Ian Morrissey, 1 Samuel K. Bouchillon, 2 Meredith Hackel, 2 Douglas J. Biedenbach, 2 Stephen Hawser, 1 Daryl Hoban 2 and Robert E.

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1 Journal of Medical Microbiology (2014), 63, DOI /jmm Evaluation of the Clinical and Laboratory Standards Institute phenotypic confirmatory test to detect the presence of extended-spectrum b-lactamases from 4005 Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates Ian Morrissey, 1 Samuel K. Bouchillon, 2 Meredith Hackel, 2 Douglas J. Biedenbach, 2 Stephen Hawser, 1 Daryl Hoban 2 and Robert E. Badal 2 Correspondence Ian Morrissey imorrissey@ihmainc.com Received 30 September 2013 Accepted 29 January IHMA Europe Sàrl, 1066 Epalinges, Switzerland 2 International Health Management Associates, Schaumburg, IL , USA A subset of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates collected for the Study for Monitoring Antimicrobial Resistance Trends that were positive for the Clinical and Laboratory Standards Institute (CLSI) extended-spectrum b-lactamase (ESBL) phenotypic confirmatory test (n53245) or had an ertapenem MIC of 0.5 mg ml 1 (n5293), or both (n5467), were analysed for ESBL genes. Most ESBL phenotype E. coli or K. pneumoniae possessed an ESBL gene (95.8 and 88.4 %, respectively), and this was 93.1 % if carbapenem-non-susceptible K. pneumoniae were removed. This rate was lower for P. mirabilis (73.4 %) and K. oxytoca (62.5 %). Virtually all ESBL-positive isolates (99.5 %) were cefotaxime non-susceptible [CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints)]. Fewer isolates (82 %) were ceftazidime non-susceptible (CLSI breakpoints). In addition, 21.1 % of E. coli,25%ofk. oxytoca and 78.7 % of P. mirabilis isolates were ceftazidime susceptible but ESBL positive. This suggests that CLSI breakpoints for ceftazidime are too high to detect ESBLs. The lower EUCAST breakpoints detected ESBLs in E. coli and K. oxytoca better, but 59.6 % of ESBL-positive isolates of P. mirabilis were ceftazidime susceptible. For isolates with ertapenem MICs 0.5 mg ml 1, more accurate ESBL phenotype analysis was observed for E. coli and K. pneumoniae (sensitivity.95 % for both, specificity 94.4 and 54.1 %, respectively). If carbapenemase-positive K. pneumoniae were excluded, the specificity increased to 78 %. The positive predictive values for the ESBL phenotypic test with E. coli and K. pneumoniae were 97.6 and 81.8 %, respectively, and negative predictive values were 75.9 and 95.2 %, respectively. We therefore suggest that it would be prudent to confirm phenotypic ESBLpositive P. mirabilis, K. pneumoniae and K. oxytoca with molecular analysis. INTRODUCTION It is well established that extended-spectrum b-lactamases (ESBLs) are an important clinical issue, and their presence can have serious medical implications and associated healthcare costs (Solomkin et al., 1990; Walsh et al., 2005). Abbreviations: CLSI, Clinical and Laboratory Standards Institute; ESBL, extended-spectrum b-lactamase; EUCAST, European Committee on Antimicrobial Susceptibility Testing; SMART, Study for Monitoring Antimicrobial Resistance Trends. Eight supplementary tables are available with the online version of this paper. Furthermore, the prevalence of ESBLs has been increasing steadily over the last 10 years or so (Leclerc et al., 2011). Since 2010, both the Clinical and Laboratory Standards Institute (CLSI, 2010) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2013) have reduced the breakpoints for most cephalosporins against Enterobacteriaceae and as a consequence no longer recommend specific testing for ESBL presence or adjustment of penicillin, cephalosporin or monobactam susceptibility accordingly (Leclerc et al., 2011), unless clinical laboratories are using automated systems that retain the older higher CLSI breakpoints (CLSI, 2012a). Although there is a G 2014 SGM Printed in Great Britain

2 Evaluation of the CLSI ESBL phenotypic test Table 1. Isolates included in the study by species, year of collection, infection and geographical region Characteristic E. coli K. oxytoca K. pneumoniae P. mirabilis Total Year Infection Intra-abdominal Urinary tract Unknown Region Africa Asia Europe Latin America Middle East North America South Pacific Overall total common recommendation not to routinely test for ESBLs from both of the two main antimicrobial resistance expert groups in the world, the decision to exclude ESBL detection is not without controversy (Livermore et al., 2012).It has also been suggested that CLSI ESBL phenotypic confirmatory tests are unable to detect the presence of ESBLs if AmpC enzymes are also present (Munier et al., 2010). Recently, the use of CLSI or EUCAST disk diffusion methodology breakpoints was shown to be better than CLSI MIC ESBL screening breakpoints for the detection of ESBLs, especially those isolates having an AmpC b-lactamase, using a panel of 236 well-characterized isolates (Polsfuss et al., 2012). As a consequence, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates (n54005) from the Study for Monitoring Antimicrobial Resistance Trends (SMART) were assessed to correlate the CLSI ESBL phenotypic test, susceptibility to cefotaxime and ceftazidime (EUCAST and CLSI breakpoints), and molecular characterization of the b-lactamase genes detected. METHODS Study isolates. All isolates in the study were from intra-abdominal ( ) or urinary tract infections ( ), and only one isolate per species per patient was accepted. All organisms were deemed clinically significant based on the local participants criteria. Isolate inclusion was independent of antimicrobial use, age or gender. The isolates were identified to species level at each site, and sent to a central laboratory (International Health Management Associates, Schaumburg, Illinois, USA) for confirmation of identification using a RapidOne panel (Remel) and antimicrobial susceptibility testing. International Health Management Associates managed the development of a centralized database of the study results. The isolates were collected between 2008 and 2011 from various countries worldwide. Isolate details are given in Table 1. Susceptibility testing. MIC values were determined using custom MicroScan dehydrated broth microdilution panels (Siemens Medical Solutions Diagnostics), following CLSI (2012b) and the manufacturer s guidelines. For the purpose of inclusion in this analysis, MIC data for the following antimicrobial agents (with their dilution ranges expressed in mg ml 21 ) were used: ertapenem, ; ceftazidime, ; ceftazidime clavulanic acid, ; cefotaxime, ; cefotaxime clavulanic acid, MICs were interpreted following CLSI (2012a) and EUCAST (2013) guidelines. CLSI phenotypic confirmatory test to detect the presence of ESBL. Following CLSI guidelines, Enterobacteriaceae were classified as ESBL producers (hereafter referred to as pesbl positive) if there was at least an eightfold reduction (i.e. three doubling dilutions) of the MIC for ceftazidime or cefotaxime in combination with clavulanic acid versus their MICs when tested alone (CLSI 2012a). Where MIC Table 2. Isolates included in the study by ertapenem MIC and pesbl positive Species Ertapenem MIC 0.5 mg ml 1 but non-esbl phenotype ESBL phenotype, including ertapenem MIC 0.5 mg ml 1 Total E. coli K. pneumoniae K. oxytoca P. mirabilis Total

3 I. Morrissey and others Table 3. Molecular detection of ESBLs in 3712 pesblpositive isolates by species Species data were off scale (i.e. the MICs of cefotaxime and/or ceftazidime alone and with clavulanic acid were greater than the highest concentration tested on the panel), disk testing with ceftazidime (30 mg) or cefotaxime (30 mg) in combination with or without clavulanic acid (10 mg) was used according to CLSI (2012a) guidelines. Molecular characterization. DNA was extracted from overnight colonies of isolates positive for the ESBL phenotypic test or with an ertapenem MIC 0.5 mg ml 21 grown on blood agar (Remel) using a QIAamp DNA Mini kit and a QIAcube instrument (Qiagen). PCR for characterization of ESBLs was performed in an ABI 9700 thermocycler (Applied Biosystems). bla genes for TEM-, SHV-, CTX-M-, OXA-48-, metallo- (IMP, VIM, SPM, SIM, GIM and NDM) and KPC-type enzymes were amplified as described previously (Ellington et al., 2007; Livermore et al., 2007; Nordmann et al., 2009; Solomkin et al., 1990; Walsh et al., 2005; Woodford et al., 2008). PCR was carried out with a Fast Cycling PCR kit (Qiagen). Purification of the PCR products was performed using Exo-SAP-IT (USB). PCR-amplified fragments were sequenced using an ABI 3730XL DNA analyser (Applied Biosystems). Nucleotide sequences were analysed with SeqScape v.7.0 (Applied Biosystems) and compared with sequences available at the National Center for Biotechnology Information website ( nih.gov). Multiplex amplification of pampc genes was performed as described previously by Pérez-Pérez & Hanson (2002). Quality control. Quality control was performed each day of testing using the CLSI-recommended quality control strains E. coli ATCC 25922, E. coli ATCC and K. pneumoniae ATCC (ESBLpositive control). Results were included in the analysis only when the corresponding quality control isolates tested within the acceptable ranges according to CLSI (2012a) guidelines. RESULTS mesbl negative [n (%)] mesbl positive [n (%)] K. pneumoniae 138 (11.7) 1045 (88.3) E. coli 102 (4.2) 2299 (95.8) K. oxytoca 24 (37.5) 40 (62.5) P. mirabilis 17 (26.6) 47 (73.4) Total 281 (7.5) 3431 (92.5) Susceptibility testing and ESBL confirmatory test Of the 3712 pesbl-positive isolates, 3671 were identified by MIC testing and 41 isolates required in addition disk susceptibility testing (data not shown). The 3712 pesblpositive isolates comprised 3245 isolates with ertapenem MICs,0.5 mg ml 21 and 467 isolates with ertapenem MICs 0.5 mg ml 21. In addition, there were 293 isolates with ertapenem MICs 0.5 mg ml 21 but that were non-esbl phenotype. These results are summarized in Table 2. These 4005 isolates were subjected to molecular analysis for detection of ESBLs and other b-lactamase genes. Molecular analysis of ESBL phenotypes The majority of the 3712 pesbl positive contained an ESBL gene (hereafter referred to as mesbl positive; 92.5 % in total). A larger proportion was observed in E. coli (95.9 %), and fewer were found in the other bacterial species, especially K. oxytoca (62.5 %) (Table 3). A breakdown of the ESBL enzymes is shown in Table S1 (available in the online Supplementary Material), with the most common ESBL being CTX-M. Further details of the ESBL enzymes detected are given in Tables S2 S6. The highest number of false-positive pesbl isolates was found in K. pneumoniae (n5138, Table 3), and 42.1 % of these possessed a carbapenemase (Tables 4 and S7). As would be expected, all 56 carbapenemase-positive K. pneumoniae isolates were ertapenem non-susceptible (results not shown), and if these were included within the pesbl-positive population, the detection success for the ESBL phenotype test for K. pneumoniae increased to 93.1 % (data not shown). Very few of the other bacterial species that were pebsl positive actually possessed a b- lactamase (Table 4), and of these the majority were AmpCs (Table S8), plus two K. oxytoca with KPC-2. None of the pebsl-positive (but mesbl-negative) isolates possessed more than one b-lactamase. Cefotaxime and ceftazidime susceptibility of ESBL phenotypes The summary MIC and susceptibility data for cefotaxime and ceftazidime against mesbl-positive Enterobacteriaceae are shown in Table 5. Both CLSI and EUCAST have identical breakpoints for cefotaxime and these classified.95 % of the mesbl-positive species as cefotaxime non-susceptible. However, for ceftazidime, detection was poor, with only 21.2 % of P. mirabilis isolates found to be non-susceptible by Table 4. Presence of other b-lactamases in mesbl-negative isolates Species mesbl-negative (n) No. of isolates [n (%)] with b-lactamase: AmpC Carbapenemase No mechanism K. pneumoniae (3.0) 56 (42.1) 73 (54.9) E. coli (9.8) 3 (2.9) 89 (87.3) K. oxytoca 24 1 (4.2) 2 (8.3) 21 (87.5) P. mirabilis 17 1 (5.9) 0 (0.0) 16 (94.1) Total (5.7) 61 (21.8) 203 (72.5) 558 Journal of Medical Microbiology 63

4 Evaluation of the CLSI ESBL phenotypic test Table 5. Summary MIC and susceptibility data for cefotaxime and ceftazidime against mesbl-positive Enterobacteriaceae Antibiotic Species (n) MIC (mg ml 1 )* CLSI breakpointsd EUCAST breakpointsd MIC 50 MIC 90 S (%) I (%) R (%) S (%) I (%) R (%) Cefotaxime Escherichia coli (2299) Klebsiella oxytoca (40) Klebsiella pneumoniae (1045) Proteus mirabilis (47) All (3431) Ceftazidime Escherichia coli (2299) Klebsiella oxytoca (40) Klebsiella pneumoniae (1045) Proteus mirabilis (47) All (3431) S, Susceptible; I, intermediate; R, resistant. *MIC 50, MIC inhibiting 50 % of the bacterial species; MIC90, MIC inhibiting 90 % of the bacterial species. DCLSI S/I/R breakpoints are 1/2/4 for cefotaxime, and 4/8/16 for ceftazidime. deucast S/I/R breakpoints are 1/2/4 for cefotaxime, and 1/2/4 for ceftazidime. CLSI breakpoints (or 40.4 % by EUCAST breakpoints). In addition, detection of mesbl-positive E. coli (79 %) or K. oxytoca (75 %) with CLSI breakpoints for ceftazidime was much weaker than that seen with cefotaxime. Only ESBLs in K. pneumoniae were adequately detected by ceftazidime MIC values (Table 5). Molecular analysis of isolates with ertapenem MICs 0.5 mg ml 1 Although ertapenem susceptibility is not related to ESBL phenotype per se, this subset was analysed because it included pesbl-negative isolates as well as pesbl-positive isolates (unlike the other bacteria evaluated in this paper). Of the 760 isolates tested, 547 were pesbl positive and 213 were pesbl negative (Table 6). Most of the isolates were E. coli or K. pneumoniae, and these were analysed further for specificity, sensitivity, positive predictive value and negative predictive value(table7).thephenotypicscreenperformedwellagainst E. coli but specificity was poor against K. pneumoniae. As indicated above this problem could be resolved by removing the carbapenemase-positive strains (Table 7). However, in practice, this would require an additional phenotypic screen. However, the CLSI phenotypic test was not as good at detecting true ESBLs from K. pneumoniae, where 137 isolates of this species (11.6 %) were incorrectly identified as ESBL positive; 56 of these were shown to possess a carbapenemase (all of which had an ertapenem MIC 0.5 mg l 21 ). From broth microdilution MICs, more than 99 % of the molecularly confirmed ESBL-positive E. coli and K. pneumoniae were found to be cefotaxime nonsusceptible. Therefore, for these species, it is probably not necessary to undertake further molecular analysis if the cefotaxime MIC is determined, unless for epidemiological or infection control purposes. In contrast, ceftazidime CLSI non-susceptibility was much weaker at predicting ESBLs with E. coli (21.1 % ESBL-positive E. coli were ceftazidime susceptible); therefore, the ceftazidime MIC should not be used as a single marker for the presence of ESBLs according to CLSI breakpoints for E. coli. The lower EUCAST breakpoints for ceftazidime, on the other hand, were able to detect 95 % of the ESBL-positive E. coli, in agreement with the findings of Polsfuss et al. (2012). This suggests that the CLSI ceftazidime breakpoints are too high to be used as a marker for ESBLs, and that they may be more useful if lowered to match those used by EUCAST. The ceftazidime DISCUSSION In this study, we analysed a total of 4005 clinical isolates of E. coli, K. oxytoca, K. pneumoniae and P. mirabilis from SMART worldwide during These isolates were screened as part of SMART for the presence of b-lactamase genes either because they were positive for ESBLa using the CLSI phenotypic test or because they had an ertapenem MIC 0.5 mg ml 21. The data indicated that, for E. coli, the CLSI phenotypic screen correctly identified 95.8 % as having an ESBL gene. Table 6. ESBL phenotypes for 760 isolates with ertapenem MICs 0.5 mg ml 1 by species Species Phenotype negative [n (%)] Phenotype positive [n (%)] Total (n) E. coli 72 (26.0) 205 (74.0) 277 K. oxytoca 3 (27.3) 8 (72.7) 11 K. pneumoniae 136 (29.0) 333 (71.0) 469 P. mirabilis 2 (66.7) 1 (33.3) 3 Total 213 (28.0) 547 (72.0)

5 I. Morrissey and others Table 7. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the CLSI phenotypic test for E. coli and K. pneumoniae with ertapenem MICs 0.5 mg ml 1 Species True positive False negative False positive True negative Sensitivity* SpecificityD PPVd NPV E. coli K. pneumoniae K. pneumoniae (carbapenemase negative) *Sensitivity5true positive/(true positive+false negative). DSpecificity5true negative/(true negative+false positive). dppv5true positive/(true positive+false positive). NPV5true negative/(true negative+false negative). susceptibility for K. pneumoniae by CLSI breakpoints, on the other hand, was adequate to detect ESBLs in this species. For K. oxytoca and P. mirabilis, a larger number of ESBL false-positive isolates was seen when using the CLSI phenotype (37.5 and 26.5 %, respectively) than for E. coli (4.2 %) and K. pneumoniae (11.7 %). There was no evidence that this was caused by potential interference with other b-lactamases, including AmpC (Munier et al., 2010). Use of cefotaxime non-susceptibility alone was able to detect.95 % of the ESBL true-positive isolates. Similarly to E. coli, ESBL-positive K. oxytoca isolates were not adequately detected by ceftazidime susceptibility using CLSI breakpoints but were with EUCAST breakpoints. For P. mirabilis, neither CLSI nor EUCAST breakpoints for ceftazidime were able to adequately detect ESBL-positive isolates (78.7 % ESBL-positive P. mirabilis isolates were susceptible according to CLSI ceftazidime breakpoints and 59.6 % by EUCAST). This result does not agree with the findings of Polsfuss et al. (2012), but in their study, only two P. mirabilis isolates were investigated, making it difficult to draw conclusions about this species. To summarize, the CLSI ESBL phenotypic test predicted the presence of an ESBL gene well for E. coli and K. pneumoniae (except those that were ertapenem nonsusceptible), as did cefotaxime susceptibility, but not ceftazidime susceptibility alone especially for P. mirabilis. False-positive results were obtained more often with P. mirabilis and K. oxytoca. Therefore, we suggest that it may be prudent to confirm phenotypically ESBL-positive P. mirabilis, K. pneumoniae and K. oxytoca by additional molecular or other analyses. It may also be worthwhile reconsidering the CLSI breakpoints for ceftazidime against Enterobacteriaceae. ACKNOWLEDGEMENTS We thank all the SMART investigators for their participation in this programme. We are also grateful to Dr Christine Lascols for her participation in the molecular analysis for this publication. The SMART surveillance programme is funded by Merck & Co.. All authors have served as scientific advisors or consultants to Merck, and received research support from Merck to conduct this study. REFERENCES CLSI (2010). Performance Standards for Antimicrobial Susceptibility Testing; 20th Informational Supplement M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute. CLSI (2012a). Performance Standards for Antimicrobial Susceptibility Testing; 22nd Informational Supplement M100-S22. Wayne, PA: Clinical and Laboratory Standards Institute. CLSI (2012b). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 9th edn, M7- A9. Wayne, PA: Clinical and Laboratory Standards Institute. Ellington, M. J., Kistler, J., Livermore, D. M. & Woodford, N. (2007). Multiplex PCR for rapid detection of genes encoding acquired metallo-b-lactamases. J Antimicrob Chemother 59, EUCAST (2013). EUCAST Clinical Breakpoints v.3. 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