Carbapenemase-negative Isolates Research Articles

In vitro activity of cefiderocol against a global collection of carbapenem-resistant Pseudomonas aeruginosa with a high level of carbapenemase diversity.

To determine the in vitro activity of cefiderocol in a global collection of carbapenem-resistant Pseudomonas aeruginosa including >200 carbapenemase-producing isolates. Isolates (n = 806) from the ERACE-PA Surveillance Program were assessed. Broth microdilution MICs were determined for cefiderocol (iron-depleted CAMHB) and comparators (CAMHB). Susceptibility was interpreted by CLSI and EUCAST breakpoints and reported as percent of isolates. The MIC distribution of cefiderocol in the entire cohort and by carbapenemase status was assessed. In the entire cohort, cefiderocol was the most active agent (CLSI 98% susceptible; EUCAST 95% susceptible; MIC50/90, 0.25/2 mg/L). Amikacin (urinary only breakpoint) was the second most active, with 70% of isolates testing as susceptible. The percentage of isolates susceptible to all other agents was low (<50%) including meropenem/vaborbactam, imipenem/relebactam, piperacillin/tazobactam and levofloxacin. Cefiderocol maintained significant activity against the most commonly encountered carbapenemases including VIM- (CLSI 97% susceptible; EUCAST 92% susceptible) and GES (CLSI 100% susceptible; EUCAST 97% susceptible)-harbouring isolates. The cefiderocol MIC distribution was similar regardless of carbapenemase status, with MIC50/90 values of 0.5/4 mg/L, 0.5/2 mg/L and 0.25/1 mg/L for MBL, serine carbapenemase and molecular carbapenemase-negative isolates, respectively. Cefiderocol displayed potent in vitro activity in this global cohort of carbapenem-resistant P. aeruginosa including >200 carbapenemase-harbouring isolates. Cefiderocol was highly active against MBL-producing isolates, where treatment options are limited. These data can help guide empirical therapy guidelines based on local prevalence of carbapenemase-producing P. aeruginosa or in response to rapid molecular diagnostics.

2755. Activity of Aztreonam-avibactam and Carbapenem-resistant Enterobacterales (CRE) Isolates Collected in a Six-year Period (2017–2022)

Abstract Background Beta-lactam/beta-lactamase inhibitor (BL/BLI) combinations and cefiderocol (FDC) have been recommended for the treatment of carbapenem-resistant Enterobacterales (CRE), but extensive comparisons of these agents are limited. We tested the activity of aztreonam-avibactam (ATM-AVI), ceftazidime-avibactam (CAZ-AVI), meropenem-vaborbactam (MEV), imipenem-relebactam (IR), all BL/BLIs, and FDC against &amp;gt; 500 CRE isolates collected in US hospitals during a 6-year period (2017–2022). Methods A total of 54,576 Enterobacterales (ENT) isolates collected during 2017–2021 were susceptibility (S) tested by reference broth microdilution methods. CRE isolates displayed meropenem and/or imipenem MIC results at &amp;gt; 1 mg/L and were submitted to whole genome sequencing for analysis of resistance mechanisms. Results The CRE isolates (n=511; 0.9% overall) were mainly KPC producers without metallo-beta-lactamases (n=346; 67.7%). Genes encoding MBLs and OXA-48–like enzymes alone were detected among 50 and 12 isolates, respectively. Another 12 isolates carried genes encoding NMC-A and SME. ATM-AVI inhibited 98.4% of the CRE isolates when applying ATM breakpoints for comparison. FDC, CAZ-AVI, MEV, and IR inhibited 94.7%, 89.2%, 86.7%, and 81.9% of the CRE isolates, respectively. Against class A serine-carbapenemase–producing isolates without MBLs, ATM-AVI, CAZ-AVI, FDC, and MEV demonstrated activity &amp;gt; 98.6%. IR inhibited 92.7% of these isolates. Except for ATM-AVI and FDC, other BL/BLI had limited activity against MBLs, ranging from 2.0% S for IR to 16% for MEV against MBL isolates. ATM-AVI inhibited 98.0% of the MBL isolates while FDC inhibited 74.0%. ATM-AVI, CAZ-AVI, IR, FDC, and MEV inhibited 94.5%, 96.7%, 90.1%, 89.0%, and 87.9% of the carbapenemase-negative isolates (n=91), respectively. Among comparators, tigecycline was the most active, inhibiting 94.7% of the CRE isolates; 84.7% of these isolates displayed a colistin intermediate MIC value of ≤ 2 mg/L. Conclusion The new BL/BLIs and FDC displayed good activity against a large collection of CRE isolates that were mainly KPC producers from US hospitals. Against MBLs, ATM-AVI was the most active agent, followed by FDC. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Joshua Maher, PhD, AbbVie: Grant/Research Support|Affinity Biosensors: Grant/Research Support|AimMax Therapeutics, Inc: Grant/Research Support|Alterity Therapeutics: Grant/Research Support|Amicrobe, Inc: Grant/Research Support|Arietis Pharma: Grant/Research Support|Armata Pharmaceuticals, Inc: Grant/Research Support|Astrellas Pharma, Inc.: Grant/Research Support|Basilea Pharmaceutica AG: Grant/Research Support|Becton Dickinson And Company: Grant/Research Support|bioMerieux, Inc: Grant/Research Support|Boost Biomes: Grant/Research Support|Diamond V: Grant/Research Support|Fedora Pharmaceuticals, Inc: Grant/Research Support|Iterum Therapeutics plc: Grant/Research Support|Johnson &amp; Johnson: Grant/Research Support|Kaleido Biosciences, Inc.: Grant/Research Support|Meiji Seika Pharma Co. Ltd.: Grant/Research Support|National Institutes of Health: Grant/Research Support|Pfizer Inc.: Grant/Research Support|Roche Holding AG: Grant/Research Support|Shionogi Inc.: Grant/Research Support|Summmit Therapeutics, Inc.: Grant/Research Support|Zoetis Inc: Grant/Research Support Katie Simpson, BS, AbbVie: Grant/Research Support Cory Hubler, BS, AbbVie: Grant/Research Support|Shionogi: Grant/Research Support Helio S. Sader, MD, PhD, FIDSA, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Distribution of β-lactamases and emergence of carbapenemases co-occurring Enterobacterales isolates with high-level antibiotic resistance identified from patients with intra-abdominal infection in the Asia-Pacific region, 2015-2018.

In this study, we aimed to assess the geographic distribution and molecular characteristics of β-lactamases among Enterobacterales isolates causing intra-abdominal infections (IAIs) from 2015 to 2018 in the Asia-Pacific region. Isolates were investigated for extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases using multiplex PCR assays and full-gene DNA sequencing. A total of 832 Enterobacterales isolates from 8 different countries with β-lactamase genes were analysed. Plasmid-mediated ESBLs and AmpC β-lactamases were encoded in 598 (71.9%) and 314 (37.7%) isolates, respectively. In 710 (85.3%) carbapenemase-negative isolates, positivity for both AmpC β-lactamases and ESBLs was identified in 51 (8.5%) Escherichia coli and 24 (3.4%) Klebsiella pneumoniae isolates. The most prevalent countries were Taiwan and Vietnam, and the co-occurrence of CMY/CTX-M in E. coli and DHA-1/ESBLs in K. pneumoniae was predominant. All isolates showed high susceptibility to colistin, but susceptibility to carbapenems varied among different resistance mechanism combinations. Among 122 (14.7%) isolates encoding carbapenemase, NDM (n=67, including 64.2% NDM-1) was the most common, followed by the OXA-48-type (n=49), KPC (n=24) and IMP (n=4). The most prevalent country was Thailand (n=44), followed by Vietnam (n=35) and the Philippines (n=21). Twenty-two isolates were found to encode multiple carbapenemases, 16 of which were collected from Thailand and harbored NDM-1, OXA-232 and CTX-M-15. Despite high susceptibility to amikacin, susceptibility to colistin was only 56%. The emergence of carbapenem-non-susceptible AmpC/ESBL co-occurring Enterobacterales and colistin non-susceptible carbapenemases co-occurring K. pneumoniae highlights potential therapeutic challenges in the Asia-Pacific region.

202. Comparative Activity of Ceftazidime-Avibactam, Imipenem-Relebactam and Meropenem-Vaborbactam Tested Against Carbapenem-Nonsusceptible Enterobacterales that Are Carbapenemase-Negative

202. Comparative Activity of Ceftazidime-Avibactam, Imipenem-Relebactam and Meropenem-Vaborbactam Tested Against Carbapenem-Nonsusceptible <i>Enterobacterales</i> that Are Carbapenemase-Negative

1218. Activity of Meropenem-Vaborbactam and Comparator Agents Against Carbapenemase-Negative, Carbapenem-Resistant Enterobacterales from US Hospitals

1218. Activity of Meropenem-Vaborbactam and Comparator Agents Against Carbapenemase-Negative, Carbapenem-Resistant <i>Enterobacterales</i> from US Hospitals

Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility.

Background and Objectives:Imipenem/relebactam (IMP/R) is a newly FDA approved β-lactam/β-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility.Materials and Methods:Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for β-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method.Results:During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebactam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla OXA-48 _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-β-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R susceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity.Conclusion:Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no activity against MBL− producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treatment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

The ERACE-PA Global Surveillance Program: Ceftolozane/tazobactam and Ceftazidime/avibactam in vitro Activity against a Global Collection of Carbapenem-resistant Pseudomonas aeruginosa

The cephalosporin-β-lactamase-inhibitor-combinations, ceftolozane/tazobactam and ceftazidime/avibactam, have revolutionized treatment of carbapenem-resistant Pseudomonas aeruginosa (CR-PA). A contemporary assessment of their in vitro potency against a global CR-PA collection and an assessment of carbapenemase diversity are warranted. Isolates determined as CR-PA by the submitting site were collected from 2019–2021 (17 centers in 12 countries) during the ERACE-PA Global Surveillance Program. Broth microdilution MICs were assessed per CLSI standards for ceftolozane/tazobactam, ceftazidime/avibactam, ceftazidime, and cefepime. Phenotypic carbapenemase testing was conducted (modified carbapenem inactivation method (mCIM)). mCIM positive isolates underwent genotypic carbapenemase testing using the CarbaR, the CarbaR NxG, or whole genome sequencing. The MIC50/90 was reported as well as percent susceptible (CLSI and EUCAST interpretation). Of the 807 isolates, 265 (33%) tested carbapenemase-positive phenotypically. Of these, 228 (86%) were genotypically positive for a carbapenemase with the most common being VIM followed by GES. In the entire cohort of CR-PA, ceftolozane/tazobactam and ceftazidime/avibactam had MIC50/90 values of 2/ > 64 and 4/64 mg/L, respectively. Ceftazidime/avibactam was the most active agent with 72% susceptibility per CLSI compared with 63% for ceftolozane/tazobactam. For comparison, 46% of CR-PA were susceptible to ceftazidime and cefepime. Against carbapenemase-negative isolates, 88 and 91% of isolates were susceptible to ceftolozane/tazobactam and ceftazidime/avibactam, respectively. Ceftolozane/tazobactam and ceftazidime/avibactam remained highly active against carbapenem-resistant P. aeruginosa, particularly in the absence of carbapenemases. The contemporary ERACE-PA Global Program cohort with 33% carbapenemase positivity including diverse enzymology will be useful to assess therapeutic options in these clinically challenging organisms with limited therapies.

Improvement of the Immunochromatographic NG-Test Carba 5 Assay for the Detection of IMP Variants Previously Undetected.

Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-Pseudomonas aeruginosa, where IMPs are most frequently detected.

Evaluation of the Immunochromatographic NG-Test Carba 5 for Rapid Identification of Carbapenemase in Nonfermenters

The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp.

In vitro activity of cefiderocol, a siderophore cephalosporin, against a recent collection of clinically relevant carbapenem-non-susceptible Gram-negative bacilli, including serine carbapenemase- and metallo-β-lactamase-producing isolates (SIDERO-WT-2014 Study)

Cefiderocol is a siderophore cephalosporin in development for treatment of infections caused by Gram-negative bacilli, including carbapenem-resistant and multidrug-resistant isolates. β-Lactamase carriage and in vitro activity of cefiderocol were determined against 1272 meropenem-non-susceptible isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii collected as part of the SIDERO-WT-2014 surveillance study. Minimum inhibitory concentration (MIC) values for cefiderocol were ≤4 µg/mL against 97.7% of tested isolates, including 100% of IMP-positive (range, 1-2 µg/mL), OXA-58-positive (MIC90, 1 µg/mL), KPC-positive (MIC90, 2 µg/mL), VIM-positive (MIC90, 2 µg/mL), and OXA-48-like-positive (MIC90, 4 µg/mL) isolates; 99.3% of carbapenemase-negative isolates (MIC90, 1 µg/mL); 97.2% of OXA-23-positive isolates (MIC90, 1 µg/mL); 95.2% of OXA-24-positive isolates (MIC90, 1 µg/mL); 91.7% of GES-positive isolates (MIC90, 4 µg/mL); and 64.3% of NDM-positive isolates (MIC90, 8 µg/mL). A total of 29 isolates (2.3%; 15 OXA-23-producers, 6 OXA-24-producers, 5 NDM-producers, and 3 carbapenemase-negative isolates) exhibited cefiderocol MIC ≥8 µg/mL, confirming there was no clear correlation between carriage of β-lactamases included in the molecular testing algorithm and elevated cefiderocol MICs. Similarly, no correlation was observed between cefiderocol MICs and truncation or loss of porin proteins in meropenem-non-susceptible isolates of E. coli and K. pneumoniae. Cefiderocol MICs were also ≤4 µg/mL against 99.3% of 136 colistin-resistant Enterobacteriaceae collected as part of the SIDERO-WT-2014 study, including isolates carrying mcr-1 (MIC90, 2 µg/mL). Cefiderocol demonstrated potent in vitro activity against a collection of carbapenemase-producing and carbapenemase-negative meropenem-non-susceptible Gram-negative bacilli for which few treatment options are available, including the majority of metallo-β-lactamase producing isolates identified.

Evaluation of the EntericBio CPE assay for the detection of carbapenemase-producing organisms.

Carbapenemase-producing organisms (CPOs) represent an increasing threat in healthcare facilities and detection of these organisms in the diagnostic laboratory can be challenging. The EntericBio CPE (EBCPE) real-time PCR assay (Serosep Ltd) was evaluated for the detection of NDM, KPC, OXA-48-like, VIM, IMP and GES carbapenemase genes from a panel of 145 multidrug-resistant organisms (29 NDM, 35 OXA-48, 21 VIM, 4 OXA-23, 3 KPC, 5 NDM+OXA-48, 3 GES-5, 1 OXA-23+NDM, 1 IMI, 2 IMP-1 and 41 multidrug-resistant carbapenemase-negative isolates). The EBCPE assay performed well, with 100 % sensitivity and specificity for the detection of all genotypes included in the assay. Turnaround time and laboratory workflow were improved compared to culture-based assays. Users must remain aware of the limitations of molecular assays for CPO detection to ensure implementation of the most suitable CPO diagnostic pathways.

Evaluation of modified carbapenem inactivation method for suspected carbapenemase among Enterobacteriaceae clinical isolates.

Modified carbapenem inactivation method (mCIM) testing was currently recommended by Clinical and Laboratory Standards Institute (CLSI) for detection of carbapenemase among Enterobacteriaceae clinical isolates. In this study, a panel of 145 clinical strains were collected for evaluating the mCIM for detection of carbapenemase. Antimicrobial susceptibility testing were performed by microbroth dilution and the results were interpreted according to CLSI guidelines. All strains were resistant to ertapenem with high MIC50 and MIC90 (64 mg/L –>128 mg/L). For blaNDM-1-positive or blaOXA-232-positive strains, the zone of inhibition of meropenem were all 6 mm despite the incubation time of 6 h, 18 h or 24 h. For 6 h, the zone of meropenem inhibition for most of carbapenemase-positive isolates were meet the positive criteria 6–15 mm. However, for carbapenemase-negative isolates, the zone of meropenem inhibition were 16–18 mm after 6 h incubation which should be considered indeterminate for standard incubating time such as 18 h or 24 h. After incubating for 18 h or 24 h, the zone of meropenem inhibition were 22–25 mm for carbapenemase-negative isolates and meet the negative criteria. Our study indicate mCIM is a simple and effective method to identify the carbapenemases producers among Enterobacteriaceae clinical isolates.

Genomics and Susceptibility Profiles of Extensively Drug-Resistant Pseudomonas aeruginosa Isolates from Spain.

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

In Vitro Susceptibility to Ceftazidime-Avibactam of Carbapenem-Nonsusceptible Enterobacteriaceae Isolates Collected during the INFORM Global Surveillance Study (2012 to 2014)

The activity of ceftazidime-avibactam was assessed against 961 isolates of meropenem-nonsusceptible Enterobacteriaceae Most meropenem-nonsusceptible metallo-β-lactamase (MBL)-negative isolates (97.7%) were susceptible to ceftazidime-avibactam. Isolates that carried KPC or OXA-48-like β-lactamases, both alone and in combination with extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamases, were 98.7% and 98.5% susceptible to ceftazidime-avibactam, respectively. Meropenem-nonsusceptible, carbapenemase-negative isolates demonstrated 94.7% susceptibility to ceftazidime-avibactam. Ceftazidime-avibactam activity was compromised only in isolates for which carbapenem resistance was mediated through metallo-β-lactamases.

Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers

To assess the performance of the agar disc diffusion method for the detection of carbapenemase-producing Enterobacteriaceae (CPE) referred to the national reference laboratories (NRLs) in Belgium and France. All Enterobacteriaceae isolates referred to the NRLs for the confirmation of CPE in 2012 were included. The inhibition zone diameters of meropenem, piperacillin/tazobactam and temocillin using CLSI disc diffusion methodology were recorded. Phenotypic and molecular detection of carbapenemases was performed on all isolates. A total of 1354 Enterobacteriaceae isolates, including 435 (32.1%) confirmed CPE isolates [OXA-48 (n = 323), KPC (n = 60), VIM (n = 32) and NDM (n = 20)] and 919 carbapenemase-negative isolates, were tested. Using recommended interpretative criteria, non-susceptibility to meropenem had poor sensitivity (52.0% by CLSI susceptibility breakpoint and 80.0% by EUCAST screening breakpoints), while non-susceptibility to piperacillin/tazobactam (according to CLSI breakpoint) or to temocillin (according to Fuchs, Barry, Thornsberry et al. Eur J Clin Microbiol 1985; 4: 30-3) was highly sensitive (99.8% and 98.2%, respectively) but poorly specific (29.4% and 42.9%, respectively) for the detection of CPE. Temocillin diameters <12 mm alone had high specificity (90.0%) and the combination of temocillin diameters ≥12 mm with piperacillin/tazobactam diameters ≥16 mm observed in 40% of all referred isolates displayed excellent negative predictive value (99.2%). In geographical areas with a high prevalence of OXA-48 producers, recommended meropenem susceptibility or screening breakpoints failed to detect CPE in a large proportion of isolates. The combination of modified zone diameter cut-offs for piperacillin/tazobactam (≥16 mm) and temocillin (≥12 mm) can be used to rule out the presence of carbapenemase and avoid unnecessary additional testing for confirmation of CPE.

MALDI-TOF MS applied to indirect carbapenemase detection: a validated procedure to clearly distinguish between carbapenemase-positive and carbapenemase-negative bacterial strains

Laboratory identification of carbapenemase-producing clinical isolates is crucial to limit the spread of the bacteria. In this study, we shall first develop the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) assay in automatic identification of carbapenemase producers. A total of 143 well-characterized isolates were studied. After an incubation of bacteria with meropenem trihydrate, the mixture was centrifuged and the supernatant analyzed by MALDI-TOF MS. A genetic algorithm model with ClinProTools software was built using spectra of 43 carbapenemase-positive isolates and 40 carbapenemase-negative isolates after 2 h of incubation. This model was externally validated using 60 test isolates. All spectra of supernatants of the carbapenemase-negative isolates showed peak profiles comparable to that of pure meropenem (m/z 384.159, 406.140, and 428.122 of its two sodium salt variants) regardless of the incubation time tested. For the carbapenemase-positive isolates, the specific peak for meropenem at m/z 384.159 disappeared during the incubation time, two products of meropenem degradation were identified with m/z 358.18 (the decarboxylated product) and 380.161 (sodium salt of the decarboxylated product), and other degradation products were observed (native molecule with disrupted amide bond with m/z 402.169, three sodium salt variants with m/z 424.151, 446.133, and 468.115). Sixty test isolates were 100% correctly classified as carbapenemase positive and carbapenemase negative with the genetic algorithm model. MALDI-TOF MS coupled with ClinProTools is capable of rapidly, accurately, and automatically identifying carbapenemase producers.

Susceptibility patterns to extended-spectrum cephalosporins among Enterobacteriaceae harbouring extended-spectrum β-lactamases using the updated Clinical and Laboratory Standards Institute interpretive criteria

We examined the effect of applying the updated 2010 Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints for extended-spectrum cephalosporins (ESCs) to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. In total, 202 ESBL-producing, plasmidic AmpC- and carbapenemase-negative isolates derived from separate patients were collected from three Greek hospitals during 2007–2011, including 150 Escherichia coli, 43 Klebsiella pneumoniae and 9 Enterobacter cloacae clinical isolates. ESBLs were detected using the ESBL CLSI confirmatory test and PCR assays. Sequencing analysis showed that 91 (45.0%) of the ESBL-producers carried the blaCTX-M-3 gene, 66 (32.7%) carried the blaCTX-M-15 gene and the remaining 45 (22.3%) carried the blaSHV-5 gene. Minimum inhibitory concentrations for cefotaxime, ceftazidime and cefepime were determined by the agar dilution method. Based on the new CLSI breakpoints, 13 (6.4%) of the ESBL-producers were susceptible to cefotaxime, 90 (44.6%) to ceftazidime and 112 (55.4%) to cefepime; as many as 145 (71.8%) were susceptible to at least one ESC. Among the 150 E. coli, 12 (8.0%), 87 (58.0%) and 79 (52.7%) were susceptible to cefotaxime, ceftazidime and cefepime, respectively, whilst among the 43 K. pneumoniae, 1 (2.3%), 3 (7.0%) and 25 (58.1%) were susceptible to the above ESCs, respectively. None of the nine E. cloacae were susceptible to cefotaxime and ceftazidime, but all except one were susceptible to cefepime. By implementation of the new 2010 CLSI breakpoints, a considerable proportion of ESBL-possessing Enterobacteriaceae would be reported as susceptible, mostly to ceftazidime and cefepime, leading to possible infection control and therapeutic implications.

Laboratory Detection of Enterobacteriaceae That Produce Carbapenemases

A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae. One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli, Citrobacter freundii, and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturer's instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae.

KMP048 Five percent of Enterobacter cloacae strains produce Extended spectrum β-lactamases (ESBLs) in Germany

We examined the effect of applying the updated 2010 Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints for extended-spectrum cephalosporins (ESCs) to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. In total, 202 ESBL-producing, plasmidic AmpC- and carbapenemase-negative isolates derived from separate patients were collected from three Greek hospitals during 2007–2011, including 150 Escherichia coli, 43 Klebsiella pneumoniae and 9 Enterobacter cloacae clinical isolates. ESBLs were detected using the ESBL CLSI confirmatory test and PCR assays. Sequencing analysis showed that 91 (45.0%) of the ESBL-producers carried the blaCTX-M-3 gene, 66 (32.7%) carried the blaCTX-M-15 gene and the remaining 45 (22.3%) carried the blaSHV-5 gene. Minimum inhibitory concentrations for cefotaxime, ceftazidime and cefepime were determined by the agar dilution method. Based on the new CLSI breakpoints, 13 (6.4%) of the ESBL-producers were susceptible to cefotaxime, 90 (44.6%) to ceftazidime and 112 (55.4%) to cefepime; as many as 145 (71.8%) were susceptible to at least one ESC. Among the 150 E. coli, 12 (8.0%), 87 (58.0%) and 79 (52.7%) were susceptible to cefotaxime, ceftazidime and cefepime, respectively, whilst among the 43 K. pneumoniae, 1 (2.3%), 3 (7.0%) and 25 (58.1%) were susceptible to the above ESCs, respectively. None of the nine E. cloacae were susceptible to cefotaxime and ceftazidime, but all except one were susceptible to cefepime. By implementation of the new 2010 CLSI breakpoints, a considerable proportion of ESBL-possessing Enterobacteriaceae would be reported as susceptible, mostly to ceftazidime and cefepime, leading to possible infection control and therapeutic implications.