped to a chromosomal location (Chenet al. 2009).

A predominant strain of K. pneumoniae ap-pears responsible for outbreaks and the inter-national spread of KPC-producing K. pneumo-niae. A specific MLST sequence type (ST),ST258, has spread in the United States. Nowthere is widespread acknowledgment that twoclades of ST258 exist (Deleo et al. 2014). A sec-ond sequence type, ST14, was also common ininstitutions in the midwestern region of theUnited States. These findings implied that cer-tain strains of K. pneumoniae may be more aptto obtain and retain the blaKPC gene.

KPC-production can confer variable levelsof carbapenem resistance with reported mini-mum inhibitory concentrations (MICs) rang-ing from �1 mg/mL (susceptible) to �16 mg/mL. Analysis of isolates displaying high-levelcarbapenem resistance showed that increasedphenotypic resistance may be caused by in-creased blaKPC gene copy number or the lossof an outer membrane porin, Omp K35 and/or Omp K36. The highest level of imipenemresistance was seen with isolates lacking bothporins and with augmented KPC enzyme pro-duction (Chen et al. 2009).

Nmc-A (non-metallo-carbapenemase-A) isa chromosomal carbapenemase originally iso-lated from Enterobacter cloacae in France. Cur-rently, reports of this particular b-lactamase are

still rare. IMI-1 was initially recovered from thechromosome of an E. cloacae isolate in thesouthwestern United States. Avariant of IMI-1,IMI-2, has been identified on plasmids isolatedfrom environmental strains of Enterobacter ab-suriae in U.S. rivers.

SME-1 (S. marcescens enzyme) was original-ly identified in an isolate of S. marcescens from apatient in London in 1982. SME-2 and SME-3were subsequently isolated in the United States,Canada, and Switzerland. Chromosomally en-coded SME-type carbapenemases continue tobe isolated at a low frequency in North America(Naas et al. 2016). Although infrequent, it iscurrently recommended to screen for SME en-zymes in carbapenem-resistant S. marcescensisolates (Bush et al. 2013).

The GES-type (Guiana extended-spectrum)b-lactamases are acquired b-lactamases recov-ered from P. aeruginosa, Enterobacteriaceae, andA. baumannii. The genes encoding these b-lac-tamase have often, but not exclusively, beenidentified within class 1 integrons residing ontransferrable plasmids. GES-1 has a similar hy-drolysis profile to other ESBLs, although theyessentially spare monobactams. Several GES b-lactamases are described with six (i.e., GES-2,GES-4, GES-5, GES-6, GES-11, and GES-14),showing detectable carbapenemase activity inthe setting of amino acid substitutions at theiractive sites (specifically at residue 104 and 170).These GES-type carbapenemases have been de-scribed in Europe, South Africa, Asia, and theMiddle East.

Class B Metallo-b-Lactamases

Class B b-lactamases are bacterial enzymes thatdegrade b-lactam antibiotics with the help of ametal cofactor (divalent zinc in the naturalform). Class B enzymes are Zn2+-dependentb-lactamases that follow a different hydrolyticmechanism than the serineb-lactamases of clas-ses A, C, and D. Although catalyzing the sameoverall reaction as serine-b-lactamases, that is,breaking the amide bond, class B MBLs arestructurally and mechanistically unrelated tothe former enzymes, and their common func-tion apparently represents an example of con-



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vergent evolution of different protein lineageswithin the bacterial domain (Mojica et al. 2015).

Organisms producing MBLs usually showresistance to penicillins, cephalosporins, carba-penems, and the clinically availableb-lactamaseinhibitors. MBL bla genes are located on thechromosome, plasmid, and integrons. Until afew years ago, the clinically important class Benzymes included those found in the nosoco-mial pathogen Stenotrophomonas maltophilia.The rapid emergence of NDM MBLs in the En-terobacteriaceae has changed this (Walsh et al.2011).

Because of the dependence on Zn2+, cataly-sis is inhibited in the presence of metal-chelat-ing agents like EDTA. MBLs are not inhibited bythe presence of commercially available b-lacta-mase inhibitors; however, hydrolytic stability ofmonobactams (i.e., aztreonam) leading to sus-ceptibility of the producing organism appearsto be preserved in the absence of concomitantexpression of other resistance mechanisms (e.g.,ESBL production). The more geographicallywidespread MBLs include IMP, VIM, andNDM (Mojica et al. 2015).

NDM-1 (New Delhi MBL) was first identi-fied in 2008. NDM-1 was first discovered inSweden in a patient of Indian descent previouslyhospitalized in India (Yong et al. 2009; Kumar-asamy et al. 2010). The patient was colonizedwith a K. pneumoniae strain and an E. coli–car-rying blaNDM-1 on transferrable plasmids. Inthe United Kingdom, an increase in the numberof clinical isolates of carbapenem-resistant En-terobacteriaceae was also seen in both 2008 and2009. A U.K. reference laboratory reported thatat least 17 of 29 patients found to be harboringNDM-1 expressing Enterobacteriaceae had ahistory of recent travel to the Indian subconti-nent with the majority having been hospitalizedin those countries.

NDM-1 shares the most homology (32.4%)with VIM-1 and VIM-2 (Yong et al. 2009). It isa 28-kDa monomeric protein that shows tightbinding to both penicillins and cephalosporins.Binding to carbapenems does not appear to beas avid as other MBLs, but catalytic efficienciesappear to be similar. Using ampicillin as a sub-strate allowed for detailed characterization of

the interactions between NDM’s active siteand b-lactams as well as improved evaluationof MBLs unique mechanism of b-lactam hy-drolysis. More recent crystal structures ofNDM-1 reveal the molecular details of howcarbapenem antibiotics are recognized by di-zinc-containing metallo-b-lactamases (King etal. 2012).

Because of its rapid international dissemi-nation and its ability to be expressed by numer-ous Gram-negative pathogens, NDM is poisedto become the most commonly isolated and dis-tributed carbapenemase worldwide. Initial re-ports frequently showed an epidemiologic linkto the Indian subcontinent where these MBLsare endemic. Indeed, retrospective analyses ofstored isolates suggest that NDM-1 may havebeen circulating in the subcontinent as early as2006. Despite initial controversy, the Balkansmay be another area of endemicity for NDM-1. Sporadic recovery of NDM-1 in the MiddleEast suggests that this region may be an addi-tional reservoir.

Like KPCs, the conveniences of internation-al travel and medical tourism have quickly pro-pelled this relatively novel MBL into a formid-able public health threat. Gram-negative bacilliharboring blaNDM have been identified world-wide (Nordmann et al. 2011).

European reports suggest that horizontaltransfer of blaNDM-1 exists within hospitals out-side of endemic areas. Of overwhelming concernare the reported cases without specific contactwith the healthcare system locally or in endemicareas, suggesting autochthonous acquisition.

Surveillance of public water supplies in In-dia indicates that exposure to NDM-1 may beenvironmental. Walsh et al. (2011) analyzedsamples of public tap water and seepage waterfrom sites around New Delhi. The results weredisheartening in that blaNDM-1 was detectedby polymerase chain reaction (PCR) in 4% ofdrinking water samples and 30% of seepagesamples. In this survey, carriage of blaNDM-1

was noted in 11 species of bacteria not previ-ously described, including virulent ones likeShigella boydii and Vibrio cholerae.

The rapid spread of NDM-1 highlights thefluidity and rapidity of gene transfer among

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bacterial species. Although blaNDM-1 was initial-ly and repeatedly mapped to plasmids isolatedfrom carbapenem-resistant E. coli and K. pneu-moniae, reports of both plasmid and chromo-somal expression of blaNDM-1 has been noted inother species of Enterobacteriaceae as well asAcinetobacter spp. and P. aeruginosa (Jones etal. 2014). Recently, bacteremia with an NDM-1 expressing V. cholerae has been described in apatient previously hospitalized in India colo-nized with a variety of Enterobacteriaceae pre-viously known to be capable of carrying plas-mids with blaNDM-1 (Darley et al. 2012).

In contrast to KPCs, the presence of a dom-inant clone among blaNDM-1-carrying isolatesremains elusive. NDM-1 expression in E. colihas been noted among sequence types previous-ly associated with the successful disseminationof other b-lactamases, including ST101 andST131. Mushtaq et al. analyzed a relatively largegroup of blaNDM-1 expressing E. coli from theUnited Kingdom, Pakistan, and India to poten-tially identify a predominant strain responsiblefor the rapid and successful spread of NDM-1(Mushtaq et al. 2011). The most frequent se-quence type identified was ST101. Anotherstudy examining a collection of carbapenem-resistant Enterobacteriaceae from India showsthe diversity of strains capable of harboringblaNDM-1. Carriage of blaNDM-1 was confirmedin ten different sequence types of K. pneumo-niae and five sequence types of E. coli. This mul-tiplicity was confirmed in a study looking at acollection of blaNDM-1 expressing Enterobacter-iaceae from around the world (Poirel et al.2011). Of most concern is that NDM-1 hasbeen identified in E. coli ST131, the strain ofE. coli credited with the global propagation ofCTX-M-15 ESBLs. Similar to KPCs, NDM-1expression portends variable levels of carbape-nem resistance, and there is often concomitantcarriage of a myriad of resistance determinantsincluding other b-lactamases and carbapene-mases as well as genes associated with resistanceto fluoroquinolones and aminoglycosides.

To date, NDM-1 remains the most commonNDM variant isolated. It is currently believedthat blaNDM-1 is a chimeric gene that may haveevolved from A. baumannii. Contributing to this

theory is the presence of complete or variationsof the insertion sequence, ISAba125, upstreamof the blaNDM-1 gene in both Enterobacteriaceaeand A. baumannii. This insertion sequence hasprimarily been found in A. baumannii.

A recent evaluation of the genetic constructassociated with blaNDM-1 has led to the discoveryof a new bleomycin resistance protein, BRPMBL.Evaluation of 23 isolates of blaNDM-1/2 harbor-ing Enterobacteriaceae and A. baumannii notedthat the overwhelming majority of them pos-sessed a novel bleomycin resistance gene, bleMBL.Coexpression of blaNDM-1 and bleMBL appears tobe mediated by a common promoter (PNDM-1),which includes portions of ISAba125. It is pos-tulated that BRPMBL expression may contributesome sort of selective advantage allowing NDM-1 to persist in the environment.

A contemporary evaluation of recently re-covered NDM-1 producing A. baumannii iso-lates from Europe shows that blaNDM-1 andblaNDM-2 genes are situated on the same chro-mosomally located transposon, Tn125. Dissem-ination of blaNDM in A. baumannii seems becaused by different strains carrying Tn125 orderivatives of Tn125, rather than plasmid-me-diated or clonal (Poirel et al. 2012).

Before the description of NDM-1, frequent-ly detected MBLs include IMP-type (imipen-em-resistant) and VIM-type (Verona integron-encoded MBL) with VIM-2 being the mostprevalent. These MBLs are embedded within avariety of genetic structures, most commonlyintegrons. When these integrons are associatedwith transposons or plasmids, they can readilybe transferred among species (Mojica et al.2015).

A more commonly recovered MBL is theVIM-type enzyme. VIM-1 was first describedin Italy in 1997 in P. aeruginosa. VIM-2 wasnext discovered in southern France in P. aerugi-nosa cultured from a neutropenic patient in1996. Although originally thought to be limitedto nonfermenting Gram-negative bacilli, VIM-type MBLs are being increasingly identified inEnterobacteriaceae as well. Many variants ofVIM have been described with VIM-2 beingthe most common MBL recovered worldwide(Mojica et al. 2015).



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Other more geographically restricted MBLsinclude (1) SPM-1, Sao Paulo MBL, which hasbeen associated with hospital outbreaks in Bra-zil; (2) GIM-1, German imipenemase, isolatedin carbapenem-resistant P. aeruginosa isolates inGermany; (3) SIM-1, Seoul imipenemase, iso-lated from A. baumannii isolates in Korea; (4)KHM-1, Kyorin Health Science MBL, isolatedfrom a Citrobacter freundii isolate in Japan (Se-kiguchi et al. 2008); (5) AIM-1, Australian imi-penemase, isolated from P. aeruginosa in Aus-tralia; (6) DIM-1, Dutch imipenemase, isolatedfrom a clinical Pseudomonas stutzeri isolatein the Netherlands; (7) SMB-1, S. marcescensMBL, in S. marcescens in Japan; (8) TMB-1,Tripoli MBL, in Achromobacter xylosoxidans inLibya; and (9) FIM-1, Florence imipenemase,from a clinical isolate of P. aeruginosa in Italy.With the notable exception of SPM-1, whichhas been introduced into European hospitalsby a Brazilian pediatric patient, these MBLshave remained confined to their countries/cit-ies of origin.

Class C Cephalosporinases

Class C enzymes include the AmpCb-lactamas-es, which are usually encoded by bla genes lo-cated on the bacterial chromosome, althoughplasmid-borne AmpC enzymes have becomemore prevalent. Organisms expressing theAmpC b-lactamase are typically resistant topenicillins, b-lactamase inhibitors (clavulanateand tazobactam), and most cephalosporins in-cluding cefoxitin, cefotetan, ceftriaxone, andcefotaxime. AmpC enzymes poorly hydrolyzecefepime, an expanded-spectrum cephalospo-rin, and are readily inactivated by carbapenems.Notably, AmpC cephalosporinases are very sus-ceptible to inactivation by avibactam. These en-zymes are absent in K. pneumoniae, Klebsiellaoxytoca, Proteus mirabilis, and Salmonella spp.as well as other bacteria (Perez et al. 2016).

Benzylpenicillin, ampicillin, amoxicillin,and cephalosporins such as cefazolin and ceph-alothin are very good inducers and good sub-strates for AmpC b-lactamase. Cefoxitin andimipenem are also strong inducers but aremuch more stable to hydrolysis. Cefotaxime,

ceftriaxone, ceftazidime, cefepime, cefuroxime,piperacillin, and aztreonam are weak inducersand weak substrates but can be hydrolyzed ifenough enzyme is expressed. Consequently,MICs of weakly inducing oxyimino-b-lactamsare dramatically increased with AmpC hyper-production. Conversely, MICs of agents thatare strong inducers show little change with reg-ulatory mutations because the level of inducedampC expression is already high.

Someb-lactamase inhibitors are also induc-ers, especially clavulanate, which has little in-hibitory effect on AmpC b-lactamase activi-ty, but can paradoxically appear to increaseAmpC-mediated resistance in an inducible or-ganism. The inducing effect of clavulanate isespecially important for P. aeruginosa, in whichclinically achieved concentrations of clavulanateby inducing AmpC expression have been shownto antagonize the antibacterial activity of ticar-cillin (Papp-Wallace et al. 2014).

Production of AmpC enzymes in clinicallyimportant Gram-negative bacteria is normallyat a low level (“repressed”), but can be “dere-pressed” by induction with certain b-lactams,particularly cefoxitin and imipenem. Sulbac-tam, but not tazobactam, is also a good inducerof AmpC b-lactamases. The genetic under-pinnings of this regulation have been the subjectof intense investigation, but are not the subjectof this review. Members of the Enterobacteria-ceae family, such as Citrobacter, Salmonella, andShigella, are clinically relevant producers ofAmpC enzymes that resist inhibition by clavu-lanate and sulbactam (Bauvois and Wouters2007).

Class D Serine Oxacillinases

Class D b-lactamases were initially categorizedas “oxacillinases” because of their ability to hy-drolyze oxacillin at a rate of at least 50% that ofbenzylpenicillin, in contrast to the relativelyslow hydrolysis of oxacillin by classes A and C.Different enzymes in this diverse class can alsoconfer resistance to penicillins, cephalosporins,extended-spectrum cephalosporins (OXA-typeESBLs), and carbapenems (OXA-type carbape-nemases) (Leonard et al. 2013).

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Oxacillinases comprise a heterogeneousgroup of class D b-lactamases, which are ableto hydrolyze amino- and carboxypenicillins.The majority of class D b-lactamases are notinhibited by commerciallyavailableb-lactamaseinhibitors but are inhibited in vitro by NaCl.OXA enzymes are insensitive to inhibition byclavulanate, sulbactam, and tazobactam, withsome exceptions; for example, OXA-2 andOXA-32 are inhibited by tazobactam, but notsulbactam and clavulanate; OXA-53 is inhibitedby clavulanate. Interestingly, sodium chloride atconcentrations .50–75 mM inhibits some car-bapenem-hydrolyzing oxacillinases (e.g., OXA-25 and OXA-26). Site-directed mutagenesisstudies suggest that susceptibility to inhibitionby sodium chloride is related to the presence of aTyr residue at position 144, which may facilitatesodium chloride binding better than the Pheresidue found in resistant oxacillinases. Exam-ples of OXA enzymes include those rapidlyemerging in A. baumannii (e.g., OXA-23) andP. aeruginosa (e.g., OXA-50).

Carbapenem-Hydrolyzing Class Db-Lactamases

More than 490 types of oxacillinases are report-ed with a minority showing low levels ofcarbapenem-hydrolyzing activity (lahey.org/Studies/other.asp#table1). This select group ofenzymes is also referred to as the carbapenem-hydrolyzing class D b-lactamases (CHDLs).CHDLs have been identified most frequentlyin Acinetobacter spp.; however, there has beenincreasing isolation among Enterobacteriaceae,specifically OXA-48 (Patel and Bonomo 2013).

With the exception of OXA-163, CHDLsefficiently inactivate penicillins, early cephalo-sporins, and b-lactam/b-lactamase inhibitorcombinations, but spare expanded-spectrumcephalosporins. Carbapenem hydrolysis effi-ciency is lower than that of other carbapene-mases, including the MBLs, and often additionalresistance mechanisms are expressed in organ-isms showing higher levels of phenotypic car-bapenem resistance. These include expressionof other carbapenemases, alterations in outermembrane proteins (e.g., CarO, OmpK36), in-

creased transcription mediated by IS elementsfunctioning as promoters, increased gene copynumber, and amplified drug efflux. Many sub-groups of CHDLs have been described. Wewill focus on those found in A. baumannii andEnterobacteriaceae: OXA-23 and -27; OXA-24/40, -25, and -26; OXA-48 variants; OXA-51,-66, and -69; OXA-58 and OXA-143.

CHDLs can be intrinsic or acquired (Pateland Bonomo 2013). A. baumannii does havenaturally occurring but variably expressed chro-mosomal CHDLs, OXA-51, OXA-66, and OXA-69. For the most part, in isolation the phenotypiccarbapenem resistance associated with these ox-acillinases is low. However, levels of carbapenemresistance appear to be increased in the presenceof specific insertion sequences promoting geneexpression. Additional resistance to extended-spectrum cephalosporins can be seen in thesetting of coexpression of ESBLs and/or othercarbapenemases (Leonard et al. 2013).

The first reported acquired oxacillinase withappreciable carbapenem-hydrolyzing activitywas OXA-23. OXA-23, or ARI-1, was identifiedfrom an A. baumannii isolate in Scotland in1993, although the isolate was first recoveredin 1985 (Paton et al. 1993). Subsequently,OXA-23 expression has been reported world-wide and both plasmid and chromosomal car-riage of blaOXA-23 are described. The OXA-23group includes OXA-27, found in a singleA. baumannii isolate from Singapore. With theexception of an isolate of P. mirabilis identifiedin France in 2002, this group of b-lactamaseshas been exclusively recovered from Acineto-bacter species. Increased expression of OXA-23has been associated with the presence of up-stream insertion sequences (e.g., ISAba1 andISAba4) acting as strong promoters.

Another group of CHDLs includes OXA-24/40, OXA-25, and OXA-26. OXA-24 andOXA-40 differ by a few amino acid substitu-tions, and OXA-25 and OXA-26 are point mu-tation derivatives of OXA-40. Although primar-ily linked with clonal outbreaks in Spain andPortugal, OXA-24/40 b-lactamases has beenisolated in other European countries and theUnited States. OXA-40 was in fact the firstCHDL documented in the United States.



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http://lahey.org/Studies/other.asp%23table1





OXA-58 has also only been detected in Acineto-bacter spp. Initially identified in France, OXA-58has been associated with institutional outbreaksand has been recovered from clinical isolates ofA. baumannii worldwide (Leonard et al. 2013).

As civilian and military personnel beganreturning from Afghanistan and the MiddleEast, practitioners noted increasing recovery ofA. baumannii from skin and soft tissue infec-tions. Drug resistance was associated with ex-pression of both OXA-23 and OXA-58. Manyisolates carrying the blaOXA-58 gene concurr-ently carry insertion sequences (e.g., ISaba1,ISAba2, or ISAba3) associated with increasedcarbapenemase production and, thus, higherlevels of carbapenem resistance. In one report,increased gene copy number was also associatedwith a higher level of enzyme production andincreased phenotypic carbapenem resistance(Hujer et al. 2006).

Spread of OXA-type carbapenemasesamong A. baumannii appears to be clonal, andin-depth reviews of the molecular epidemiologyand successful dissemination of these clones hasbeen published. Two MLST schemes with threeloci in common exist for A. baumannii—thePubMLST scheme and the Pasteur scheme.Both schemes assign different sequence typesinto clonal complexes. Sequence types and clon-al complexes (CCs) from both schemes can befurther categorized into the international (Eu-ropean) clones I, II, and III. It should be noted,however, that the molecular taxonomyof A. bau-mannii continues to evolve. OXA-23 producingA. baumannii predominantly belong to inter-national clones I and II with a notable propor-tion being part of CC92 (PubMLST). Similarly,A. baumannii isolates associated with epidemicspread of OXA-24/40 in Portugal and Spainappear to be incorporated in international cloneII and ST56 (PubMLST). OXA-58 expressingA. baumannii have been associated with inter-national clones I, II, and III and a variety ofunrelated sequence types (Hujer et al. 2006).

OXA-48 was originally identified in a car-bapenem-resistant isolate of K. pneumoniae inTurkey. Early reports suggested that this enzymewas geographically restricted to Turkey. In thepast few years, however, the enzyme has been

recovered from variety of Enterobacteriaceaeand has successfully circulated outside of Turkeywith reports of isolation in the Middle East,North Africa, Europe, and, most recently, theUnited States. The Middle East and North Africamay be secondary reservoirs for these CHDLs.Indeed, the introduction of OXA-48 expressingEnterobacteriaceae in some countries has beenfrom patients from the Middle East or NorthernAfrica. In the United States, the first clinical cas-es were associated with ST199 and ST43.

At least six OXA-48 variants (e.g., OXA-48,OXA-162, OXA-163, OXA-181, OXA-204, andOXA-232) have been identified. OXA-48 is byfar the most globally dispersed and its epidemi-ology has been recently reviewed. Unlike KPCsand NDM-1, which have been associated with avariety of plasmids, a single 62-kb self-conjuga-tive IncL/M-type plasmid has contributed to alarge proportion of the distribution of blaOXA-48

in Europe. Sequencing of this plasmid (pOXA-48a) notes that blaOXA-48 had been integratedthrough the acquisition of a Tn1999 compositetransposon. blaOXA-48 appears to be associatedwith a specific insertion sequence, IS1999. Avariant of Tn1999, Tn1999.2, has been identi-fied among isolates from Turkey and Europe.Tn1999.2 harbors an IS1R element within theIS1999. OXA-48 appears to have the highest af-finity for imipenem of the CHDLs specificallythose harboring blaOXA-48 within a Tn1999.2composite transposon. Three isoforms of theTn1999 transposon have been described.

Although much of the spread of OXA-48is attributed to a specific plasmid, outbreakevaluations show that a variety of strains havecontributed to dissemination of this emergingcarbapenemase in K. pneumoniae. The sameK. pneumoniae sequence type, ST395, harbor-ing blaOXA-48 was identified in Morocco, France,and the Netherlands. ST353 was associated withan outbreak of OXA-48 producing K. pneumo-niae in London (Woodford et al. 2011) andST221 with an outbreak of OXA-48 in Ireland.OXA-48 production in K. pneumoniae, likeKPC-expressing K. pneumoniae, has also beenassociated with ST14 (Poirel et al. 2004a) anda recent outbreak in Greece was associatedwith ST11. blaOXA-48 is remarkably similar to

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blaOXA-54, a b-lactamase gene intrinsic to She-wanella oneidensis (Poirel et al. 2004b). Shewa-nella spp. are relatively ubiquitous waterborneGram-negative bacilli and are proving to be apotential environmental reservoir for OXA-48like carbapenemases as well as other resistancedeterminants.

OXA-163, a single amino acid variant ofOXA-48, was identified in isolates of K. pneu-moniae and E. cloacae from Argentina and isunique in that it has activity against expand-ed-spectrum cephalosporins (Poirel et al.2011). OXA-163 also has been identified inEgypt, which has a relatively prevalence ofOXA-48, in patients without epidemiologiclinks to Argentina (Abdelaziz et al. 2012).

OXA-181 was initially identified among car-bapenem-resistant Enterobacteriaceae collectedfrom India (Castanheira et al. 2011). OXA-181differs from OXA-48 by four amino acids; how-ever, it appears to be nestled in an entirely dif-ferent genetic platform. The blaOXA-181 gene hasbeen mapped to a different group of plasmids,the ColE family, and has been associated with analternative insertion sequence, ISEcp1. The lat-ter insertion sequence has been associated withthe acquisition of other b-lactamases includingCTX-M-like ESBLs. Like, OXA-48, it appearsthat OXA-181 may have evolved from a water-borne environmental species Shewanella xiame-nensis. OXA-204 differs from OXA-48 by twoamino acid substitution. It was recently identi-fied in a clinical K. pneumoniae isolate fromTunisia. Its genetic construct appears to be sim-ilar to that of OXA-181. OXA-232 was recentlyidentified among K. pneumoniae isolates inFrance. OXA-143 is a novel plasmid-borne car-bapenem-hydrolyzing oxacillinase recoveredfrom clinical A. baumannii isolates in Brazil.Information regarding its significance and prev-alence continues to evolve.

CONCLUSIONS

This review briefly recapitulates our under-standing of the history of this complex familyof enzymes, reviews the mechanisms by whichthese hydrolases inactivate b-lactams, andhighlights the current challenges that threaten

our b-lactam armamentarium, especially thecarbapenemases KPCs, NDMs, and OXAs. In80 years, we have learned that the constant evo-lution of substrate specificity meets each newb-lactam introduced. The “long view” predictsthese enzymes will continue to evolve in novelforms. Perhaps, we will eventually understandwhy this occurs and how to prevent this. Thefuture of research in this arena needs to beopen and mindful to new approaches. Wehave still not defined all the correlates ofactivity and resistance or have answered whynovel structural variants emerge. The currentchallenges will have to be faced with new tech-nologies.

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published online October 14, 2016Cold Spring Harb Perspect Med Robert A. Bonomo -Lactamases: A Focus on Current Challengesβ

Subject Collection Antibiotics and Antibiotic Resistance

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The Whys and Wherefores of Antibiotic

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Mode of Action and Resistance−−Pleuromutilins: Potent Drugs for Resistant Bugs

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-Lactamases: A Focus on Current ChallengesβRobert A. Bonomo

Appropriate Targets for Antibacterial DrugsLynn L. Silver Mechanism of Action and Resistance

Approved Glycopeptide Antibacterial Drugs:

et al.Daina Zeng, Dmitri Debabov, Theresa L. Hartsell,

of ResistancePleuromutilins: Mode of Action and Mechanisms Lincosamides, Streptogramins, Phenicols, and

et al.Stefan Schwarz, Jianzhong Shen, Kristina Kadlec,

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AriasWilliam R. Miller, Arnold S. Bayer and Cesar A.

Health PathogensResistance to Macrolide Antibiotics in Public

al.Corey Fyfe, Trudy H. Grossman, Kathy Kerstein, et

ResistancePolymyxin: Alternative Mechanisms of Action and

al.Michael J. Trimble, Patrik Mlynárcik, Milan Kolár, et

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through Resistance to the Next GenerationAntibacterial Antifolates: From Development

AndersonAlexavier Estrada, Dennis L. Wright and Amy C.

Overview-Lactamase Inhibitors: Anβ-Lactams and β

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Lipopolysaccharide Biosynthetic Enzyme LpxCAntibacterial Drug Discovery Targeting the

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Rifamycins, Alone and in CombinationDavid M. Rothstein

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