extended-spectrum -lactamases in the 21st century ... · ability to hydrolyze speciﬁc...

CLINICAL MICROBIOLOGY REVIEWS,0893-8512/01/$04.00�0 DOI: 10.1128/CMR.14.4.933–951.2001

Oct. 2001, p. 933–951 Vol. 14, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Extended-Spectrum �-Lactamases in the 21st Century:Characterization, Epidemiology, and Detection

of This Important Resistance ThreatPATRICIA A. BRADFORD*

Wyeth-Ayerst Research, Pearl River, New York

INTRODUCTION AND HISTORY..........................................................................................................................933CHARACTERIZATION OF ESBLs .........................................................................................................................934

Functional and Molecular Grouping ...................................................................................................................934Susceptibility and Biochemical Characteristics .................................................................................................934

TYPES OF ESBLs ......................................................................................................................................................934TEM..........................................................................................................................................................................934Inhibitor-Resistant �-Lactamases ........................................................................................................................934SHV...........................................................................................................................................................................936CTX-M......................................................................................................................................................................936OXA ..........................................................................................................................................................................939Other ESBLs ...........................................................................................................................................................939

ESBL DETECTION METHODS..............................................................................................................................940Clinical Microbiology Techniques ........................................................................................................................941Molecular Detection Methods...............................................................................................................................944Medical Significance of Detection of ESBLs.......................................................................................................944

EPIDEMIOLOGY.......................................................................................................................................................945CONCLUSION............................................................................................................................................................946ACKNOWLEDGMENTS ...........................................................................................................................................946REFERENCES ............................................................................................................................................................946

INTRODUCTION AND HISTORY

Emergence of resistance to �-lactam antibiotics began evenbefore the first �-lactam, penicillin, was developed. The first�-lactamase was identified in Escherichia coli prior to the re-lease of penicillin for use in medical practice (1). The age ofpenicillin saw the rapid emergence of resistance in Staphylo-coccus aureus due to a plasmid-encoded penicillinase. This�-lactamase quickly spread to most clinical isolates of S. aureusas well as other species of staphylococci.

Many genera of gram-negative bacteria possess a naturallyoccurring, chromosomally mediated �-lactamase. These en-zymes are thought to have evolved from penicillin-bindingproteins, with which they show some sequence homology. Thisdevelopment was likely due to the selective pressure exerted by�-lactam-producing soil organisms found in the environment(61). The first plasmid-mediated �-lactamase in gram-nega-tives, TEM-1, was described in the early 1960s (48). TheTEM-1 enzyme was originally found in a single strain of E. coliisolated from a blood culture from a patient named Temonierain Greece, hence the designation TEM (96). Being plasmidand transposon mediated has facilitated the spread of TEM-1to other species of bacteria. Within a few years after its firstisolation, the TEM-1 �-lactamase spread worldwide and is nowfound in many different species of members of the familyEnterobacteriaceae, Pseudomonas aeruginosa, Haemophilus in-

fluenzae, and Neisseria gonorrhoeae. Another common plasmid-mediated �-lactamase found in Klebsiella pneumoniae and E.coli is SHV-1 (for sulphydryl variable). The SHV-1 �-lacta-mase is chromosomally encoded in the majority of isolates ofK. pneumoniae but is usually plasmid mediated in E. coli.

Over the last 20 years, many new �-lactam antibiotics havebeen developed that were specifically designed to be resistantto the hydrolytic action of �-lactamases. However, with eachnew class that has been used to treat patients, new �-lactama-ses emerged that caused resistance to that class of drug. Pre-sumably, the selective pressure of the use and overuse of newantibiotics in the treatment of patients has selected for newvariants of �-lactamase. One of these new classes was theoxyimino-cephalosporins, which became widely used for thetreatment of serious infections due to gram-negative bacteriain the 1980s.

Not surprisingly, resistance to these expanded-spectrum�-lactam antibiotics due to �-lactamases emerged quickly. Thefirst of these enzymes capable of hydrolyzing the newer �-lac-tams, SHV-2, was found in a single strain of Klebsiella ozaenaeisolated in Germany (81). Because of their increased spectrumof activity, especially against the oxyimino-cephalosporins,these enzymes were called extended-spectrum �-lactamases(ESBLs). Today, over 150 different ESBLs have been de-scribed. These �-lactamases have been found worldwide inmany different genera of Enterobacteriaceae and P. aeruginosa.This review will focus on the characterization of ESBLs, theimportance of detection of these enzymes, and their epidemi-ology.

* Mailing address: Wyeth-Ayerst Research, 401 N. Middletown Rd.,Pearl River, NY 10965. Phone: (845) 732-4386. Fax: (845) 732-5671.E-mail: [email protected].

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CHARACTERIZATION OF ESBLs

Functional and Molecular Grouping

The majority of ESBLs contain a serine at the active site andbelong to Ambler’s molecular class A (4). Class A enzymes arecharacterized by an active-site serine, a molecular mass ofapproximately 29,000 Da, and the preferential hydrolysis ofpenicillins (95). Class A �-lactamases include such enzymes asTEM-1, SHV-1, and the penicillinase found in S. aureus. Themolecular classification scheme is still used to characterize�-lactamases; however, it does not sufficiently differentiate themany different types of class A enzymes. The classificationscheme of Richmond and Sykes was based on the substrateprofile and the location of the gene encoding the �-lactamase(145). This classification scheme was developed before ESBLsarose, and it did not allow for the differentiation between theoriginal TEM and SHV enzymes and their ESBL derivatives.More recently, a classification scheme was devised by Bush,Jacoby, and Medeiros that uses the biochemical properties ofthe enzyme plus the molecular structure and nucleotide se-quence of the genes to place �-lactamases into functionalgroups (32). Using this scheme, ESBLs are defined as �-lacta-mases capable of hydrolyzing oximino-cephalosporins that areinhibited by clavulanic acid and are placed into functionalgroup 2be (32).

Susceptibility and Biochemical Characteristics

ESBLs contain a number of mutations that allow them tohydrolyze expanded-spectrum �-lactam antibiotics. WhileTEM- and SHV-type ESBLs retain their ability to hydrolyzepenicillins, they are not catalytically as efficient as the parentenzymes (33). In addition, the expansion of the active site thatallows the increased activity against expanded-spectrum ceph-alosporins may also result in the increased susceptibility ofESBLs to �-lactamase inhibitors (74). ESBLs are not activeagainst cephamycins, and most strains expressing ESBLs aresusceptible to cefoxitin and cefotetan. However, it has beenreported that ESBL-producing strains can become resistant tocephamycins due to the loss of an outer membrane porinprotein (92, 121, 181).

TYPES OF ESBLs

Most ESBLs are derivatives of TEM or SHV enzymes (32,74). There are now �90 TEM-type �-lactamases and �25SHV-type enzymes (for amino acid sequences for TEM, SHV,and OXA extended-spectrum and inhibitor-resistant �-lacta-mases, see http://www.lahey.org/studies/webt.htm). With bothof these groups of enzymes, a few point mutations at selectedloci within the gene give rise to the extended-spectrum phe-notype. TEM- and SHV-type ESBLs are most often found inE. coli and K. pneumoniae; however, they have also been foundin Proteus spp., Providencia spp., and other genera of Entero-bacteriaceae.

TEM

TEM-1 is the most commonly encountered �-lactamase ingram-negative bacteria. Up to 90% of ampicillin resistance inE. coli is due to the production of TEM-1 (85). This enzyme is

also responsible for the ampicillin and penicillin resistance thatis seen in H. influenzae and N. gonorrhoeae in increasing num-bers. TEM-1 is able to hydrolyze penicillins and early cepha-losporins such as cephalothin and cephaloridine. TEM-2, thefirst derivative of TEM-1, had a single amino acid substitutionfrom the original �-lactamase (10). This caused a shift in theisoelectric point from a pI of 5.4 to 5.6, but it did not changethe substrate profile. TEM-3, originally reported in 1989, wasthe first TEM-type �-lactamase that displayed the ESBLphenotype (157). In the years since that first report, over 90additional TEM derivatives have been described (for aminoacid sequences for TEM, SHV, and OXA extended-spectrumand inhibitor-resistant �-lactamases, see http://www.lahey.org/studies/webt.htm). Some of these �-lactamases are inhibitor-resistant enzymes, but the majority of the new derivatives areESBLs.

As shown in Fig. 1, the amino acid substitutions that occurwithin the TEM enzyme occur at a limited number of posi-tions. The combinations of these amino acid changes result invarious subtle alterations in the ESBL phenotypes, such as theability to hydrolyze specific oxyimino-cephalosporins such asceftazidime and cefotaxime, or a change in their isoelectricpoints, which can range from a pI of 5.2 to 6.5 (Table 1). Anumber of amino acid residues are especially important forproducing the ESBL phenotype when substitutions occur atthat position. They include glutamate to lysine at position 104,arginine to either serine or histidine at position 164, glycine toserine at position 238, and glutamate to lysine at position 240(Fig. 1). In addition to �-lactamases TEM-1 through TEM-92shown in Fig. 1 and Table 1, there has been a report of anaturally occurring TEM-like enzyme, TEM-AQ, that con-tained a number of amino acid substitutions and one aminoacid deletion that have not been noted in other TEM enzymes(127).

It is interesting that laboratory mutants of TEM-1 that con-tain mutations at positions other than the ones described innature have been constructed (18, 130, 180, 182). It has beensuggested that the naturally occurring TEM-type ESBLs arethe result of fluctuating selective pressure from several �-lac-tams within a given institution rather than selection with asingle agent (18). Although TEM-type �-lactamases are mostoften found in E. coli and K. pneumoniae, they are also foundin other species of gram-negative bacteria with increasing fre-quency. TEM-type ESBLs have been reported in genera ofEnterobacteriaceae such as Enterobacter aerogenes, Morganellamorganii, Proteus mirabilis, Proteus rettgeri, and Salmonella spp.(19, 91, 101, 120, 128, 166). Furthermore, TEM-type ESBLshave been found in non-Enterobacteriaceae gram-negative bac-teria. The TEM-42 �-lactamase was found in a strain of P.aeruginosa (103). Additionally, a recent report found theTEM-17 �-lactamase being expressed from a plasmid in ablood culture isolate of Capnocytophaga ochracea (146).

Inhibitor-Resistant �-Lactamases

Although the inhibitor-resistant �-lactamases are not ESBLs,they are often discussed with ESBLs because they are alsoderivatives of the classical TEM- or SHV-type enzymes. In theearly 1990s �-lactamases that were resistant to inhibition byclavulanic acid were discovered. Nucleotide sequencing re-

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vealed that these enzymes were variants of the TEM-1 orTEM-2 �-lactamase. These enzymes were at first given thedesignation IRT for inhibitor-resistant TEM �-lactamase;however, all have subsequently been renamed with numericalTEM designations. There are at least 19 distinct inhibitor-resistant TEM �-lactamases (for amino acid sequences forTEM, SHV and OXA extended-spectrum and inhibitor resis-tant �-lactamases, see http://www.lahey.org/studies/webt.htm).Inhibitor-resistant TEM �-lactamases have been found mainlyin clinical isolates of E. coli, but also some strains of K. pneu-moniae, Klebsiella oxytoca, P. mirabilis, and Citrobacter freundii(31, 83). Although the inhibitor-resistant TEM variants areresistant to inhibition by clavulanic acid and sulbactam,thereby showing clinical resistance to the �-lactam–�-lacta-mase inhibitor combinations of amoxicillin-clavulanate, ticar-cillin-clavulanate, and ampicillin-sulbactam, they remain sus-ceptible to inhibition by tazobactam and subsequently thecombination of piperacillin and tazobactam (23, 37). To date,

these �-lactamases have primarily been detected in France anda few other locations within Europe (37). In a recent survey ofamoxicillin-clavulanate-resistant E. coli in a hospital in France,Leflon-Guibout et al. found that up to 41% of these isolatesproduced inhibitor-resistant TEM variants (82). Althoughthese enzymes have not yet been reported in isolates originat-ing in the United States, it is likely that they will eventually bedetected here as well.

As shown in Fig. 2, point mutations that lead to the inhibi-tor-resistant phenotype occur at a few specific amino acidresidues within the structural gene for the TEM enzyme, Met-69, Arg-244, Arg-275, and Asn-276 (16, 66, 191). The sites ofthese amino acid substitutions are distinct from those that leadto the ESBL phenotype. Laboratory mutants that containamino acid substitutions which are common to both the IRTand the ESBL phenotype have been constructed (159). Thesestrains were found to possess either the ESBL or IRT pheno-type, but not both. However, the TEM-50 enzyme, which had

FIG. 1. Amino acid substitutions in TEM ESBL derivatives. The amino acids listed within the grey bar are those found in the structural geneof the TEM-1 �-lactamase (162). The amino acid numbering is according to the scheme of Ambler et al. (5). Substitutions found in TEM-typeESBL derivatives are shown under the amino acids of TEM-1. TEM-type variants may contain more than one amino acid substitution. �, TEM-2is not an ESBL but is included in the figure as a derivative of TEM-1. The Gln39Lys substitution does not contribute to the ESBL phenotype, buta number of ESBLs are derived from TEM-2. ��, TEM-50 and TEM-68 contain amino acid substitutions that are common to both the ESBL andthe IRT phenotypes. Only the amino acid substitutions that are common to TEM-type ESBLs are shown in this figure.

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amino acid substitutions common to both the ESBL and in-hibitor-resistant TEMs, was recently identified. This enzymewas resistant to inhibition by clavulanate, but it also conferreda slight resistance to the expanded-spectrum cephalosporins(154). This could indicate the possibility of a new group of�-lactamases with a complex phenotype sharing some charac-teristics of ESBLs and inhibitor-resistant enzymes. In additionto the variants of TEM, inhibitor-resistant variants of SHV-1and the related enzyme OHIO-1 have been detected (22, 137).

SHV

The SHV-1 �-lactamase is most commonly found in K. pneu-moniae and is responsible for up to 20% of the plasmid-medi-ated ampicillin resistance in this species (172). In many strainsof K. pneumoniae, blaSHV-1 or a related gene is integrated intothe bacterial chromosome (85). Although it has been hypoth-esized that the gene encoding SHV-1 may exist as part of atransposable element, it has never been proven (75). Unlikethe TEM-type �-lactamases, there are relatively few deriva-tives of SHV-1 (Table 2). Furthermore, the changes that havebeen observed in blaSHV to give rise to the SHV variants occurin fewer positions within the structural gene (Fig. 3). Themajority of SHV variants possessing an ESBL phenotype arecharacterized by the substitution of a serine for glycine atposition 238. A number of variants related to SHV-5 also havea substitution of lysine for glutamate at position 240. It isinteresting that both the Gly238Ser and Glu240Lys amino acidsubstitutions mirror those seen in TEM-type ESBLs. Theserine residue at position 238 is critical for the efficient hydro-lysis of ceftazidime, and the lysine residue is critical for theefficient hydrolysis of cefotaxime (69).

To date, the majority of SHV-type derivatives possess theESBL phenotype. However, one variant, SHV-10, is reportedto have an inhibitor-resistant phenotype. This enzyme appearsto be derived from SHV-5 and contains one additional aminoacid substitution of glycine for serine 130 (137). It is interestingthat the inhibitor-resistant phenotype conferred by theSer140Gly mutation seems to override the strong ESBL phe-notype usually seen in enzymes containing the Gly238Ser andthe Glu240Lys mutations seen in other SHV-5-type enzymes.The majority of SHV-type ESBLs are found in strains of K.pneumoniae. However, these enzymes have also been found inCitrobacter diversus, E. coli, and P. aeruginosa (27, 51, 108, 139).

CTX-M

In recent years a new family of plasmid-mediated ESBLs,called CTX-M, that preferentially hydrolyze cefotaxime hasarisen. They have mainly been found in strains of Salmonellaenterica serovar Typhimurium and E. coli, but have also beendescribed in other species of Enterobacteriaceae (Table 3).They include the CTX-M-type enzymes CTX-M-1 (formerlycalled MEN-1), CTX-M-2 through CTX-M-10 (9, 11, 12, 13,21, 29, 58, 59, 64, 148; A. Oliver, J. C. Perez-Dıaz, T. M. Coque,F. Baquero, and R. Canton, 40th Intersci. Conf. Antmicrob.Agents Chemother., abstr. 1480, 2000) as well as Toho en-zymes 1 and 2 (72, 88).

These enzymes are not very closely related to TEM or SHV�-lactamases in that they show only approximately 40% iden-tity with these two commonly isolated �-lactamases (174). Pre-viously, the most closely related enzymes outside this familywere thought to be the chromosomally encoded class A cepha-losporinases found in K. oxytoca, C. diversus, Proteus vulgaris,

TABLE 1. Characteristics of TEM-type �-lactamasesa

pI EnzymesEnzyme type

Broad spectrum ESBL IRT

5.2 TEM-12, TEM-55, TEM-57, TEM-58 XTEM-30, TEM-31, TEM-35, TEM-36, TEM-37, TEM-38, TEM-41, TEM-45,

TEM-51, TEM-73, TEM-74X

5.3 TEM-25 X5.4 TEM-1 X

TEM-7, TEM-19, TEM-20, TEM-65 XTEM-32, TEM-33, TEM-34, TEM-39, TEM-40, TEM-44 X

5.42 TEM-29 X5.55 TEM-5, TEM-17 X5.59 TEM-9 X5.6 TEM-2 X

TEM-10, TEM-11, TEM-13, TEM-26, TEM-63 XTEM-50 X XTEM-59 X

5.7 TEM-68 X X5.8 TEM-42 X5.9 TEM-4, TEM-6, TEM-8, TEM-27, TEM-72 X6.0 TEM-15, TEM-47, TEM-48, TEM-49, TEM-52, TEM-66, TEM-92 X6.1 TEM-28, TEM-43 X6.3 TEM-3, TEM-16, TEM-21, TEM-22 X6.4 TEM-56, TEM-60 X6.5 TEM-24, TEM-46, TEM-61 X

Not determined TEM-14, TEM-53, TEM-54 XTEM-76, TEM-77, TEM-78, TEM-79, TEM-81, TEM-82, TEM-83, TEM-84 X

a Amino acid sequences for TEM, SHV, and OXA extended-spectrum and inhibitor-resistant �-lactamases may be found at http://www.lahey.org/studies/webt.htm.All enzymes listed are naturally occurring mutants.


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and Serratia fonticola (73 to 77% homology) (13, 19). However,it was recently reported by Humeniuk et al. that there is a highdegree of homology between the chromosomal AmpC enzymeof Kluyvera ascorbata (designated Klu-1 and Klu-2) and theCTX-M-type enzymes, suggesting that the latter probably orig-inated from this species (G. Humeniuk, G. Arlet, R. Labia, P.Grimont, and A. Philippon, Abstr. Reunion Interdis. Chimi-other. Anti-infect., abstr. 20/C4, 2000) A phylogenetic study ofthe CTX-M family of �-lactamases showed four major types:the CTX-M-1 type, including CTX-M-1, and CTX-M-3; theCTX-M-2 type, including CTX-M-2, CTX-M-4, CTX-M-5,CTX-M-6, CTX-M-7, and Toho-1; Toho-2; and CTX-M-8, thelatter two groups containing only one member to date (21).The evolutionary distances between each of these groupingssuggest an early divergence from a common ancestor (21).

Kinetic studies have shown that the CTX-M-type �-lactam-ases hydrolyze cephalothin or cephaloridine better than ben-zylpenicillin and they preferentially hydrolyze cefotaxime overceftazidime (29, 174). Although there is some hydrolysis ofceftazidime by these enzymes, it is usually not enough to pro-vide clinical resistance to organisms in which they reside. It has

FIG. 2. Amino acid substitutions in TEM IRT derivatives. The amino acids listed within the grey bar are those found in the structural gene ofthe TEM-1 �-lactamase (162). The amino acid numbering is according to the scheme of Ambler et al. (5). Substitutions found in TEM-type IRTderivatives are shown under the amino acids of TEM-1. TEM-type variants may contain more than one amino acid substitution. ��, TEM-50 andTEM-68 contain amino acid substitutions that are common to both the ESBL and the IRT phenotypes. Only the amino acid substitutions that arecommon to TEM-type IRTs are shown in this figure.

TABLE 2. Characteristics of SHV-type �-lactamasesa

pI Enzymes

Enzyme type

Broadspectrum ESBL Inhibitor

resistant

7.0 OHIO-1, LEN-1 XSHV-3, SHV-14 X

7.5 SHV-24 X7.6 SHV-1, SHV-11 X

SHV-2, SHV-2a, SHV-6, SHV-8,SHV-13, SHV-19, SHV-20,SHV-21, SHV-22

X

7.8 SHV-4, SHV-7b, SHV-18 X8.2 SHV-5, SHV-9, SHV-12 X

SHV-10 X

a Amino acid sequences for TEM, SHV, and OXA extended-spectrum andinhibitor-resistant �-lactamases may be found at http://www.lahey.org/studies/webt.htm. All enzymes listed are naturally occurring mutants.

b SHV-7 was reported to have a pI of 7.6 (27), but further examination of thisenzyme indicates that the pI is most likely to be 7.8. This enzyme shows aninteresting phenomenon in that the isoelectric point varies depending on the dayof testing and the purity of the enzyme (unpublished observations).


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been suggested that the serine residue at position 237, which ispresent in all of the CTX-M enzymes, plays an important rolein the extended-spectrum activity of the CTX-M-type �-lacta-mases (174). Although it has been shown not to be essential,the Arg-276 residue lies in a position equivalent to Arg-244 inTEM- or SHV-type ESBLs, as suggested by molecular model-ing, and may also play a role in the hydrolysis of oxyimino-cephalosporins (56). Recent crystallographic data for theToho-1 enzyme suggested that there was increased flexibility ofthe interacting �3 strand and � loop of this enzyme in com-parison to other class A �-lactamases. Furthermore, the lack ofhydrogen bonds in the vicinity of the � loop could account forthe extended-spectrum phenotype (71). In addition to therapid hydrolysis of cefotaxime, another unique feature of these

enzymes is that they are inhibited better by the �-lactamaseinhibitor tazobactam than by sulbactam and clavulanate (29,88, 148, 174).

Strains expressing CTX-M-type �-lactamases have been iso-lated from many parts of the world, but have most often beenassociated with focal outbreaks in eastern Europe (29, 57, 64),South America, and Japan (88). There have been a few reportsof these enzymes in isolates from patients in western Europe,mostly in isolates from immigrants from the outbreak areas(173). However, Sabete et al. recently reported that 23 strainsof E. coli and Salmonella isolated in Spain expressed the CTX-M-9 �-lactamase, suggesting that there may be an endemicfocus of this enzyme in western Europe as well (148). More-over, a CTX-M-3-producing strain of Enterobacter cloacae was

FIG. 3. Amino acid substitutions in SHV ESBL derivatives. The amino acids listed within the grey bar are those found in the structural geneof the SHV-1 �-lactamase (25). The amino acid numbering is according to the scheme of Ambler et al. (5). Substitutions found in SHV-type ESBLderivatives are shown under the amino acids of SHV-1. SHV-type variants may contain more than one amino acid substitution. �, SHV-11 is notan ESBL but is included in the figure as a derivative of SHV-1.

TABLE 3. Characteristics of CTX-M-type ESBLs

�-Lactamase Alternative name pI Country of origin Bacterial species Reference(s)

CTX-M-1 MEN-1 8.9 Germany, Italy E. coli 12, 13CTX-M-2 7.9 Argentina S. entericaa 11, 13CTX-M-3 8.4 Poland C. freundii, E. coli 64CTX-M-4 8.4 Russia S. enterica 57, 59CTX-M-5 CTX-M-3 8.8 Latvia S. enterica 29CTX-M-6 8.4 Greece S. enterica 58, 173CTX-M-7 CTX-M-5 8.4 Greece S. enterica 58, 173CTX-M-8 7.6 Brazil P. mirabilis, E. cloacae, E. aerogenes,

C. amalonaticus21

CTX-M-9 8.0 Spain E. coli 148CTX-M-10 8.1 Spain E. coli Oliver et al.b

Toho-1 7.8 Japan E. coli 72Toho-2 7.7 Japan E. coli 88

a All strains of S. enterica were serovar Typhimurium.b A. Oliver, J. C. Perez-Dıaz, T. M. Coque, F. Baquero, and R. Canton, 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1480, 2000.


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recently isolated in France (50). Several institutions in theareas where outbreaks have occurred reported that the CTX-M-type enzyme is the most frequently isolated ESBL amongclinical isolates in their laboratories (148).

Interestingly, a number of these enzymes have been foundamong isolates of Salmonella enterica serovar Typhimurium(11, 29, 57, 58, 173). Large outbreaks of isolates of S. entericaserovar Typhimurium expressing CTX-M �-lactamases haveoccurred in both South America and eastern Europe. Theseisolates have also been found to express a variety of CTX-M-type variants. Therefore, it is unlikely that a single origin forthe occurrence and propensity of this type of �-lactamaseamong S. enterica serovar Typhimurium can be found.

OXA

The OXA-type enzymes are another growing family of ES-BLs. These �-lactamases differ from the TEM and SHV en-zymes in that they belong to molecular class D and functionalgroup 2d (32). The OXA-type �-lactamases confer resistanceto ampicillin and cephalothin and are characterized by theirhigh hydrolytic activity against oxacillin and cloxacillin and thefact that they are poorly inhibited by clavulanic acid (32). TheOXA �-lactamase family was originally created as a pheno-typic rather than a genotypic group for a few �-lactamases thathad a specific hydrolysis profile. Therefore, there is as little as20% sequence homology among some of the members of thisfamily. However, recent additions to this family show somedegree of homology to one or more of the existing members ofthe OXA �-lactamase family.

While most ESBLs have been found in E. coli, K. pneu-moniae, and other Enterobacteriaceae, the OXA-type ESBLshave been found mainly in P. aeruginosa (Table 4). Several ofthe OXA-type ESBLs have been derived from OXA-10 (OXA-11, -14, -16, and -17) (44, 45, 65, 104). OXA-14 differs fromOXA-10 by only one amino acid residue, OXA-11 andOXA-16 differ by two, and OXA-13 and OXA-19 differ by nine(Table 4). Among the enzymes related to OXA-10, the ESBLvariants have one of two amino acid substitutions: an aspara-gine for serine at position 73, or an aspartate for glycine atposition 157. In particular, the Gly157Asp substitution may benecessary for high-level resistance to ceftazidime (44). It ap-

pears that either of these mutations may be required to conferthe ESBL phenotype on the OXA-type variant. In addition tothe OXA-10 group, OXA-15 is a derivative of OXA-2, andOXA-18 is not directly derived from other OXA-type enzymes(closest relative is OXA-9, with 42% homology) (Table 4)(131).

The OXA-type ESBLs provide weak resistance to oxyimino-cephalosporins when cloned into E. coli, but provide fairlyhigh-level resistance in P. aeruginosa transconjugants (65). Incontrast to the majority of the OXA-type ESBLs, which conferresistance to ceftazidime, the OXA-17 �-lactamase confersresistance to cefotaxime and ceftriaxone but provides onlymarginal protection against ceftazidime (44). With respect to�-lactamase inhibitors, the original OXA enzymes were char-acterized by their lack of inhibition by clavulanic acid; how-ever, the OXA-18 �-lactamase was reported to be inhibited bythis compound (131). One additional OXA-type enzyme hasbeen identified, OXA-21 (184). This enzyme was found in astrain of Acinetobacter baumannii and is the first incidence ofan OXA-type enzyme’s originating in this organism. Becausethe clinical isolate of A. baumannii also expressed two other�-lactamases, it is unclear whether OXA-21 is an ESBL or anoriginal-spectrum enzyme (184).

In addition to the OXA-type ESBLs, a number of recentOXA derivatives that are not ESBLs have also been described.These include OXA-20 (110), OXA-22 (115), OXA-24 (24),OXA-25, -26, and -27 (2), and OXA-30 (155). Many of thenewer members of the OXA �-lactamase family have beenfound in bacterial isolates originating in Turkey and in France.It is not certain whether these two countries represent foci ofstrains harboring these enzymes or if they represent the localeof the investigators studying these �-lactamases.

Other ESBLs

While the majority of ESBLs are derived from TEM or SHV�-lactamases and others can be categorized with one of thenewer families of ESBLs, a few ESBLs have been reported thatare not closely related to any of the established families of�-lactamases (Table 5). The PER-1 �-lactamase was first dis-covered in strains of P. aeruginosa isolated from patients inTurkey (113). Later, it was also found among isolates of S.

TABLE 4. Characteristics of OXA-type ESBLs

�-Lactamase Derivation pI Amino acid substitutions vs. OXA-10 Country of origin Bacterial species Reference

OXA-11 OXA-10 6.4 Asn143Ser, Gly157Asp Turkey P. aeruginosa 65OXA-13 OXA-10 8.0 Ile10Thr, Gly20Ser, Asp55N, Asn73Ser,

Thr107Ser, Tyr174Phe, Glu229Gly,Ser245Asn, Glu259Ala

France P. aeruginosa 104

OXA-14 OXA-10 6.2 Gly157Asp Turkey P. aeruginosa 45OXA-15 OXA-2 8.7, 8.9 doublet NAa Turkey P. aeruginosa 46OXA-16 OXA-10 6.2 Ala124Thr, Gly157Asp Turkey P. aeruginosa 47OXA-17 OXA-10 6.1 Asn73Ser Turkey P. aeruginosa 44OXA-18 OXA-9, OXA-12 5.5 NA France P. aeruginosa 131OXA-19 OXA-10 7.6 Ile10Thr, Gly20Ser, Asp55Asn,

Thr107Ser, Gly157Asp, Tyr174Phe,Glu229Gly, Ser245Asn, Glu259Ala


OXA-28 OXA-10 7.6 Ile10Thr, Gly20Ser, Thr107Ser,Trp154Gly, Gly157Asp, Tyr174Phe,Glu229Gly, Ser245Asn, Glu259Ala


a NA, not applicable; these enzymes do not originate from OXA-10.


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enterica serovar Typhimurium and A. baumanii (176, 177, 179).The PER-1 �-lactamase is widespread across Turkey and isfound in up to 60% of ceftazidime-resistant strains of A. bau-manii, which represent 46% of total isolates (179). A commonplasmid encoding PER-1 was found in multiple nosocomialisolates of S. enterica serovar Typhimurium, suggesting that thestrains acquired the resistance plasmids in the hospital setting(176). A related enzyme, PER-2, which has 86% amino acidhomology with PER-1, was found among S. enterica serovarTyphimurium strains in Argentina. (14). It is interesting thatPER-1 is found almost exclusively in Turkey, while PER-2 hasbeen found almost exclusively in South America.

Another enzyme that is somewhat related to PER-1 is theVEB-1 �-lactamase (135). VEB-1 was first found in a singleisolate of E. coli in a patient from Vietnam, but was subse-quently also found in a P. aeruginosa isolate from a patientfrom Thailand (109). A third related enzyme is CME-1, whichwas isolated from Chryseobacterium meningosepticum (147). Afourth enzyme in this group is TLA-1, which was identified inan E. coli isolate from a patient in Mexico (153). The PER-1,PER-2, VEB-1, CME-1, and TLA-1 �-lactamases are relatedbut show only 40 to 50% homology. These enzymes all conferresistance to oxyimino-cephalosporins, especially ceftazidime,and aztreonam. They also show some homology to the chro-mosomal cephalosporinases in Bacteroides spp. and may haveoriginated from this genus (147).

An unusual feature of SFO-1, which is highly related to aclass A �-lactamase from Serratia fonticola, is that it is a trans-ferable �-lactamase that can be induced to high-level produc-tion of �-lactamase by imipenem (94). The plasmid carryingthe gene encoding the SFO-1 �-lactamase also carries theampR regulatory gene that is necessary for the induction ofclass C �-lactamases. However, unlike class C �-lactamases,SFO-1 cannot hydrolyze cephamycins and is inhibited well byclavulanic acid (94). GES-1 is another uncommon ESBL en-zyme that is not closely related to any other plasmid-mediated�-lactamase but does show 36% homology to a carbenicillinasefrom Proteus mirabilis (136).

A dendrogram of the phylogeny of ESBL sequences isshown in Fig. 4. The TEM and SHV families are tightly clus-tered and are related to each other. All of the class A ESBLsare more closely related to each other than they are to any ofthe class D OXA-type enzymes.

ESBL DETECTION METHODS

The increased prevalence of Enterobacteriaceae producingESBLs creates a great need for laboratory testing methods thatwill accurately identify the presence of these enzymes in clin-ical isolates. Although most ESBLs confer resistance to one or

FIG. 4. Phylogeny of ESBLs. Representative sequences of variousESBLs were obtained from GenBank. The PC1 (class A, S. aureusenzyme), IMP-1 (class B, metallo-enzyme), and ACT-1 (class C,AmpC-type enzyme) �-lactamases were included for comparison. Sig-nal peptides were identified with SPSScan and removed prior to align-ment. Sequences were aligned using Clustal X (168). Trees were con-structed with Clustal X, which uses the neighbor-joining method, witha bootstrap value of 1,000. The IMP-1 sequence was used to root thetree. Trees were visualized with TREEVIEW (118).

TABLE 5. Characteristics of novel, unrelated ESBLs

�-Lactamase Closest relative pI Preferred substratea Country of origin Bacterial species Reference

BES-1 Penicllinase from Yersiniaenterocolitica

7.5 CTX, CAZ, ATM Brazil S. marcescens 20

FEC-1 8.2 CTX Japan E. coli 93GES-1 Penicillinase from P. mirabilis 5.8 CAZ French Guiana K. pneumoniae 136CME-1 VEB-1 �9.0 CAZ Isolated from

reference strainChryseobacterium meningosepticum 147

PER-1 PER-2 5.4 CAZ France P. aeruginosa 113PER-2 PER-1 5.4 CAZ Argentina S. enterica serovar Typhimurium 14SFO-1 AmpA from S. fonticola 7.3 CTX Japan E. cloacae 94TLA-1 CME-1 9.0 CAZ, CTX, ATM Mexico E. coli 153VEB-1 PER-1, PER-2 5.35 CAZ, ATM Vietnam/Thailand E. coli 135

a CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam.


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more of the oxyimino-�-lactam antibiotics, the �-lactamasedoes not always increase the MICs to high enough levels to becalled resistant by the National Committee for Clinical Labo-ratory Standards (NCCLS) interpretive guidelines (78, 111).The sensitivity and specificity of a susceptibility test to detectESBLs vary with the cephalosporin tested. A number of inves-tigators have suggested that either dilution tests or disk diffu-sion susceptibility tests performed with cefpodoxime detectedmore ESBLs than other cephalosporins such as ceftazidime,cefotaxime, and ceftriaxone (52, 100). However, more recentdata suggest that susceptibility testing with cefpodoxime canlead to a high number of false-positives if the current NCCLSinterpretive criteria are applied (F. C. Tenover, P. Raney, P. P.Williams, K. L. Brittan, C. D. Steward, S. K. Fridkin, R. P.Gaynes, and J. E. McGowan, Jr., 40th Intersci. Conf. Antimi-crob. Agents Chemother., abstr. 1606, 2000). The NCCLS iscurrently reevaluating the testing procedures and interpretivecriteria that should be used for the detection of ESBLs.

The failure of either MIC or disk tests alone to accuratelydetect the presence of an ESBL in all strains of E. coli and K.pneumoniae has been well documented (52, 73). In a recentsurvey conducted through the World Health Organization,5.4% of laboratories using disk diffusion tests found an ESBL-producing challenge strain to be susceptible to all cephalospo-rins (165). In that study, Tenover et al. reported that only 2 ofthe 130 laboratories surveyed specifically reported the isolateas an ESBL producer (165). It also appears that there is adifference in the ability of various susceptibility-testing meth-ods used for detecting cephalosporin resistance in an ESBL-producing strain. Steward et al. reported the results of a pro-ficiency test assessing the ability of hospital laboratoriesparticipating in Project ICARE (Intensive Care AntimicrobialResistance Epidemiology) to detect specific types of antimi-crobial resistance (160). Only 35% of laboratories using theVitek system reported an ESBL challenge strain of K. pneu-moniae as being resistant to ceftazidime and ceftriaxone. Incontrast, 100% of the laboratories using the MicroScan systemreported the same strain as being resistant. However, only 29%of the laboratories using MicroScan reported the strain asbeing resistant to ceftriaxone (160).

This lack of sensitivity and specificity in traditional suscep-tibility tests to detect ESBLs has prompted the search for anaccurate test to detect the presence of ESBLs in clinical iso-lates. In the years since ESBLs were first described, a numberof different testing methods have been suggested.

Clinical Microbiology Techniques

Clinical microbiology tests employ a �-lactamase inhibitor,usually clavulanate, in combination with an oxyimino-cephalo-sporin such as ceftazidime or cefotaxime. In these tests, theclavulanate inhibits the ESBL, thereby reducing the level ofresistance to the cephalosporin.

Several ESBL detection tests that have been proposed arebased on the Kirby-Bauer disk diffusion test methodology. Oneof the first detection tests to be described was the double-diskapproximation test described by Jarlier et al. (76). In this test,the organism is swabbed onto a Mueller-Hinton agar plate. Asusceptibility disk containing amoxicillin-clavulanate is placed

in the center of the plate, and disks containing one of theoxyimino-�-lactam antibiotics are placed 30 mm (center tocenter) from the amoxicillin-clavulanate disk. As shown in Fig.5, enhancement of the zone of inhibition of the oxyimino-�-lactam caused by the synergy of the clavulanate in the amoxi-cillin-clavulanate disk is a positive result (76). This test remainsa reliable method for the detection of ESBLs. However, it hasbeen suggested that the sensitivity of this test can be increasedby reducing the distance between the disks to 20 mm (169,171). The use of cefpodoxime as the expanded-spectrum ceph-alosporin of choice for use in double-disk tests for ESBL de-tection has been suggested (41). Alternatively, the addition ofclavulanate (4 �g/ml) to the Mueller-Hinton agar can be usedto potentiate the zone of inhibition of one or more diskscontaining expanded-spectrum cephalosporins (67).

A similar test was designed by Jacoby and Han, in which 20�g of sulbactam was added to susceptibility disks containingone of the oxyimino-�-lactam antibiotics (73). An increase of 5mm in the zone of inhibition in a disk containing sulbactamcompared to the drug alone was considered a positive test.Although many ESBL-producing strains were detected withthis method, a significant number of strains were not. In addi-tion, a number of AmpC-producing strains also showed anenhancement of the zone diameter with the addition of sul-bactam (73). Recently, several commercial manufacturers havedeveloped disks that contain an expanded-spectrum cephalo-sporin plus clavulanate. A differential between results obtainedwith 10-�g disks containing cefpodoxime, ceftazidime, or ce-fotaxime with or without the addition of 1 �g of clavulanatewas shown to accurately detect the presence of an ESBL (35,105).

Another method suggested for the detection of ESBLs is thethree-dimensional test described by Thomson and Sanders(169). In this test, following inoculation of the test organismonto the surface of a Mueller-Hinton agar plate, a slit is cutinto the agar, into which a broth suspension of the test organ-ism is introduced. Subsequently, antibiotic disks are placed onthe surface of the plate 3 mm from the slit. Distortion ordiscontinuity in the expected circular zone of inhibition is con-sidered a positive test (169). This test was determined to bevery sensitive in detecting ESBLs, but it is more technicallychallenging and labor intensive than other methods. All of thetests utilizing one of the variations of a disk diffusion techniquerequire some interpretation and therefore should be per-formed by clinical microbiologists experienced in reading thesetests.

It has also been suggested that dilution tests performed withan expanded-spectrum cephalosporin with and without the ad-dition of clavulanic acid or another �-lactamase inhibitor beused for the detection of ESBLs in a clinical isolate. In general,these tests look for a reduction in the MIC of the cephalospo-rin in the presence of a �-lactamase inhibitor. However, thequestion of which cephalosporin to use has not been defini-tively resolved (170).

Currently, the NCCLS recommends an initial screening bytesting for growth in a broth medium containing 1 �g/ml of oneof five expanded-spectrum �-lactam antibiotics. A positive re-sult is to be reported as suspicious for the presence of an ESBL(111). This screen is then followed by a phenotypic confirma-tory test that consists of determining MICs of either ceftazi-


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dime or cefotaxime with and without the presence of clavulanicacid (4 �g/ml). A decrease in the MIC of �3 twofold dilutionsin the presence of clavulanate is indicative of the presence ofan ESBL. If an ESBL is detected, the strain should be reportedas nonsusceptible to all expanded-spectrum cephalosporinsand aztreonam regardless of the susceptibility testing result(111).

Several commercial manufacturers have developed ESBLdetection tests that can be used along with MIC test methodsalready in place in the clinical laboratory. Etest ESBL strips(AB Biodisk, Solna, Sweden) are two-sided strips that contain

a gradient of ceftazidime on one end and ceftazidime plusclavulanate on the other end. As shown in Fig. 5, a positive testfor an ESBL is a �3-dilution reduction in the MIC of ceftazi-dime in the presence of clavulanic acid. This test was shown tobe more sensitive than the double-disk approximation test indetecting ESBLs in clinical isolates (39). This method is con-venient and easy to use, but it is sometimes difficult to read thetest when the MICs of ceftazidime are low because the clavu-lanate sometimes diffuses over to the side that contains cefta-zidime alone (Fig. 5) (183).

The automated microbial susceptibility test system Vitek

FIG. 5. Double-disk diffusion and Etest ESBL detection tests. (A) The double-disk diffusion ESBL detection test as suggested by Jarlier et al.is shown (76). A disk containing amoxicillin-clavulanate (AMC) is placed in proximity to a disk containing ceftazidime (CAZ) or anotheroxyimino-cephalosporin. The clavulanate in the amoxicillin-clavulanate disk diffuses through the agar and inhibits the �-lactamase surrounding theceftazidime disk. Enhancement of the zone of the ceftazidime disk on the side facing the amoxicillin-clavulanate disk is interpreted as a positivetest. (B) Etest ESBL strip (AB Biodisk, Solna, Sweden). The zone of inhibition is read from two halves of the strip containing ceftazidime alone(TZ) or ceftazidime plus clavulanate (TZL). A reduction in the MIC of ceftazidime of �3 dilutions in the presence of clavulanate is interpretedas a positive test. (C) The Etest ESBL strip is sometimes difficult to interpret with weak enzyme producers such as the strain expressing TEM-12shown in this panel. The clavulanate from the ceftazidime plus clavulanate half of the strip diffuses into the agar and interferes with the readingof the MICs for the half of the strip containing ceftazidime alone.


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(Biomerieux, Hazlewood, Mo.) has also produced an ESBLtest that utilizes either ceftazidime or cefotaxime alone and incombination with clavulanic acid (4 �g/ml). A predeterminedreduction in growth in wells containing clavulanate comparedto those containing drug alone indicates the presence of anESBL. In a study of Klebsiella spp. and E. coli expressingwell-characterized �-lactamases, Sanders et al. showed that theVitek ESBL test was 99% sensitive and specific for the detec-tion of ESBLs (149). Furthermore, updated computer algo-rithms in the new Vitek system have also been shown to cat-egorize the �-lactamases present in many gram-negativeclinical isolates based on the phenotype of susceptibility pat-terns with various �-lactam antibiotics (150).

While each of these tests has its merit, none of these meth-ods can accurately detect all strains producing ESBLs. Vercau-teren et al. showed that the Etest ESBL test with ceftazidimeonly detected 81% of ESBLs tested in their laboratory, com-pared to 97 and 91% for the double-disk test and the three-dimensional test, respectively (183). Tzelepi et al. have re-ported that the Vitek ESBL detection test failed to detect themajority of ESBL-producing strains of Enterobacter spp. (171).In a recent survey of detection of ESBLs in clinical isolates,Tenover et al. found that only 18% of laboratories correctlyidentified challenge organisms as potential ESBL producersusing susceptibility to one or more expanded-spectrum �-lac-tam antibiotics as the method of detection (164). Furthermore,a survey in Europe found that 37% of ESBL-producing organ-

isms were mistakenly reported as being susceptible to expand-ed-spectrum cephalosporins (86).

The merits and shortcomings of each of the detection testsare outlined in Table 6. Of the tests that have been developedto date, the double-disk approximation test recommended byJarlier et al. (76), and the broth-dilution MIC reductionmethod (NCCLS confirmatory test) (111) are the easiest andmost cost-effective methods for use by many clinical laborato-ries. However, none of the detection tests that are based on thephenotype of the �-lactamase produced are 100% sensitive orspecific for the accurate detection of ESBLs among clinicalisolates of gram-negative bacteria. The need for improved de-tection of ESBLs in clinical isolates is well recognized (123).

It should also be noted that caution must be employed wheninterpreting ESBL detection tests because there have beenreports of false-positive results for ESBL phenotypic screeningtests that can occur with strains that do not possess an ESBL.Several groups have reported that the high-level expression ofSHV-1 in K. pneumoniae can cause the MIC of ceftazidime torise to levels at which an ESBL would be suspected (99, 129,141). In addition, Rasheed et al. reported that the productionof SHV-1 in a strain of K. pneumoniae that was also lacking anouter membrane porin protein caused a false-positive in ESBLdetection tests that looked at the differential between MICs ofoxyimino-�-lactam antibiotics with and without clavulanate(139). The presence of an ESBL can also be masked by theexpression of an AmpC-type enzyme in the same strain (28).

TABLE 6. ESBL detection techniques

Technique type Test Advantages Disadvantages Reference(s)

Clinical microbiology Standard NCCLS interpretivecriteria

Easy to use, performed inevery lab

ESBLs not always “resistant” 78, 111

NCCLS ESBL confirmatorytest

Easy to use and interpret Sensitivity depends on choice ofoxyimino-cephalosporin

111

Double-disk approximationtest

Easy to use, easy to interpret Distance of disk placement foroptimal sensitivity notstandardized

76, 169, 71

Three-dimensional test Sensitive, easy to interpret Not specific for ESBLS, laborintensive

169

Etest ESBL strips Easy to use Not always easy to interpret, not assensitive as double-disk test

183

Vitek ESBL test Easy to use, easy to interpret Reduced sensitivity 149, 164

Molecular detection DNA probes Specific for gene family (e.g.,TEM or SHV)

Labor intensive, cannot distinguishbetween ESBLs and non-ESBLs,cannot distinguish betweenvariants of TEM or SHV

7, 55, 70

PCR Easy to perform, specific forgene family (e.g., TEM orSHV)

Cannot distinguish between ESBLsand non-ESBLs, cannotdistinguish between variants ofTEM or SHV

42, 90, 116

Oligotyping Detects specific TEMvariants

Requires specific oligonucleotideprobes, labor intensive, cannotdetect new variants

117

PCR-RFLP Easy to perform, can detectspecific nucleotide changes

Nucleotide changes must result inaltered restriction site fordetection

116

PCR-SSCP Can distinguish between anumber of SHV variants

Requires special electrophoresisconditions

106, 107

LCR Can distinguish between anumber of SHV variants

Requires a large number ofoligonucleotide primers

80

Nucleotide sequencing The gold standard, candetect all variants

Labor intensive, can be technicallychallenging, can be difficult tointerpret manual methods

25


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Molecular Detection Methods

The tests described above only presumptively identify thepresence of an ESBL. The task of identifying which specificESBL is present in a clinical isolate is more complicated. In theearly days of studying ESBLs, determination of the isoelectricpoint was usually sufficient to identify the ESBL that waspresent. However, with �90 TEM-type �-lactamases, many ofwhich possess identical isoelectric points, determination of theESBL by isoelectric point is no longer possible. A similarsituation is found in the SHV, CTX-M, and OXA families ofESBLs.

Early detection of �-lactamase genes was performed usingDNA probes that were specific for TEM and SHV enzymes (7,55, 70). However, using DNA probes can sometimes be ratherlabor intensive. The easiest and most common molecularmethod used to detect the presence of a �-lactamase belongingto a family of enzymes is PCR with oligonucleotide primersthat are specific for a �-lactamase gene. Oligonucleotide prim-ers can be chosen from sequences available in public databasessuch as Genbank (GenBank, National Center for Biotechnol-ogy Information, http://www.ncbi.nlm.nih.gov/Genbank/index.html). These primers are usually chosen to anneal to regionswhere various point mutations are not known to occur. How-ever, PCR will not discriminate among different variants ofTEM or SHV. Several molecular methods that will aid in thedetection and differentiation of ESBLs without sequencinghave been suggested.

The first molecular method for the identification of �-lacta-mase was the oligotyping method developed by Ouellette et al.,which was used to discriminate between TEM-1 and TEM-2(117). This method used oligonucleotide probes that are de-signed to detect point mutations under stringent hybridizationconditions. Subsequently, Mabilat and Courvalin developedadditional oligonucleotide probes to detect mutations at sixpositions within the blaTEM gene (89). Using this method,several new TEM variants were identified within a set of clin-ical isolates. The probes used in oligotyping tests for TEM�-lactamases have been labeled either with a radioisotope orwith biotin (89, 167). Another approach for molecular charac-terization of the TEM �-lactamase gene was to add restrictionfragment length polymorphism analysis to PCR (PCR-RFLP)(6). In this test, amplified PCR products were subjected todigestion with several restriction endonucleases, and the sub-sequent fragments were separated by electrophoresis. Thesizes of the fragments generated by each restriction enzymeindicate point mutations within the blaTEM structural gene.

A number of different tests have been proposed for thedetection and identification of SHV derivatives. The simplestof these was suggested by Nuesch-Inderbinen et al. and em-ploys PCR-RFLP (116). Following PCR, the amplified DNA isdigested with restriction enzyme NheI, which detects the G-to-A nucleotide change that gives rise to the glycine-to-serinesubstitution at position 238 that is common to many of theearly SHV-type ESBLs. Although this method cannot deter-mine which SHV-type ESBL is present, it can detect the spe-cific mutation at position 238 (116). Another method used tocharacterize SHV-type ESBLs is PCR single-strand conforma-tional polymorphism (PCR-SSCP) analysis. This method hasbeen used to detect a single base mutation at specific locations

within the blaSHV gene (106, 107). In this test, a 475-bp am-plimer is generated using oligonucleotide primers that are in-ternal to the coding sequence of the blaSHV gene, digested withrestriction enzyme PstI. The fragments are then denatured andseparated on a 20% polyacrylamide gel. Genes for SHV-1, -2,-3, -4, -5, and -7 �-lactamases can be identified by the electro-phoretic pattern of the digested amplimer (106, 107). With theidentification of a number of additional SHV-type �-lactamasegenes, PCR-RFLP was developed to help with the identifica-tion of some of the newer SHV variants (38). Following PCR,Chanawong et al. used a variety of restriction endonucleases todetect 12 mutations at 11 positions within the blaSHV structuralgene. The combination of PCR-SSCP with PCR-RFLP allowsthe identification of 17 different SHV genes (38).

Another method proposed for the identification of SHVgenes is the use of ligase chain reaction (LCR) (80). LCRallows the discrimination of DNA sequences that differ by asingle base pair by the use of a thermostable ligase with fouroligonucleotide primers that are complimentary to the targetsequence and hybridize adjacent to each other. A single basemismatch in the oligonucleotide junction will not be ligatedand subsequently amplified. In this LCR test, the target DNAcontaining the blaSHV gene is denatured in a thermocycler andannealed with biotinylated oligonucleotide primers that detectmutations at four positions. The LCR product is detected by anenzymatic reaction using NADPH-alkaline phosphatase. Thismethod was able to detect seven of the SHV variants.

For OXA-10-derived ESBLs, the presence of an OXA-typegene in clinical isolates of P. aeruginosa was first detected usinga colony hybridization technique (178). Subsequently, positiveisolates were subjected to PCR with specific OXA primers andthen digested with restriction endonucleases that would distin-guish several groups of related OXA enzymes based on thesizes of the restriction fragments. While this technique doesnot completely identify which OXA gene is present in a strain,it can distinguish the ESBL OXA-type �-lactamases from non-ESBLs that are also related to OXA-10 (178).

Nucleotide sequencing remains the standard for determina-tion of the specific �-lactamase gene present in a strain. How-ever, this too can give variable results depending on themethod used (25). It is possible that some of the variabilityseen in the sequences for some of the SHV �-lactamases wasdue to compressions and difficulty in reading traditional se-quencing autoradiographs, rather than actual differences in thesequence (25).

Medical Significance of Detection of ESBLs

It is generally thought that patients having infections causedby an ESBL-producing organism are at an increased risk oftreatment failure with an expanded-spectrum �-lactam antibi-otic. Therefore, it is recommended that any organism that isconfirmed for ESBL production according to NCCLS criteriabe reported as resistant to all expanded-spectrum �-lactamantibiotics, regardless of the susceptibility test result (111).While some ESBL-producing strains have overt resistance toexpanded-spectrum �-lactam antibiotics, many isolates will notbe phenotypically “resistant” according to guidelines such asthose previously used by the NCCLS. Therefore, it is importantfor the clinical microbiology lab to be aware of isolates that


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may show increased MICs of oxyimino-cephalosporins eventhough they may not be reported as resistant, as this mightsuggest the presence of an ESBL. It is also important for theclinical microbiology lab to then implement one or more meth-ods to detect ESBLs. In contrast, the susceptibility test resultsof the �-lactam–�-lactamase inhibitor combinations can bereported as is. There have been several reports that theseinhibitor combinations may provide a viable alternative for thetreatment of infections caused by ESBL-producing organisms(124, 125).

The concern for the accurate detection of ESBLs is twofold.First, there is an increasing prevalence of ESBLs worldwide(see below). Second, many strains producing ESBLs demon-strate an inoculum effect in that the MICs of expanded-spec-trum cephalosporins rise as the inoculum increases (36, 74,158). Medeiros and Crellin found that the MICs of most ceph-alosporins rose dramatically when the inoculum of susceptibil-ity tests was raised from 105 to 107 CFU/ml (97). This in vivoinoculum effect has also been demonstrated in animal modelsof endocarditis and intra-abdominal abscesses (34, 53, 144).There are many types of infections in which the bacterial loadcould reach these levels. Therefore, it is imperative that thedetection of ESBLs accurately reflect the level of resistancethat would be achieved by strains expressing these enzymes invivo.

EPIDEMIOLOGY

ESBLs are now a problem in hospitalized patients world-wide. The ESBL phenomenon began in western Europe, mostlikely because expanded-spectrum �-lactam antibiotics werefirst used there clinically. However, it did not take long beforeESBLs had been detected in the United States and Asia. Theprevalence of ESBLs among clinical isolates varies from coun-try to country and from institution to institution. In the UnitedStates, occurrence of ESBL production in Enterobacteriaceaeranges from 0 to 25%, depending on the institution, with thenational average being around 3% (CDC National NosocomialInfections Surveillance, http://www.cdc.gov/ncidod/hip/SUR-VEILL/NNIS.HTM) Among isolates of K. pneumonia, the per-centage of ceftazidime resistance ranges from 5 to 10% fornon-intensive care unit (non-ICU) and ICU isolates, respec-tively (D. Mathai, R. N. Jones, M. Stilwell, and M. A. Pfaller,40th Intersci. Conf. Antimicrob. Agents Chemother., abstr.1027, 2000). Some hospitals with low levels of ESBLs may notfind it cost-effective to test for ESBLs on a routine basis (52).However, these institutions should monitor the rates of resis-tance in their own hospitals and be aware of an increase inresistance.

In Europe, the prevalence of ESBL production among iso-lates of Enterobacteriaceae varies greatly from country to coun-try. In the Netherlands, a survey of 11 hospital laboratoriesshowed that �1% of E. coli and K. pneumoniae strains pos-sessed an ESBL (161). However, in France, as many as 40% ofK. pneumoniae isolates were found to be ceftazidime resistant(30). Across Europe, the incidence of ceftazidime resistanceamong K. pneumoniae strains was 20% for non-ICU isolatesand 42% for isolates from patients in the ICU (Mathai et al.,abstr. 1027). In Japan, the percentage of �-lactam resistancedue to ESBL production in E. coli and K pneumoniae remains

very low. In a recent survey of 196 institutions across thecountry, �0.1% of E. coli and 0.3% of K. pneumoniae strainspossessed an ESBL (187). Elsewhere in Asia, the percentage ofESBL production in E. coli and K. pneumoniae varies, from4.8% in Korea (119) to 8.5% in Taiwan (188) and up to 12%in Hong Kong (68).

It is interesting that specific ESBLs appear to be unique toa certain country or region. For example, TEM-10 has beenresponsible for several unrelated outbreaks of ESBL-produc-ing organisms in the United States for a number of years (26,112, 143, 175). However, TEM-10 has only recently been re-ported in Europe with the same frequency (8, 84). Similarly,TEM-3 is common in France, but has not been detected in theUnited States (114, 156). In recent years, there have beenreports of outbreaks of TEM-47-producing organisms in Po-land (62), and the prevalence of TEM-52 in Korea is unique tothat country (119). Another recent survey of Korea revealedthat the SHV-12 and SHV-2a �-lactamases are the most com-mon ESBLs found in Korea (79). In contrast, the SHV-5 �-lac-tamase is commonly encountered worldwide and has beenreported in Croatia, France, Greece, Hungary, Poland, SouthAfrica, the United Kingdom, and the United States (15, 43, 54,63, 133, 152, 163, 181).

A common theme among hospitals plagued by organismsthat produce ESBLs is the high volume and indiscriminateadministration of expanded-spectrum cephalosporins (140,142). Specific risk factors include length of hospital stay, se-verity of illness, time in the ICU, intubation and mechanicalventilation, urinary or arterial catheterization, and previousexposure to antibiotics (126, 140). Many of the patients in-fected with ESBLs are found in ICUs, but they can occur insurgical wards as well as most other areas of the hospital.ESBLs are also being isolated with increasing frequency frompatients in extended-care facilities (27, 143, 186). In addition,whereas early outbreaks of ESBL-producing strains werecaused by isolates that produced only a single �-lactamase,more recently outbreaks have been caused by organisms withmultiple �-lactamases (26, 27, 189). This combination of non-ESBL class A enzymes and AmpC-type enzymes along withESBLs often compounds the resistance, so that many of thesestrains are now resistant to �-lactam–�-lactamase inhibitorcombinations, cephamycins, and even carbapenems in additionto the oxyimino-cephalosporins and aztreonam (28). In addi-tion, there is a high association with ciprofloxacin resistance instrains that produce ESBLs (122).

Many hospitals have experienced outbreaks of ESBL-pro-ducing organisms. These outbreaks are often fueled by thelarge number of patient transfers between units and betweenhospitals (87). It was found that barrier precautions were oftendifficult to enforce with a mobile patient population. Eventu-ally, many of the reported outbreaks were successfully man-aged using infection control methods (87), restriction of theuse of oxyimino-cephalosporins (125, 138), and antibiotic cy-cling (49, 77). A successful approach to the control of thespread of ESBL-producing organisms involved switching todifferent classes of broad-spectrum antibiotics for the treat-ment of serious infections (140). The two most successful re-placement antibiotics have been imipenem and piperacillin-tazobactam (98, 124, 125, 142, 143).

In the mid-1990s, Rice et al. reported that an outbreak of


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TEM-6-producing ceftazidime-resistant K. pneumoniae in aVeterans Administration hospital was successfully controlledafter the institution switched from ceftazidime to piperacillin-tazobactam for empiric therapy for gram-negative infections(142). Although the ceftazidime-resistant strains causing theoutbreak were originally resistant to piperacillin-tazobactam,they saw a rapid decrease in the isolation of K. pneumoniaestrains resistant to both ceftazidime and piperacillin-tazobac-tam. The incidence of ESBL-producing K. pneumoniae hasremained low since that time in that institution (142). Thisphenomenon of a reduction in the resistance rate to piperacil-lin-tazobactam following the switch from expanded-spectrumcephalosporin use to piperacillin-tazobactam has been con-firmed by several other investigators (124, 125). Moreover, ithas been reported that the use of �-lactam–�-lactam inhibitorcombinations results in a protective effect, in that they areassociated with a lower incidence of colonization with anESBL-producing isolate (132).

Many investigators are using molecular methods such aspulsed-field gel electrophoresis (PFGE) to examine epidemi-ology with the strains involved in outbreaks of infectionscaused by ESBLs (30, 40, 54). Other methods for studying theepidemiology of these strains include plasmid profiles, ribotyp-ing, random amplified polymorphic DNA (RAPD), and arbi-trarily primed PCR (17, 43, 151, 185, 190). These outbreaksoften start in an ICU and then spread to other parts of thehospital by the usual transmission routes (17). Very often, theexact source of outbreaks caused by ESBL-producing organ-isms is never identified. However, some interesting epidemiol-ogy of these resistant bacteria has been reported. In one hos-pital in France, ceftazidime-resistant K. pneumoniae expressingSHV-5 was isolated from six peripartum women and two ne-onates. Plasmid and PFGE profiles of the strains revealed thatall of the strains were identical to a strain that was culturedfrom contaminated ultrasonography coupling gel (54). An-other study demonstrated that cockroaches infesting a neona-tal ICU in South Africa carried the same PFGE strain types ofESBL-producing K. pneumoniae that were responsible for anoutbreak of infections and high mortality rate among neonatesin that institution (40).

ESBLs are most often encoded on plasmids, which can easilybe transferred between isolates. In an outbreak of ESBL-pro-ducing K. pneumoniae and E. coli in Chicago, it was shown thata common plasmid expressing TEM-10 was found in isolatesfrom numerous patients in several hospitals and nursing homes(26, 186). Because this plasmid was found in multiple differentstrain types, as demonstrated by PFGE, it was presumed thatthis promiscuous plasmid expressing TEM-10 was transferredto the normal flora of some of the patients. In another reportfrom France, a 180-kb self-transmissible plasmid expressingTEM-24 was found in four different species of Enterobacteri-aceae (E. coli, K. pneumoniae, E. aerogenes, and P. rettgeri)isolated from a single patient (91).

CONCLUSION

In the last 15 years, ESBLs have gone from being an inter-esting scientific observation to a reality of great medical im-portance. The introduction of the oxyimino-�-lactam antibiot-ics was met with the emergence of new �-lactamases. Some of

these new �-lactamases, like the TEM- and SHV-type ESBLs,result from simple point mutations in existing �-lactamasegenes that lead to a changed substrate profile. Other new�-lactamases, such as the CTX-M-type enzymes, have beenborrowed from the chromosomally encoded �-lactamases thatoccur naturally in other species of Enterobacteriaceae. The de-velopment and spread of ESBLs have most likely been causedby the overuse of expanded-spectrum cephalosporins in thehospital setting.

Numerous methods have been proposed for the detection ofESBLs in clinical isolates. Regardless of the method used fordetection, it is important to note that none of the methods thatrely on phenotypic expression of the �-lactamase will detectevery ESBL-producing isolate. Nevertheless, increased aware-ness of the ESBL problem among clinical microbiology labo-ratory and infection control personnel will help in the inter-pretation of these tests.

Current therapy for strains of Enterobacteriaceae that ex-press ESBLs is limited to such broad-spectrum agents as imi-penem. However, there have already been reports of therapeu-tic failures of this drug with strains that produce multiple�-lactamases (3). There are limited therapeutic options left forsome of these organisms. Strains expressing extended-spec-trum �-lactamases will present a host of challenges for clinicalmicrobiologists and clinicians alike as we head into the 21stcentury.

ACKNOWLEDGMENTS

I thank Ellen Murphy for the sequence alignments and creating thedendrogram, Steven J. Projan for critical review of the manuscript, andMelissa Visalli and David Correa for help in gathering references.

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