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Extended-Spectrum ß-Lactamases: a Clinical Update

Infectious Disease Division, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,¹ Infectious Disease Division, Louis Stokes VA Medical Center, Cleveland, Ohio²

SUMMARY

INTRODUCTION

DEFINITION OF EXTENDED-SPECTRUM ß-LACTAMASES

DIVERSITY OF ESBL TYPES

SHV

TEM

CTX-M and Toho ß-Lactamases

OXA

PER

VEB-1, BES-1, and Other ESBLs

EPIDEMIOLOGY OF ESBL-PRODUCING ORGANISMS

Global Epidemiology

Europe.

North America.

South and Central America.

Africa and the Middle East.

Australia.

Asia.

Molecular Epidemiology of Nosocomial Infections with ESBL-Producing Organisms

Risk Factors for Colonization and Infection with ESBL Producers

Nursing Homes and ESBL Producers

Community-Acquired Infections

INFECTION CONTROL IMPLICATIONS OF ESBL-PRODUCING ORGANISMS

Use of Typing Methods

Modes of Spread of ESBL-Producing Organisms within Hospitals

Infection Control When ESBL-Producing Organisms Have Not Been Endemic

Infection Control When ESBL-Producing Organisms Are Already Endemic

DETECTION OF ESBLs IN THE CLINICAL MICROBIOLOGY LABORATORY

Need for Clinical Microbiology Laboratories To Detect ESBL Production by Members of the Enterobacteriaceae

CLSI Recommended Methods for ESBL Detection

Screening for ESBL producers. (i) Disk diffusion methods.

(ii) Screening by dilution antimicrobial susceptibility tests.

Phenotypic Confirmatory Tests for ESBL Production

Cephalosporin/clavulanate combination disks.

Broth microdilution.

Quality control when performing screening and phenotypic confirmatory tests.

Implications of positive phenotypic confirmatory tests.

False positives and false negatives obtained with phenotypic confirmatory tests.

Commercially Available Methods for ESBL Detection

Etest for ESBLs.

Vitek ESBL cards.

MicroScan panels.

BD Phoenix Automated Microbiology System.

Other Methods for ESBL Detection

Cephalosporin/clavulanate combination disks on Iso-Sensitest agar.

Double-disk diffusion test.

Agar supplemented with clavulanate.

Disk replacement method.

Three-dimensional test.

Difficult Organisms

Klebsiella oxytoca.

Enterobacteriaceae other than Escherichia coli and Klebsiella spp.

TREATMENT AND OUTCOME OF INFECTIONS WITH ESBL-PRODUCING ORGANISMS

In Vitro Studies

Antibiotic Choice for Serious Infections

Carbapenem-Resistant Klebsiella pneumoniae Isolates

Outcome of Serious ESBL- and Non-ESBL-Producing Infections

CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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The introduction of the third-generation cephalosporins into clinical practice in the early 1980s was heralded as a major breakthrough in the fight against ß-lactamase-mediated bacterial resistance to antibiotics. These cephalosporins had been developed in response to the increased prevalence of ß-lactamases in certain organisms (for example, ampicillin hydrolyzing TEM-1 and SHV-1 ß-lactamases in Escherichia coli and Klebsiella pneumoniae) and the spread of these ß-lactamases into new hosts (for example, Haemophilus influenzae and Neisseria gonorrhoeae). Not only were the third-generation cephalosporins effective against most ß-lactamase-producing organisms but they had the major advantage of lessened nephrotoxic effects compared to aminoglycosides and polymyxins.

The first report of plasmid-encoded ß-lactamases capable of hydrolyzing the extended-spectrum cephalosporins was published in 1983 (197). The gene encoding the ß-lactamase showed a mutation of a single nucelotide compared to the gene encoding SHV-1. Other ß-lactamases were soon discovered which were closely related to TEM-1 and TEM-2, but which had the ability to confer resistance to the extended-spectrum cephalosporins (62, 373). Hence these new ß-lactamases were coined extended-spectrum ß-lactamases (ESBLs). In the first substantial review of ESBLs in 1989, it was noted by Philippon, Labia, and Jacoby that the ESBLs represented the first example in which ß-lactamase-mediated resistance to ß-lactam antibiotics resulted from fundamental changes in the substrate spectra of the enzymes (308).

In 2001, the ESBLs were reviewed in this journal by Patricia Bradford (51). The body of knowledge pertaining to ESBLs has grown rapidly since that time (Fig. 1). A PubMed search using the key-words extended-spectrum ß-lactamase reveals more than 1,300 relevant articles, with more than 600 published since the time Bradford's review was written.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Explosion of knowledge on extended-spectrum ß-lactamases.

The purpose of this review is to focus on the clinical aspects of ESBL production, with an emphasis on the implications for clinical microbiology laboratories, hospital molecular epidemiology laboratories, infection control practitioners, and infectious disease physicians.

DEFINITION OF EXTENDED-SPECTRUM ß-LACTAMASES

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ß-Lactamases are most commonly classified according to two general schemes: the Ambler molecular classification scheme and the Bush-Jacoby-Medieros functional classification system (10, 66, 338). The Ambler scheme divides ß-lactamases into four major classes (A to D). The basis of this classification scheme rests upon protein homology (amino acid similarity), and not phenotypic characteristics. In the Ambler classification scheme, ß-lactamases of classes A, C, and D are serine ß-lactamases. In contrast, the class B enzymes are metallo-ß-lactamases. The Bush-Jacoby-Medeiros classification scheme groups ß-lactamases according to functional similarities (substrate and inhibitor profile). There are four main groups and multiple subgroups in this system. This classification scheme is of much more immediate relevance to the physician or microbiologist in a diagnostic laboratory because it considers ß-lactamase inhibitors and ß-lactam substrates that are clinically relevant.

There is no consensus of the precise definition of ESBLs. A commonly used working definition is that the ESBLs are ß-lactamases capable of conferring bacterial resistance to the penicillins, first-, second-, and third-generation cephalosporins, and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by ß-lactamase inhibitors such as clavulanic acid. For the purpose of this review, the term ESBL will be taken to mean those ß-lactamases of Bush-Jacoby-Medeiros group 2be and those of group 2d which share most of the fundamental properties of group 2be enzymes (66).

The 2be designation shows that these enzymes are derived from group 2b ß-lactamases (for example, TEM-1, TEM-2, and SHV-1); the e of 2be denotes that the ß-lactamases have an extended spectrum. Group 2b enzymes hydrolyze penicillin and ampicillin, and to a lesser degree carbenicillin or cephalothin (66). They are not able to hydrolyze extended-spectrum cephalosporins or aztreonam to any significant degree. TEM-1 is the most common plasmid-mediated ß-lactamase of ampicillin resistant enteric gram-negative bacilli (for example, Escherichia coli), while SHV-1 is produced by the vast majority of Klebsiella pneumoniae (219). TEM-2 is a less common member of the same group with identical biochemical properties to TEM-1. The ESBLs derived from TEM-1, TEM-2, or SHV-1 differ from their progenitors by as few as one amino acid. This results in a profound change in the enzymatic activity of the ESBLs, so that they can now hydrolyze the third-generation cephalosporins or aztreonam (hence the extension of spectrum compared to the parent enzymes).

With the exception of OXA-type enzymes (which are class D enzymes), the ESBLs are of molecular class A, in the classification scheme of Ambler. They are able to hydrolyze the penicillins, narrow-spectrum and third-generation cephalosporins, and monobactams. The ESBLs have hydrolysis rates for ceftazidime, cefotaxime, or aztreonam (aminothiazoleoxime ß-lactam antibiotics) at least 10% that for benzylpenicillin. They are inhibited by clavulanic acid (66).

This property differentiates the ESBLs from the AmpC-type ß-lactamases (group 1) produced by organisms such as Enterobacter cloacae which have third-generation cephalosporins as their substrates but which are not inhibited by clavulanic acid. Selection of stably derepressed mutants which hyperproduce the AmpC-type ß-lactamases has been associated with clinical failure when third-generation cephalosporins are used to treat serious infections with Enterobacter spp. (86, 92, 192). In general, the fourth-generation cephalosporin, cefepime, is clinically useful against organisms producing Amp C-type ß-lactamases (354), but may be less useful in treating ESBL-producing organisms (440). This is discussed in more detail later in this article. Additionally, the metalloenzymes (group 3) produced by organisms such as Stenotrophomonas maltophilia can hydrolyze third-generation cephalosporins (and carbapenems), but are inhibited by EDTA (a heavy metal chelator) but not clavulanic acid (413a).

Some enzymes generally regarded as ESBLs (for example, TEM-7 and TEM-12) do not rigorously meet the hydrolysis criteria above. However, large increases in hydrolysis rates for ceftazidime are seen compared to the parent TEM-1 and TEM-2 enzymes, resulting in increased MICs of ceftazidime for organisms bearing such ß-lactamases. Hence, these TEM ß-lactamases are included in group 2be and are widely regarded as ESBLs (66).

In common with the ESBLs are other groups of ß-lactamases (2d, 2e, and 2f) that hydrolyze cephalosporins and are inhibited by clavulanic acid. However, group 2e ß-lactamases (for example, the inducible cephalosporinases of Proteus vulgaris) hydrolyze cefotaxime well but lack good penicillin-hydrolyzing activity, and do not have a high affinity for aztreonam, in contrast to the cephalosporinases in group 1. Group 2f ß-lactamases (for example, Sme-1 from Serratia marcescens) are carbapenem-hydrolyzing enzymes that are weakly inhibited by clavulanic acid. Extension of the spectrum of OXA-type ß-lactamases (group 2d) towards the extended-spectrum cephalosporins has been observed, and many authorities regard some of these enzymes as ESBLs (243).

DIVERSITY OF ESBL TYPES

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In 1983, a Klebsiella ozaenae isolate from Germany was discovered which possessed a ß-lactamase which efficiently hydrolyzed cefotaxime, and to a lesser extent ceftazidime (197). Sequencing showed that the ß-lactamase differed from SHV-1, by replacement of glycine by serine at the 238 position. This mutation alone accounts for the extended-spectrum properties of this ß-lactamase, designated SHV-2. Within 15 years of the discovery of this enzyme, organisms harboring SHV-2 were found in every inhabited continent (288), implying that selection pressure from third-generation cephalosporins in the first decade of their use was responsible. SHV-type ESBLs have been detected in a wide range of Enterobacteriaceae and outbreaks of SHV-producing Pseudomonas aeruginosa and Acinetobacter spp. have now been reported (170, 321)

TEM-1, TEM-2, and TEM-13 are not ESBLs. However, in 1987 Klebsiella pneumoniae isolates detected in France as early as 1984 were found to harbor a novel plasmid-mediated ß-lactamase coined CTX-1 (62, 373). The enzyme was originally named CTX-1 because of its enhanced activity against cefotaxime. The enzyme, now termed TEM-3, differed from TEM-2 by two amino acid substitutions (377).

In retrospect, TEM-3 may not have been the first TEM-type ESBL. Klebsiella oxytoca, harboring a plasmid carrying a gene encoding ceftazidime resistance, was first isolated in Liverpool, England, in 1982 (117). The responsible ß-lactamase was what is now called TEM-12. Interestingly, the strain came from a neonatal unit which had been stricken by an outbreak of Klebsiella oxytoca producing TEM-1. Ceftazidime was used to treat infected patients, but subsequent isolates of Klebsiella oxytoca from the same unit harbored the TEM-type ESBL (117). This is a good example of the emergence of ESBLs as a response to the selective pressure induced by extended-spectrum cephalosporins.

Well over 100 TEM-type ß-lactamases have been described, of which the majority are ESBLs. Their isoelectric points range from 5.2 to 6.5. The amino acid changes in comparison with TEM-1 or TEM-2 are documented at http://www.lahey.org/studies/temtable.htm.

A number of TEM derivatives have been found which have reduced affinity for ß-lactamase inhibitors. These enzymes have been reviewed elsewhere (78). With very few exceptions (see below), TEM-type enzymes which are less susceptible to the effects of ß-lactamase inhibitors have negligible hydrolytic activity against the extended-spectrum cephalosporins and are not considered ESBLs.

However, interesting mutants of TEM ß-lactamases are being recovered that maintain the ability to hydrolyze third-generation cephalosporins but which also demonstrate inhibitor resistance. These are referred to as complex mutants of TEM (CMT-1 to -4) (128, 273, 322, 372). A unique TEM-derived enzyme, TEM-AQ, has been found in Italy (301). This enzyme has an amino acid deletion not seen in other TEM enzymes plus several amino acid substitutions.

Toho-1 and Toho-2 are ß-lactamases related structurally to CTX-M-type ß-lactamases. (Toho refers to the Toho University School of Medicine Omori Hospital in Tokyo, where a child was hospitalized who was infected with Toho-1 ß-lactamase-producing Escherichia coli.) Like most CTX-M-type ß-lactamases, the hydrolytic activity of the Toho-1 and Toho-2 enzymes is more potent against cefotaxime than ceftazidime (204, 227).

It appears that the CTX-M-type ß-lactamases are closely related to ß-lactamases of Kluyvera spp. (109, 112, 171, 278, 319, 353). For example, a chromosomally encoded ß-lactamase gene of Kluyvera georgiana encoded an extended-spectrum ß-lactamase, KLUG-1, which shares 99% amino acid identity with CTX-M-8 (319). CTX-M-type ß-lactamases have 40% or less identity with TEM and SHV-type ESBLs.

The number of CTX-M-type ESBLs is rapidly expanding. They have now been detected in every populated continent (9, 22, 23, 49, 58, 70, 71, 81, 90, 120, 140, 190, 226, 253, 284, 288, 305, 333, 353, 414, 428, 437). For some years, CTX-M ESBLs were predominantly found in three geographic areas: South America, the Far East, and Eastern Europe. In Western Europe and North America, CTX-M-type ß-lactamases have previously appeared to be infrequent (105). However, in recent years, a number of authors have reported the advent of CTX-M-type ESBLs in these regions (9, 43, 253, 254, 263, 313, 324). Given the widespread findings of CTX-M-type ESBLs in China and India, it could be speculated that CTX-M-type ESBLs are now actually the most frequent ESBL type worldwide.

The relationship between antibiotic consumption and occurrence of CTX-M-type ß-lactamases has not been studied, although the prevalence of the enzymes in agents of community-acquired diarrhea raises speculation that oxyimino cephalosporins available outside the hospital (such as ceftriaxone) may be important. Interestingly, identical ß-lactamases have been discovered in widely separated parts of the world (for example, CTX-M-3 has been discovered in Poland and Taiwan), suggesting independent evolution of these enzymes (144, 429). Clonal spread of CTX-M-type ß-lactamase producing bacteria has been well-documented (144).

Most OXA-type ß-lactamases do not hydrolyze the extended-spectrum cephalosporins to a significant degree and are not regarded as ESBLs. However, OXA-10 hydrolyzes (weakly) cefotaxime, ceftriaxone, and aztreonam, giving most organisms reduced susceptibility to these antibiotics. Other OXA ESBLS include: OXA-11, -14, -16, -17, -19, -15, -18, -28, -31, -32, -35, and -45 (396). These confer frank resistance to cefotaxime and sometimes ceftazidime and aztreonam (97-100, 156, 396). The simultaneous production of a carbapenem-hydrolyzing metalloenzyme and an aztreonam hydrolyzing OXA enzyme can readily lead to resistance to all ß-lactam antibiotics (396).

The OXA-type ESBLs were originally discovered in Pseudomonas aeruginosa isolates from a single hospital in Ankara, Turkey. In France, a novel derivative of OXA-10 (numbered OXA-28) was found in a Pseudomonas aeruginosa isolate (317). A novel ESBL (OXA-18) and an extended-spectrum derivative of the narrow-spectrum OXA-13 ß-lactamase (numbered OXA-19) have also been discovered in France in Pseudomonas aeruginosa isolates (309). The evolution of ESBL OXA-type ß-lactamases from parent enzymes with narrower spectra has many parallels with the evolution of SHV- and TEM-type ESBLs. Unfortunately there are very few epidemiologic data on the geographical spread of OXA-type ESBLs.

Although PER-1-producing organisms have been predominantly found in Turkey, a Pseudomonas aeruginosa outbreak in Italy occurred with no apparent contacts with Turkey (225). Worryingly, a Pseudomonas aeruginosa strain producing both PER-1 and the carbapenemase VIM-2 has been detected in Italy (115). The coexistence of these enzymes renders an organism resistant to virtually all ß-lactam antibiotics. PER-1 has also been found in Proteus mirabilis and Alcaligenes faecalis in Italy (283, 300). Isolates of Pseudomonas aeruginosa producing PER-1 have been detected in France, Italy, and Belgium (87, 102, 282). Additionally, a high prevalence of PER-1 in Acinetobacter spp. from Korea has been noted (203, 434).

VEB-1 has greatest homology with PER-1 and PER-2 (38%) (323). It confers high-level resistance to ceftazidime, cefotaxime, and aztreonam, which is reversed by clavulanic acid. The gene encoding VEB-1 was found to be plasmid mediated; such plasmids also confer resistance to non-ß-lactam antibiotics. The patient from whom the ß-lactamase was originally described was a Vietnamese infant hospitalized in France (323). An identical ß-lactamase has also been found in Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter sakazakii, and Pseudomonas aeruginosa isolates in Thailand (142, 266, 397). Other VEB enzymes have also been detected in Kuwait and China (185, 325).

GES (76, 118, 320, 327, 410, 413, 416, 418), BES (45), TLA (8, 369), SFO (240), and IBC (133, 138, 191, 208, 241, 410-412) are other examples of non-TEM, non-SHV ESBLs and have been found in a wide range of geographic locations.

EPIDEMIOLOGY OF ESBL-PRODUCING ORGANISMS

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There is considerable geographical difference in the occurrence of ESBLs in European countries. Within countries, hospital-to-hospital variability in occurrence may also be marked (20). In a 1997-1998 survey of 433 isolates from 24 intensive care units in western and southern Europe, 25% of klebsiellae possessed ESBLs (20). A similar survey was performed by the same group in 1994; the overall proportion of klebsiellae which possessed ESBLs did not differ significantly between the two time periods, but the percentage of intensive care units which recorded ESBL-producing klebsiellae rose significantly from 74% to >90% (20, 221). Another large study from more than 100 European intensive care units found that the prevalence of ESBLs in klebsiellae ranged from as low as 3% in Sweden to as high as 34% in Portugal (158). A third study, which included both intensive care unit and non-intensive care unit isolates from 25 European hospitals, found that 21% of Klebsiella pneumoniae isolates had reduced susceptibility to ceftazidime (usually indicative of ESBL production, although it is acknowledged that other mechanisms of resistance may be responsible) (129). In Turkey, a survey of Klebsiella spp. from intensive care units from eight hospitals showed that 58% of 193 isolates harbored ESBLs (151).

North America. First reports of ESBL-producing organisms in the United States occurred in 1988 (181). In 1989, significant infections with TEM-10-producing Klebsiella pneumoniae were noted in Chicago by Quinn and colleagues (331). Other early reports of outbreaks mainly described infections with TEM-type ESBLs (particularly TEM-10, TEM-12, and TEM-26) (246, 270, 337, 341-343, 358, 402). However, outbreaks with SHV-type ESBLs have also been described (177). CTX-M-type ESBLs have recently been described in the United States and Canada (50, 253, 313).

Assessment of the prevalence of ESBL-producing organisms in the United States has been hampered by reliance of statistics examining resistance of organisms to third-generation cephalosporins, where resistance is defined as an MIC of 32 µg/ml (ceftazidime) or 64 µg/ml (cefotaxime/ceftriaxone). Since many ESBL-producing organisms have MICs for third-generation cephalosporins between 2 and 16 µg/ml, the prevalence of ESBL-producing organisms in the United States may have been underestimated in the past. Moland and colleagues have shown that ESBL-producing isolates were found in 75% of 24 medical centers in the United States (254). In a survey of nearly 36,000 isolates from intensive care units in North America, nonsusceptibility of Klebsiella pneumoniae to third-generation cephalosporins averaged 13% (273). The percentage of isolates which were susceptible fell by 3% over the years 1994 to 2000 (273). National Nosocomial Infection Surveillance (NNIS) figures for the period January 1998 to June 2002 reveal that 6.1% of 6,101 Klebsiella pneumoniae isolates from 110 intensive care units were resistant to third-generation cephalosporins (268). In at least 10% of intensive care units, resistance rates exceeded 25%. In non-intensive care unit inpatient areas, 5.7% of 10,733 Klebsiella pneumoniae isolates were ceftazidime resistant (268). In outpatient areas, just 1.8% of 12,059 Klebsiella pneumoniae isolates and 0.4% of 71,448 Escherichia coli isolates were ceftazidime resistant (268).

South and Central America. In 1988 and 1989 isolates of Klebsiella pneumoniae from Chile and Argentina were reported as harboring SHV-2 and SHV-5 (74). In retrospect, however a Klebsiella pneumoniae isolate kept lyophilized since 1982, during a preclinical study of cefotaxime in Buenos Aires, also proved to be a producer of an SHV-5 ESBL (J. M. Casellas, Abstracts of the International Congress of Chemotherapy, Birmingham, England, 1999). In 1989 an outbreak of multiresistant Salmonella enterica serovar Typhimurium infections occurred in 12 of 14 Argentinian provinces. From these isolates a new non-SHV, non-TEM ESBL named CTX-M-2 was identified (26, 28, 29, 333). Organisms with CTX-M-2 have spread throughout many parts of South America (333). Other CTX-M enzymes (CTX-M-8, -9, and -16) have been discovered in Brazil (44, 46). Curiously, TEM-type ESBLs have been very rarely reported from South America. As noted above, two novel non-TEM, non-SHV ESBLs have been recently reported from South America: GES-1, isolated from an infant previously hospitalized in French Guiana (320), and BES-1, from an ESBL-producing Serratia marcescens isolate from a hospital in Rio de Janeiro (45).

ESBLs have been found in 30 to 60% of klebsiellae from intensive care units in Brazil, Colombia, and Venezuela (245, 281, 306, 307, 349, 350). Reports of ESBL-producing organisms also exist from Central America and the Caribbean Islands (84, 114, 146, 370, 371).

Africa and the Middle East. Several outbreaks of infections with ESBL-producing Klebsiella have been reported from South Africa (93, 188, 310, 367), but no national surveillance figures have been published. However, it has been reported that 36.1% of Klebsiella pneumoniae isolates collected in a single South African hospital in 1998 and 1999 were ESBL producers (33). ESBLs have also been documented in Israel, Saudi Arabia, and a variety of North African countries (3, 25, 35, 36, 47, 122, 247, 272). Outbreaks of Klebsiella infections with strains resistant to third-generation cephalosporins have been reported in Nigeria and Kenya without documentation of ESBL production (5, 264). A novel CTX-M enzyme (CTX-M-12) has been found in Kenya (190). Characterization of ESBLs from South Africa has revealed TEM and SHV types (especially SHV-2 and SHV-5) (160, 315). A nosocomial outbreak of infections with Pseudomonas aeruginosa, expressing GES-2 has been described in South Africa (326).

Australia. The first ESBLs to be detected in Australia were isolated from a collection of gentamicin-resistant Klebsiella spp. collected between 1986 and 1988 from Perth (259). These were characterized as being of SHV derivation (260). In the last decade, ESBL-producing organisms have been detected in every state of Australia and in the Northern Territory (33, 121, 169, 348, 359). Outbreaks of infection have occurred in both adult and pediatric patients. Overall, it appears that the proportion of Klebsiella pneumoniae isolates which are ESBL producers in Australian hospitals is about 5% (33).

Asia. In 1988, isolates of Klebsiella pneumoniae from China which contained SHV-2 were reported (181). Further reports of other SHV-2-producing organism in China occurred in 1994 (83). In reports comprising limited numbers of isolates collected in 1998 and 1999, 30.7% of Klebsiella pneumoniae isolates and 24.5% of Escherichia coli isolates were ESBL producers (33). In a major teaching hospital in Beijing, 27% of Escherichia coli and Klebsiella pneumoniae blood culture isolates collected from 1997 through 1999 were ESBL producers (116). Of isolates collected from Zhejiang Province, 34% of Escherichia coli isolates and 38.3% of Klebsiella pneumoniae isolates were ESBL producing (438).

National surveys have indicated the presence of ESBLs in 5 to 8% of Escherichia coli isolates from Korea, Japan, Malaysia, and Singapore but 12 to 24% in Thailand, Taiwan, the Philippines, and Indonesia. Rates of ESBL production by Klebsiella pneumoniae have been as low as 5% in Japan (215, 427) and 20 to 50% elsewhere in Asia. However, there are clearly differences from hospital to hospital: it has been reported that a quarter of all Klebsiella pneumoniae isolates from a hospital in Japan in 1998 and 1999 were ESBL producers (33).

ESBLs of the SHV-2, SHV-5, and SHV-12 lineage initially dominated in those studies in which genotypic characterization has been carried out (209). Newly described SHV-type ESBLs have recently been reported from Taiwan and Japan (82, 202). However, the appearance of CTX-M ESBLs in India (189, 318) and China (81, 414, 425), and more frequent reports of outbreaks of infection with CTX-M-type ESBLs in Japan (200, 226), Korea (284), and Taiwan (436), raise suspicions that these may indeed be the dominant ESBL types in Asia. Plasmid-mediated non-TEM, non-SHV ESBLs, showing homology to the chromosomal ß-lactamases of Klebsiella oxytoca (Toho-1 and Toho-2), have been detected in Japan (173, 227). A new non-TEM, non-SHV ESBL (VEB-1) has been reported from Thailand and Vietnam (141, 142, 266, 323).

Many ESBL genes are on large plasmids; even prior to the advent of ESBLs, large multiresistance plasmids were more common in klebsiellae than Escherichia coli (219). Of importance may be the well-noted adaptation of klebsiellae to the hospital environment. Klebsiellae survive longer than other enteric bacteria on hands and environmental surfaces, facilitating cross-infection within hospitals (75).

In 100% of the more than 50 studies previously mentioned, at least two patients were colonized or infected with genotypically similar strains, implying patient-to-patient transmission of the strain. A number of outbreaks have been decribed with dissemination of a single clone of genotypically identical organism (131, 132, 143, 273). Clones have been found to persist for more than 3 years (57).

However, in many hospitals a more complex molecular epidemiologic picture has emerged (20). Recent reports have described the clonal dissemination of at least five different ESBL-producing Klebsiella strains in the same unit at the same time (128). Additionally, members of a single epidemic strain may carry different plasmids (carrying different ESBL genes) (128). Furthermore, genotypically nonrelated strains may produce the same ESBL due to plasmid transfer from species to species (38, 128). Finally, although the same ESBL may be prevalent in a particular unit of a hospital, they may be mediated by different plasmids (53). This may imply independent evolution via the effects of antibiotic pressure, or plasmid transfer from organism to organism.

Intensive care units are often the epicenter of ESBL production in hospitals—in one large outbreak, more than 40% af all the hospital's ESBL-producing organisms were from patients in intensive care units (147). As was noted in the pre-ESBL era, neonatal intensive care units can also be a focus of infections with multiply resistant klebsiellae (6, 38, 121, 217, 348, 386, 387, 408). Intensive care units in tertiary referral hospitals may acquire patients already colonized with ESBL-producing organisms, thereby triggering an outbreak of infection (147, 363, 364).

Transfer of genotypically related ESBLs from hospital to hospital within a single city (40, 256, 351, 439), from city to city (439), and from country to country (128, 147, 365, 439) has been documented. A notable clone has been an SHV-4-producing, serotype K-25 isolate of Klebsiella pneumoniae which has spread to multiple hospitals in France and Belgium (439). Another notable dissemination has been of a TEM-24-producing Enterobacter aerogenes clone in France, Spain, and Belgium (68, 108, 119). Intercontinental transfer has also been described (365).

Although ESBL-producing organisms can be introduced into intensive care units, epidemics of infection from intensive care units to other parts of the hospital have been well documented to occur (37, 182, 363). Likewise, ESBLs may spontaneously evolve outside of the intensive care unit. Units noted to have been affected by outbreaks include neurosurgical (37), burns (339), renal (131), obstetrics and gynecology (132), hematology and oncology (163, 270), and geriatric units (149, 268). Nursing homes and chronic care facilities may also be a focus of infections with ESBL-producing organisms. In these settings, clonal spread has also been documented (54, 343, 419).

Heavy antibiotic use is also a risk factor for acquisition of an ESBL-producing organism (16, 205, 299). Several studies have found a relationship between third-generation cephalosporin use and acquisition of an ESBL-producing strain (16, 19, 116, 126, 164, 193, 196, 205, 210, 291, 302, 358). Other studies, which were underpowered to show statistical significance, showed trends towards such an association (the P values in all three studies were between 0.05 and 0.10) (103, 299, 419). Furthermore, a tight correlation has existed between ceftazidime use in individual wards within a hospital and prevalence of ceftazidime-resistant strains in those wards (341). In a survey of 15 different hospitals, an association existed between cephalosporin and aztreonam usage at each hospital and the isolation rate of ESBL-producing organisms at each hospital (329, 357).

Use of a variety of other antibiotic classes has been found to be associated with subsequent infections due to ESBL-producing organisms. These include quinolones (103, 205, 419), trimethoprim-sulfamethoxazole (103, 205, 419), aminoglycosides (19, 205), and metronidazole (205). Conversely, prior use of ß-lactam/ß-lactamase inhibitor combinations, penicillins, or carbapenems seems not to be associated with frequent infections with ESBL-producing organisms.

In a point prevalence study in the skilled care floor of a Chicago nursing home, 46% of residents were colonized with ESBL-producing organisms (all Escherichia coli) (419). These patients had been in the nursing home, without intercurrent hospitalization, for a mean of more than 6 months. Patients from this nursing home, as well as seven other nursing homes, served as a reservoir for introduction of ESBL-producing organisms into an acute-care hospital (419).

Within nursing homes, antibiotic use is a risk factor for colonization with ESBL-producing organisms. Antibiotic use is frequent in nursing homes; in one recent study, 38% of nursing home residents had taken a systemic antibiotic in the last month (375). Use of third-generation cephalosporins has been identified as a predisposing event in some (343), but not all studies (419). In contrast to the situation in acute-care hospitals, use of orally administered antibiotics (ciprofloxacin and/or trimethoprim-sulfamethoxazole may also be a risk for colonization with an ESBL-producing strain (419). Nursing home residents would appear to have several additional risk factors for infection with ESBL-producing organisms. They are prone to exposure to the microbial flora of other residents, especially if they are incontinent and require frequent contact with health care providers. It has been well documented that handwashing rates are low among nursing home personnel (111). Urinary catheterization and decubitus ulcers are frequent (375), and have been associated with colonization of non-ESBL-producing, antibiotic-resistant gram-negative bacilli (258, 368).

Several community-acquired pathogens that commonly cause diarrhea have been found to be ESBL producers. ESBL-producing Salmonella infections remain a concern in many parts of the world (3, 24, 26, 56, 135, 174, 201, 257, 303). Additionally, a Shigella flexneri islate from the stool of an Algerian child admitted to hospital with dysentery and harboring SHV-2 has been detected (130). SHV-11 has been detected in Shigella dysenteriae (1) as have a variety of TEM- and CTX-M-type ESBLs in Shigella sonnei (195, 284, 332). Finally, Vibrio cholerae isolates and Shiga toxin-producing Escherichia coli isolates which are ESBL producers have been found (172, 305).

In the last 3 years there have been several reports of true community-acquired infection or colonization with ESBL-producing Escherichia coli (42, 59, 88, 249, 261, 312, 345, 423). These reports have come from Spain, Israel, the United Kingdom, Canada, and Tanzania. Typically, patients have developed urinary tract infection with CTX-M-producing Escherichia coli. Some urinary tract infections have been associated with bacteremia. The majority of isolates have been resistant to commonly used first-line agents for urinary tract infection such as trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and ceftriaxone. Rodriguez-Bano, in Seville, Spain, performed a case-control study examining risk factors for ESBL-producing Escherichia coli infections in nonhospitalized patients and found that diabetes mellitus, prior quinolone use, recurrent urinary tract infections, prior hospital admissions, and older age were independent risk factors (345). Pitout, in Calgary, Canada, showed that 22 cases of ESBL-producing Escherichia coli infection occurred per year per 100,000 population greater than 65 years of age (312). The cause of this sudden upsurge in community-acquired infections with ESBL-producing organisms is not yet clear, but associations with foodstuffs, animal consumption of antibiotics, and frequent patient contact with health care facilities need to be explored.

INFECTION CONTROL IMPLICATIONS OF ESBL-PRODUCING ORGANISMS

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Before the advent of molecular biologic techniques to assess the genetic relationships between nosocomially acquired organisms, typing methods that assessed phenotypic differences between organisms were widely used. At least seven phenotypic methods could potentially be used to type Klebsiella pneumoniae isolates harboring ESBLs. These include biotyping (assessing the potential clonal relationship between organisms by way of observing common biochemical reactions, colonial morphology, or environmental tolerances) (18) and assessment of the antimicrobial susceptibility test pattern. Neither test has particularly good discriminatory power. Occasionally, stored isolates of organisms may lose transferrable genetic elements (for example, plasmids) which confer antibiotic resistance and appear to have a different antibiotic susceptibility pattern than when the isolate was examined fresh (242).

Serotyping is potentially useful in discriminating ESBL-producing klebsiellae. The klebsiellae typically express both lipopolysaccharide (O antigen) and capsule polysaccharide (K antigen) on the surface (159). Seventy-seven K antigen types form the basis of an internationally recognized capsule antigen scheme (280). The drawback of this method is the large number of serological cross-reactions that occur among the 77 capsule types. Thus, individual sera have to be absorbed with the cross-reacting K antigens. Moreover, the antisera are not commercially available and the typing procedure is cumbersome because of the time needed to perform the test. Finally the test is susceptible to subjectivity bacause of weak reactions that are not always easy to interpret (316). In contrast to the large number of capsular serotypes, only nine lipopolysaccharide O groups have been recognized. Since there are only nine O types compared with 77 K types, O typing is clearly less discriminatory than K typing. Furthermore, traditional methods of O typing are hampered by the heat stability of capsular polysaccharide (159). Recently, an inhibition enzyme-linked immunosorbent assay method has been developed which overcomes this technical problem (159). A number of studies have evaluated use of serotyping ESBL-producing klebsiellae using the K antigens (18, 32, 57, 62, 147, 153, 211, 363, 364, 439). No studies to date evaluated O types of ESBL-producing klebsiellae; however, a combination of K and O typing is likely to be a very discriminatory nonmolecular method of typing ESBL-producing klebsiellae.

Phage typing, bacteriocin typing, analytical isoelectric focusing, and multilocus enzyme electrophoresis are other methods which have been used to discriminate ESBL-producing strains (27, 175, 277, 316).

Since the vast majority of ESBLs are plasmid mediated, plasmid profile analysis has been applied to the epidemiologic study of ESBL-producing organisms. A simple method is to determine the number and size of the plasmids carried by the organism by preparing a plasmid extract and subjecting it to routine agarose gel electrophoresis. The reproducibility and discriminatory power of plasmid analysis can be improved by first digesting the plasmids with restriction enzymes and then performing agarose gel electrophoresis. This procedure and the analysis of the size and number of the resulting restriction fragments are referred to as restriction enzyme analysis of plasmids. A drawback in plasmid profile analysis is that plasmids may be lost after storage. It should be noted that plasmid extraction methods may yield different results if the efficiency of extraction is not optimal.

Most researchers use molecular methods to determine the relatedness of ESBL-producing organisms. Pulsed-field gel electrophoresis of chromosomal DNA is probably the most widely used method of genotyping ESBL-producing organisms (15, 17, 57, 68, 85, 113, 128, 132, 149, 207, 218, 222, 234, 235, 256, 268, 298, 328, 334, 341, 348, 351, 358, 374, 381, 408, 426, 431, 439). These references describe the restriction enzymes used for various organisms harboring ESBLs. Ribotyping, a southern blot analysis in which strains are characterized by the restriction fragment lengh polymorphisms associated with the ribosomal operons, is potentially very useful in typing ESBL-producing organisms, especially when automated ribotyping systems are used. Multiple variations of PCR have been applied to the typing of ESBL-producing organisms. These are randomly amplified polymorphic DNA, which is also known as arbitrarily primed PCR, and PCR based on repetitive chromosomal sequences. Of these, use of arbitrarily primed PCR has been by far the most popular method used to evaluate the genetic relatedness of ESBL-producing strains. The randomly amplified polymorphic method is based on the observation that short primers (around 10 base pairs), whose sequence is not directed to any known genetic locus, will regardless hybridize at random chromosomal sites with sufficient affinity to permit the initiation of polymerization. If two such sites are located within a few kilobases of each other on opposite DNA strands and in the proper orientation, amplification of the intervening fragment will occur. The number and locations of these random sites (and therefore the number and sizes of fragments) will vary among different strains of the same species (13).

Restriction site insertion PCR is a recently developed technique to detect mutations of the SHV genes to identify ESBLs. Restriction site insertion PCR uses amplification primers designed with one to three base mismatches near the 3' end to engineer a desired restriction site. Chanawong et al. (80) demonstrated that the combination of PCR-restriction fragment length polymorphism and restriction site insertion PCR techniques can be readily applied to the epidemiological study of SHV ß-lactamases. Using the combination of the techniques, eight different ß-lactamases were reliably distinguished. Another useful tool for the detection of certain SHV variants is the combination of PCR-single strand conformational polymorphism and PCR-restriction fragment length polymorphism (79). Using PCR-single strand conformational polymorphism and PCR-restriction fragment length polymorphism with DdeI and NheI digestion, the genes encoding SHV-1, SHV-2a, SHV-3, SHV-4, SHV-5, SHV-11, and SHV-12 were distinguisable (79).

Ligase chain reaction is a recently developed technique also used to discriminate SHV variants. Ligase chain reaction uses a thermostable ligase and biotinylated primers. It can detect single base pair changes (194). Kim and Lee evaluated seven Escherichia coli strains known to produce different SHV enzymes and 46 clinical isolates, and found that ligase chain reaction typing simply and rapidly defined the SHV types (194).

A recently described method marries the sensitivity of PCR with fluorescently labeled probes. Randeggar and Haechler developed a technique using real-time PCR monitored with fluorescently labeled hybridization probes followed by melting curve analysis (335). Their technique was able to differentiate SHV variants in five well-characterized Escherichia coli strains and six clinical isolates, and to discriminate between non- ESBLs and ESBLs. It remains to be seen whether this technique, termed the SHV melting curve mutation detection method, will identify all SHV variants.

Present evidence suggests that transient carriage on the hands of health care workers is a more important means of transfer from patient to patient. Hand carriage has been documented by most (121, 166, 348) but not all (339, 387) investigators who have sought it. In these instances, the hand isolates were genotypically identical to isolates which caused infection in patients. Hand carriage by health care workers is usually eliminated by washing with chlorhexidine or alcohol-based antiseptics. However, the authors know of one example of prolonged, persistent skin carriage in a nurse with chronic dermatitis. The use of artificial nails may also promote long-term carriage and has been associated with at least one outbreak (152). Gastrointestinal tract carriage has been documented in health care workers (131, 166, 378), but is astonishingly rare and seldom prolonged, except with ESBL-producing Salmonella species (157, 247).

The hands of health care workers are presumably colonized by contact with the skin of patients whose skin is colonized with the organism (131). It is important to recognize that many patients may have asymptomatic colonization with ESBL-producing organisms without signs of overt infection. These patients represent an important reservoir of organisms. For every patient with clinically significant infection with an ESBL-producing organism, at least one other patient exists in the same unit with gastrointestinal tract colonization with an ESBL producer (161, 222). In some hyperendemic intensive care units and transplants units, 30 to 70% of patients have gastrointestinal tract colonization with ESBL producers at any one time (150, 299, 378).

Since gastrointestinal tract colonization with klebsiellae occurs in every person, selective media must be used in order to assess the carriage of ESBL producers in stool. Examples of such media include Drigalski agar supplemented with cefotaxime 0.5 mg/liter (378), MacConkey agar supplemented with ceftazimide 4 mg/liter (299) and nutrient agar supplemented with ceftazimide 2 mg/liter, vancomycin 5 mg/liter, and amphotericin B 1,667 mg/liter (150). Prior gastrointestinal carriage of ESBL-producing Klebsiella pneumoniae is an independent variable associated with infection with ESBL-producing Klebsiella pneumoniae (298). At least 80% of patients with infection with ESBL-producing Klebsiella pneumoniae can be documented to have prior gastrointestinal tract carriage (222, 298). Patients who develop infection usually do so within weeks of acquiring gastrointestinal tract colonization (range, 0 to 90 days) (222, 298). There does not appear to be any variation in particular kinds of infection (bacteremia, pneumonia, etc.) and incidence of prior gastrointestinal tract colonization (298).

Therefore, control of the initial outbreak of ESBL-producing organisms in a hospital or specialized unit of a hospital is of critical importance (Table 1).

Although common environmental sources of infection have rarely been discovered, when they are recognized their impact on arresting an outbreak of infection with a multiresistant organism can be dramatic. Three examples of such an intervention have been described in the context of controlling outbreaks of infection with ESBL-producing organisms. Gaillot (132) found that contaminated gel used for ultrasonography was contaminated with ESBL-producing organisms. Replacement of this gel quickly curtailed the outbreak. Branger (57) found that a poorly maintained bronchoscope was colonized with ESBL-producing organisms and could be linked to respiratory tract infections with the same strain. Repair and proper maintenance of the bronchoscope stopped nosocomial transmission of the organism. Finally, Rogues (346) found colonization of four of 12 glass mercury thermometers with ESBL-producing Klebsiella pneumoniae and axillary colonization with the same strain in two patients. Disinfection of the thermometers curtailed the outbreak.

Contact isolation implies use of gloves and gowns when contacting the patient. Several studies have documented that this practice alone can lead to reduction in horizontal spread of ESBL-producing organisms. However, compliance with these precautions needs to be high in order to maximize the effectiveness of these precautions. Furthermore, we recommend that patients who have gastrointestinal tract colonization as well as those with frank infection should undergo contact isolation. It has been noted that standard methods of hand washing, screening for colonization, and patient isolation may not always be effective in controlling outbreaks of ESBL-producing organisms (230). Macrae and colleagues (230) were forced to close a ward tempoararily in order to adequately control an outbreak which had been unresponsive to conventional measures.

A number of groups have previously attempted selective digestive decontamination as a means of interrupting transmission of ESBL-producing organisms. Erythromycin-based therapies have not been effective (107, 110). However, three groups successfully used selective digestive decontamination with polymyxin, neomycin, and nalidixic acid (63), colistin and tobramycin (388), or norfloxacin (294) to interrupt outbreaks of infection with ESBL-producing infections that had not been completely controlled using traditional infection control measures. It should be noted that in many hospitals at least 15 to 30% of ESBL-producing organisms (20, 183, 206, 292, 435) are quinolone resistant and therefore unlikely to suppressed by use of norfloxacin prophylaxis. Additionally, multidrug-resistant isolates are unlikely to respond to selective digestive decontamination using aminoglycosides.

An alternative approach to digestive tract decolonization has been decolonization of the nasopharynx. A recent study has utilized a nasal spray with povidone-iodine as a means of decolonizing the upper respiratory tract (167). In this study (performed in a neurologic rehabilitation unit), only 1 of 10 patients had gastrointestinal carriage with an ESBL-producing organism but all had nasotracheal colonization. Upper airway decolonization led to management of an outbreak (167).

The methods used to achieve control over endemic ESBL producers have included close attention to practices that may lead to breakdowns in good infection control (223). This includes a review of procedures that lead to nurses, physicians and ancillary staff (e.g., radiography technicians) failing to use contact isolation precautions (223). Although infection control procedures continue to play a central role, changes in antibiotic policy may play an even greater role in this setting (352). Indeed in one institution, no effort was made to change infection control procedures (341). Instead, at this hospital, ceftazidime use decreased and piperacillin-tazobactam was introduced in the formulary. A number of authors have shown that ceftazidime restriction alone is insufficient to control continued infections with ESBL-producing organisms (334, 343, 387). Rahal et al. (334) were forced to withdraw cephalosporins as an entire class in order to exact control over endemic ESBL producers.

Some authors have suggested that use of ß-lactam/ß-lactamase inhibitor combinations, rather than cephalosporins, as workhorse empirical therapy for infections suspected as being due to gram-negative bacilli, may facilitate control of ESBL producers (296, 311, 341). The mechanism by which these drugs may reduce infections with ESBL producers is not certain. It should be noted, however, that many organisms now produce multiple ß-lactamases (24, 53, 81, 366), which may reduce the effectiveness of ß-lactam/ß-lactamase inhibitor combinations. It remains to be shown whether a strategy based on the empiric use of carbapenems would be effective.

DETECTION OF ESBLs IN THE CLINICAL MICROBIOLOGY LABORATORY

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ESBL detection originated because some ESBL-producing organisms appeared susceptible to cephalosporins using conventional breakpoints. How frequently are ESBL-producing organisms susceptible to cephalosporins? The answer to this question depends on which breakpoints are used. National differences are quite considerable. For example, susceptibility to cefotaxime may be reported for an organism with MICs ranging from 1 µg/ml to 8 µg/ml, depending on the country. Variation in reporting of resistance is from 2 µg/ml to 64 µg/ml. The most liberal interpretation of cephalosporin susceptibility has been that of the Clinical and Laboratory Standards Institute (CLSI,