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Prevalence of extended-spectrum β-lactamases in group-1 β-lactamase-producing isolates

2001; Elsevier BV; Volume: 7; Issue: 5 Linguagem: Inglês

10.1046/j.1198-743x.2001.00255.x

1469-0691

Carmen Varela, Antonio Oliver, Teresa M. Coque, Fernando Baquero, Rafael Cantón,

Enterobacteriaceae and Cronobacter Research

Resistance to extended-spectrum cephalosporins and aztreonam emerges in Enterobacter spp., Citrobacter freundii, Serratia spp., Morganella morganii and Pseudomonas aeruginosa due to a mutation in a chromosomal gene that normally prevents high-level expression of this organism's chromosomal β-lactamase [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar]. This mutation results in high-level production of the chromosomal Bush Group 1 β-lactamase (AmpC). A different mechanism of resistance to extended-spectrum cephalosporins has also been recognized in these species. This involves the acquisition of plasmid encoding extended-spectrum β-lactamases (ESBLs). These enzymes, which have been mainly described in Escherichia coli and Klebsiella pneumoniae, usually derive from the classical TEM-1, TEM-2 and SHV-1 β-lactamases [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar, 2Jacoby GA Medeiros AA More extended spectrum β‐lactamases.Antimicrob Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (654) Google Scholar]. The occurrence of ESBLs in Enterobacteriaceae that possess group 1 β-lactamase is increasingly reported worldwide, particularly in Enterobacter spp. [3Gianneli D Tzelepi E Tzouvelekis LS Mentis AF Nikolopoulou C Dissemination of cephalosporin‐resistant Serratia marcescens strains producing a plasmidic SHV type beta‐lactamase in Greek hospitals.Eur J Clin Microbiol Infect Dis. 1994; 13: 764-767Crossref PubMed Scopus (24) Google Scholar, 4Coudron PE Moland ES Sanders CC Occurrence and detection of extended‐spectrum beta‐lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find.J Clin Microbiol. 1997; 35: 2593-2597PubMed Google Scholar, 5Gniadkowski M Schneider I Palucha A Jungwirth R Mikiewicz B Bauernfeind A Cefotaxime‐resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX‐M‐3 cefotaxime‐hydrolyzing β‐lactamase that is closely related to the CTX‐M‐1/MEN‐1 enzyme.Antimicrob Agents Chemother. 1998; 42: 827-832PubMed Google Scholar, 6Pitout JD Thomson KS Hanson ND Ehrhardt AF Coudron P Sanders CC Plasmid‐mediated resistance to expanded‐spectrum cephalosporins among Enterobacter aerogenes strains.Antimicrob Agents Chemother. 1998; 42: 596-600PubMed Google Scholar, 7Silva J Aguilar C Becerra Z López‐Antunano F García R Extended‐spectrum beta‐lactamases in clinical isolates of enterobacteria in Mexico.Microb Drug Resist. 1999; 5: 189-193Crossref PubMed Scopus (19) Google Scholar, 8Tzelepi E Giakkoupi P Sofianou D Loukova V Kemeroglou A Tsakris A Detection of extended‐spectrum β‐lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.J Clin Microbiol. 2000; 38: 542-546PubMed Google Scholar]. In these species, the detection of ESBL-producing isolates by methods based on the inhibitory effects of clavulanic acid could be difficult, because of the superimposed resistance phenotype due to ESBLs and AmpC hyperproduction, and it is dependent on the level of chromosomal enzyme production [9Bush K Is it important to identify extended‐spectrum β‐lactamase‐producing isolates?.Eur J Clin Microbiol Infect Dis. 1996; 15: 361-364Crossref PubMed Scopus (75) Google Scholar]. From a clinical point of view, the discrimination between ESBL and overproduced group 1 β-lactamases in these species may not be critical. Nevertheless, the detection of such ‘hidden’ ESBLs is still of epidemiologic importance for the hospital environment, because it implies the possibility of plasmid transmission among different isolates in addition to patient-to-patient transmission of the ESBL-producing strains. In order to investigate the prevalence of ESBLs among group 1 β-lactamase-producing isolates, 277 Enterobacteriaceae strains (107 Enterobacter cloacae, 34 Enterobacter aerogenes, 51 Serratia spp., 26 C. freundii, 54 M. morganii and five Providencia stuartii) and 193 P. aeruginosa strains were consecutively and prospectively collected during a 6-month period in a Spanish teaching hospital (January to June, 1999). Only one isolate per patient and bacterial species was selected, to avoid repetition of strains. Preliminary identification and antimicrobial susceptibility testing were performed by using the semi-automated microdilution Wider System (Francisco Soria Melguizo, SA Madrid, Spain) [10Cantón R Pérez‐Vázquez M Oliver A et al.Evaluation of the Wider system, a new computer‐assisted image‐processing device for bacterial identification and susceptibility testing.J Clin Microbiol. 2000; 38: 1339-1346PubMed Google Scholar]. Moreover, the NCCLS agar dilution method [11National Committee for Clinical Laboratory Standards Performance Standards for Antimicrobial Susceptibility Testing; eleventh informational supplement. M100‐S11. NCCLS, Wayne, PA2001Google Scholar] was used to determine the MIC values for different β-lactam antibiotics. The inoculum effect on antibiotic MICs was determined using a 103 CFU/mL inoculum (low inoculum) and a 107 CFU/mL inoculum (high inoculum). The corresponding manufacturers provided all antibiotics as powders. The double disk synergy (DDS) test was performed to assess ESBL expression [12Jarlier V Nicolas MH Fournier G Philippon A Extended spectrum β‐lactamases conferring transferable resistance to newer β‐lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns.Rev Infect Dis. 1988; 10: 867-878Crossref PubMed Scopus (1334) Google Scholar]. The strains that showed synergy between oximino-cephalosporins or aztreonam and clavulanic acid (DDS-positive test) were considered to produce ESBL enzymes. Induction of the AmpC β-lactamase was also determined by a double disk test [13Leich C Boonlayangoor S β‐lactamases tests.in: Isenberg HD Clinical Microbiology Procedures Handbook. 1. American Society for Microbiology, Washington, DC1992: 5.3.1-5.3.8Google Scholar]. Conjugation experiments were performed with nalidixic acid-resistant E. coli BM21 strain as the recipient. Transconjugants were selected on MacConkey agar (Difco, Detroit, MI, USA) containing nalidixic acid (64 mg/L) and cefotaxime (2 mg/L). ESBL production in the transconjugants was confirmed with the DDS test. Isoelectric focusing was performed by electrophoresis of ultrasonic cell extracts on polyacrylamide gels containing ampholytes with pHs that ranged from 3 to 9 in a PhastSystem apparatus (Pharmacia Biotech, Uppsala, Sweden) [14Huovinen S Rapid isolectric focusing of plasmid‐mediated β‐lactamases with Pharmacia PhastSystem.Antimicrob Agents Chemother. 1988; 32: 1730-1732Crossref PubMed Scopus (26) Google Scholar]. β-Lactamases with known pIs (TEM-1, 5.4; TEM-4, 5.9; TEM-3, 6.3; SHV-2, 7.6; SHV-4, 7.8; CTX-M-10, 8.1; SHV-5, 8.2) were focused in parallel as controls. Gels were stained with a 0.2 mg/mL nitrocefin solution (Oxoid, Basingstoke, UK) to identify the β-lactamase bands. ESBL bla genes were amplified by using specific primers for blaTEM [15Mabilat C Goussard S Sougakoff W Spencer RC Courvalin P Direct sequencing of the amplified structural gene and promoter for the extended‐broad‐spectrum beta‐lactamase TEM‐9 (RHH‐1) of Klebsiella pneumoniae.Plasmid. 1990; 23: 27-34Crossref PubMed Scopus (88) Google Scholar] and blaSHV [16Nuesch‐Inderbinen MT Hachler H Kayser FH Detection of genes coding for extended‐spectrum SHV beta‐lactamases in clinical isolates by a molecular genetic method, and comparison with the E test.Eur J Clin Microbiol Infect Dis. 1996; 15: 398-402Crossref PubMed Scopus (216) Google Scholar]. In addition, forward primer 5′-GCT GAT-GAG CGC TTT GCG-3′ (nucleotide positions 193-210 of blaCTX-M-1) and reverse primer 5′-TTA CAA ACC GTT GGT-GAC G-3′ (last 19 nucleotides of blaCTX-M-1, including the stop codon) specific to CTX-M-1 and related enzymes (CTX-M-3 and CTX-M-10) were used [5Gniadkowski M Schneider I Palucha A Jungwirth R Mikiewicz B Bauernfeind A Cefotaxime‐resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX‐M‐3 cefotaxime‐hydrolyzing β‐lactamase that is closely related to the CTX‐M‐1/MEN‐1 enzyme.Antimicrob Agents Chemother. 1998; 42: 827-832PubMed Google Scholar, 17Oliver A Pérez‐Díaz JC Coque TM Baquero F Cantón R Nucelotide sequence and characterization of a novel cefotaxime‐hydrolysing β‐lactamase (CTX‐M‐10) isolated in Spain.Antimicrob Agents Chemother. 2001; 45: 616-620Crossref PubMed Scopus (94) Google Scholar, 18Tzouvelekis LS Tzelepi E Tassios PT Legakis NJ CTX‐M‐type beta‐lactamases: an emerging group of extended‐spectrum enzymes.Int J Antimicrob Agents. 2000; 14: 137-142Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar]. Eighteen per cent (51 of 277) of Enterobacteriaceae and 15.5% (30 of 193) of P. aeruginosa isolates were resistant to third-generation cephalosporins. The highest values were observed among Enterobader spp. (24.1%) and C. freundii (23.0%) isolates. Only three E. cloacae isolates (2.1% of all Enterobacter isolates tested) and one C. freundii (3.8%) isolate showed a DDS-positive test and were considered to produce ESBL enzymes. None of the Serratia spp., M. morganii, Providencia spp. and P. aeruginosa isolates displayed a DDS-positive test. It is remarkable that ceftazidime-clavulanate synergy, commonly used in commercial systems to detect ESBL-producing strains, was less suitable for detecting these β-lactamases, as a lower DDS-positive result was observed with this cephalosporin. In contrast, cefotaxime-clavulanate and cefepime-clavulanate synergies were more useful for detecting ESBLs among group 1 β-lactamase producers (Figure 1). The greater efficacy of these cephalosporins compared to ceftazidime could be related to the specific ESBL produced. In fact, all ESBL-producing isolates were PCR positive for blaCTX-M and not for blaTEM or blaSHV genes. In addition, cefepime, a fourth-generation cephalosporin, in contrast to those of the third generation, retains activity against AmpC-derepressed Enterobacteriaceae isolates [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar], and it could be more useful for detecting ESBL production in these isolates [8Tzelepi E Giakkoupi P Sofianou D Loukova V Kemeroglou A Tsakris A Detection of extended‐spectrum β‐lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.J Clin Microbiol. 2000; 38: 542-546PubMed Google Scholar]. Ceftazidime was specifically less affected in all four isolates with ESBL enzymes. This was also observed with the transconjugant isolates and the inoculum effect experiments (Table 1). As previously noted [8Tzelepi E Giakkoupi P Sofianou D Loukova V Kemeroglou A Tsakris A Detection of extended‐spectrum β‐lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.J Clin Microbiol. 2000; 38: 542-546PubMed Google Scholar], closer application of cefepime and clavulanate disks, 20-25 mm instead of 30 mm, enhanced the detection of ESBL-producing isolates, as a clear synergy inhibition zone was observed (Figure 1).Table 1MICs of β-lactam and non-β-lactam antibiotics against ESBL-producing isolates and their corresponding transconjugants, and the effect of different inoculum size on MIC values of several β-lactam antibiotics MIC (mg/L)MIC (mg/L)Antibiotic (inoculum size, CFU/mL)E. cloacae 99043823E. coli TC-99043823E. cloacae 99074125E. coli TC-99074125E. cloacae 99071078-1E. coli TC-99071078-1C. freundii 99071078-2E. coli TC-99071078-2Amoxicillin (105)>128>128>128>128>128>128>128>128Amoxicillin/clavulanate (105)6483243216648Ticarcillin (105)>128>128>128>128>128>128>128>128Piperacillin (105)12864>128>128>128>128>128>128Piperacillin/tazobactam(103)0.52224222(105)0.54244422(107)28488848Cefuroxime (105)256>256>256>256>256>256>256>256Cefotaxime(103)248464323216(105)88168128646464(107)>256>256256256>256>256>256>256Ceftazidime(103)0.110.50.58142(105)0.2110.516242(107)142232484Cefepime(103)0.212416444(105)0.522432888(107)>256>256256>256>256>256256>256Aztreonam(103)0.2424161688(105)0.544432321616(107)32128128128>256>256>256>256Imipenem (105)0.50.10.50.20.10.10.20.1Gentamicin (105)<1<1<1<1<1<1<1<1Tobramycin (105)<1<1<1<1<1<1<1<1Amikacin (105)<4<4<4<4<4<4<4<4Co-trimoxazole (105)<1/19<1/19<1/19<1/19<1/19<1/19<1/19<1/19 Open table in a new tab On the other hand, the disk induction approximation test demonstrated that all ESBL-producing isolates were inducible, and no AmpC hyperproduction was noted. It is interesting to note that, excluding all four ESBL-producing isolates, 11.7% of third-generation cephalosporin-resistant Enterobacteriaceae isolates still demonstrated a positive induction test. This discordance, which could reflect an AmpC partial depression or the absence of a major porin, has been extensively described in P. aeruginosa isolates [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar]. It is worth noting that all ESBL-producing isolates have a transferable β-lactamase of pi 8.1. The ESBL gene was amplified by PCR using specific primers for blaCTX-M genes for the CTX-M-1 and related enzymes, which may correspond to the CTX-M-10 newly described by our group [17Oliver A Pérez‐Díaz JC Coque TM Baquero F Cantón R Nucelotide sequence and characterization of a novel cefotaxime‐hydrolysing β‐lactamase (CTX‐M‐10) isolated in Spain.Antimicrob Agents Chemother. 2001; 45: 616-620Crossref PubMed Scopus (94) Google Scholar]. This CTX-M enzyme is widely distributed in our hospital, as nearly 50% of our ESBL-producing E. coli strains carried the same enzyme of pi 8.1 (data not shown). CTX-M ESBL is a heterogeneous family, which is endemic in South America and is now increasingly being recognized in Spain and other European countries [5Gniadkowski M Schneider I Palucha A Jungwirth R Mikiewicz B Bauernfeind A Cefotaxime‐resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX‐M‐3 cefotaxime‐hydrolyzing β‐lactamase that is closely related to the CTX‐M‐1/MEN‐1 enzyme.Antimicrob Agents Chemother. 1998; 42: 827-832PubMed Google Scholar, 17Oliver A Pérez‐Díaz JC Coque TM Baquero F Cantón R Nucelotide sequence and characterization of a novel cefotaxime‐hydrolysing β‐lactamase (CTX‐M‐10) isolated in Spain.Antimicrob Agents Chemother. 2001; 45: 616-620Crossref PubMed Scopus (94) Google Scholar, 18Tzouvelekis LS Tzelepi E Tassios PT Legakis NJ CTX‐M‐type beta‐lactamases: an emerging group of extended‐spectrum enzymes.Int J Antimicrob Agents. 2000; 14: 137-142Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 19Sabate M Tarrago Navarro F Miro E Verges C Barbe J Prats G Cloning and sequencing of the gene encoding a novel cefotaxime‐hydrolyzing β‐lactamase (CTX‐M‐9) from E. coli in Spain.Antimicrob Agents Chemother. 2000; 44: 1970-1973Crossref PubMed Scopus (126) Google Scholar]. AH ESBL-producing isolates were resistant according to NCCLS criteria [11National Committee for Clinical Laboratory Standards Performance Standards for Antimicrobial Susceptibility Testing; eleventh informational supplement. M100‐S11. NCCLS, Wayne, PA2001Google Scholar] to amoxicillin, amoxicillin-clavulanate, ticarcillin, piperacillin, cefuroxime, and cefotaxime, but susceptible to piperacillin-tazobactam (Table 1). In the absence of DDS and disk induction approximation tests, the latter combination could be essential to discriminate between AmpC hyperproduction and ESBL synthesis, as the former but not the latter commonly confers resistance to this combination [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar]. Considering the ceftazidime MICs, it is remarkable that the E. cloacae ESBL-producing isolates fall completely into the ceftazidime NCCLS susceptible category (MIC 0.2-4 mg/L) (Figure 2) [11National Committee for Clinical Laboratory Standards Performance Standards for Antimicrobial Susceptibility Testing; eleventh informational supplement. M100‐S11. NCCLS, Wayne, PA2001Google Scholar]. Following the NCCLS criteria, change to the resistance category is only considered when ESBLs are detected in Klebsiella spp. or E. coli isolates, but not when these enzymes are detected in other Enterobacteriaceae. However, this change, justified for the TEM- and SHV-type ESBL producers, could be unreliable for ceftazidime in the presence of CTX-M-type enzymes. In fact, remarkable increases in cefepime, aztreonam and cefotaxime MICs were observed with the increase of the inoculum size in all wild-type isolates and the corresponding transconjugants, but were barely observed with ceftazidime (Table 1), as this antibiotic is a poor substrate for CTX-M enzymes [5Gniadkowski M Schneider I Palucha A Jungwirth R Mikiewicz B Bauernfeind A Cefotaxime‐resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX‐M‐3 cefotaxime‐hydrolyzing β‐lactamase that is closely related to the CTX‐M‐1/MEN‐1 enzyme.Antimicrob Agents Chemother. 1998; 42: 827-832PubMed Google Scholar, 17Oliver A Pérez‐Díaz JC Coque TM Baquero F Cantón R Nucelotide sequence and characterization of a novel cefotaxime‐hydrolysing β‐lactamase (CTX‐M‐10) isolated in Spain.Antimicrob Agents Chemother. 2001; 45: 616-620Crossref PubMed Scopus (94) Google Scholar, 18Tzouvelekis LS Tzelepi E Tassios PT Legakis NJ CTX‐M‐type beta‐lactamases: an emerging group of extended‐spectrum enzymes.Int J Antimicrob Agents. 2000; 14: 137-142Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 19Sabate M Tarrago Navarro F Miro E Verges C Barbe J Prats G Cloning and sequencing of the gene encoding a novel cefotaxime‐hydrolyzing β‐lactamase (CTX‐M‐9) from E. coli in Spain.Antimicrob Agents Chemother. 2000; 44: 1970-1973Crossref PubMed Scopus (126) Google Scholar]. As previously pointed out [17Oliver A Pérez‐Díaz JC Coque TM Baquero F Cantón R Nucelotide sequence and characterization of a novel cefotaxime‐hydrolysing β‐lactamase (CTX‐M‐10) isolated in Spain.Antimicrob Agents Chemother. 2001; 45: 616-620Crossref PubMed Scopus (94) Google Scholar], the report of isolates harboring CTX-M β-lactamases as susceptible or resistant to ceftazidime can only be resolved with clinical observations. Interestingly, and in contrast to the majority of ESBL-producing isolates [1Livermore DM β‐Lactamases in laboratory and clinical resistance.Clin Microbiol Rev. 1995; 8: 557-584Crossref PubMed Google Scholar, 2Jacoby GA Medeiros AA More extended spectrum β‐lactamases.Antimicrob Agents Chemother. 1991; 35: 1697-1704Crossref PubMed Scopus (654) Google Scholar], the association between aminoglycoside and/or co-trimoxazole resistance and ESBL production was not found (Table 1). It is not surprising that none of the P. aeruginosa isolates produced ESBLs, as these enzymes are less frequently observed in these isolates than in Enterobacteriacaeae. To date, TEM-, SHV- and OXA-type ESBLs have been identified in P. aeruginosa, and most isolates have been recovered in Turkey [20Nordmann P Guibert M Extended spectrum‐betalactamases in Pseudomonas aeruginosa.J Antimicrob Chemother. 1998; 42: 128-131Crossref PubMed Google Scholar]. On the other hand, the prevalence of ESBLs among group 1 β-lactamase-producing Enterobacteriaceae during the studied 6-month period in our hospital was low (1.4%). Nevertheless, this result represented 7.8% of all third-generation cephalosporin-resistant group 1 β-lactamase-producing Enterobacteriaceae. Considering the prevalence of ESBLs in E. cloacae, three of 107 isolates (2.8%), this value was similar to that previously observed in other studies [4Coudron PE Moland ES Sanders CC Occurrence and detection of extended‐spectrum beta‐lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find.J Clin Microbiol. 1997; 35: 2593-2597PubMed Google Scholar, 7Silva J Aguilar C Becerra Z López‐Antunano F García R Extended‐spectrum beta‐lactamases in clinical isolates of enterobacteria in Mexico.Microb Drug Resist. 1999; 5: 189-193Crossref PubMed Scopus (19) Google Scholar] but lower than that found by Tzelepi et al [8Tzelepi E Giakkoupi P Sofianou D Loukova V Kemeroglou A Tsakris A Detection of extended‐spectrum β‐lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.J Clin Microbiol. 2000; 38: 542-546PubMed Google Scholar], who recently observed 32% of E. cloacae strains with ESBLs over a 3-month period. The prevalence of these organisms in our hospital seems to have been quite stable during the last decade, as only 11 E. cloacae and three E. aerogenes isolates with ESBLs have been detected during this period. The possibility of clonal relatedness of all these isolates requires further study. Nevertheless, E. cloacae isolates were recovered from unrelated patients attending different units. From a clinical point of view, the discrimination between ESBLs and overproduced group 1 β-lactamases may not be critical, but it is important for epidemiologic purposes. The possibilities of plasmid transmission among different isolates and patient-to-patient transmission of ESBL-producing isolates support the continuous monitoring of these isolates. This approach could enhance the epidemiologic importance of detecting these enzymes among group 1 β-lactamase-producing isolates.