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Increasing numbers of variants of the carbapenem-hydrolyzing class D β-lactamase OXA-48 are identified in Enterobacterales worldwide. Among them, OXA-181 and OXA-232 are of particular interest, as they differ from each other by a single amino acid substitution at position 214 (R in OXA-181 and S in OXA-232) that results in reduced carbapenem-hydrolyzing activity for OXA-232. To investigate the role of amino acid position 214 (AA214), the X-ray structure of OXA-232 was determined and AA214 of OXA-48 and of OXA-232 was replaced by G, L, D, E, S, R, and K using site-directed mutagenesis. These mutants were phenotypically characterized, and three mutants of OXA-232 were purified to study their steady-state kinetic properties. The X-ray structure of OXA-232 along with molecular modeling studies showed that the interaction via a salt bridge between R214 and D159 in OXA-48 is not possible with the G214 or S214 mutation. In contrast, with K214, which is also positively charged, the interaction with D159 is maintained. With the E214 mutant, an alternative binding conformation of imipenem that is not compatible with a nucleophilic attack by S70 was evidenced. Thus, imipenem has a very poor apparent affinity for the E214 mutant because of its nonproductive binding mode. Similarly, we could explain the lack of temocillin hydrolysis by the OXA-232-S214E mutant, which is due to the unfavorable interaction between the negatively charged R1 substituent of temocillin with the E214 residue. Overall, we demonstrate that AA214 in OXA-48-like β-lactamases is critical for the carbapenemase activity., (Copyright © 2020 American Society for Microbiology.)

The recently released CHROMagar™ ESBL/CHROMagar™ mSuperCARBA bi-plate medium was evaluated for the detection of ESBL- and carbapenemase-producing Enterobacteriaceae. Spiked stools were used to mimic in vivo stool colonization. Two-hundred enterobacterial isolates were tested. Respective sensitivities of 93.9% and 97.8% were obtained for the detection of ESBL and carbapenemase producers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene mcr-1 (mostly in Escherichia coli ) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals., (Copyright © 2019 Volland et al.)

The immunochromatographic assay, NG-test Carba 5 (NG Biotech), has been evaluated for detection of carbapenemase-producing Enterobacterales (CPE) from spiked blood cultures ( n  = 205). It detected and discriminated in less than 30 minutes KPC, IMP, VIM, NDM, and OXA-48-like producers with a sensitivity and specificity of 97.7% and 96.1%, respectively. Thus, it might help the rapid optimization of treatment of bloodstream infections due to CPE., (Copyright © 2019 American Society for Microbiology.)

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has led to the increased use of colistin, which has resulted in the emergence of colistin-resistant Enterobacteriaceae worldwide. One of the most threatening scenarios is the dissemination of colistin resistance in CPE, particularly the plasmid-encoded resistance element MCR. Thus, it has now become mandatory to possess reliable media to screen for colistin-resistant Gram-negative bacterial isolates, especially Enterobacteriaceae In this study, we evaluated the performances of the Superpolymyxin medium (ELITechGroup) and the ChromID Colistin R medium (bioMérieux) to screen for colistin-resistant Enterobacteriaceae from spiked rectal swabs. Stool samples were spiked with a total of 94 enterobacterial isolates ( Escherichia coli , Klebsiella pneumoniae , Salmonella enterica , Enterobacter cloacae ), including 53 colistin-resistant isolates. ESwabs (Copan Diagnostics) were then inoculated with those spiked fecal suspensions, and culture proceeded as recommended by both manufacturers. The sensitivity of detection of colistin-resistant Enterobacteriaceae was 86.8% (95% confidence interval [95% CI] = 74.0% to 94.0%) using both the Superpolymyxin medium and the ChromID Colistin R plates. Surprisingly, the isolates that were not detected were not the same for both media. The specificities were high for both media, at 97.9% (95% CI = 87.3% to 99.9%) for the Superpolymyxin medium and 100% (95% CI = 90.4% to 100%) for the ChromID Colistin R medium. Both commercially available media, ChromID Colistin R and Superpolymyxin, provide useful tools to screen for colistin-resistant Enterobacteriaceae from patient samples (rectal swabs) regardless of the level and mechanism of colistin resistance., (Copyright © 2018 American Society for Microbiology.)

OXA-48-like beta-lactamase producing bacteria are now endemic in several European and Mediterranean countries. Among this carbapenemase family, the OXA-48 and OXA-181 variants predominate, whereas other variants such as OXA-204 are rarely reported. Here, we report the molecular epidemiology of a collection of OXA-204-positive enterobacterial isolates (n = 29) recovered in France between October 2012 and May 2014. This study describes the first outbreak of OXA-204-producing Enterobacteriaceae in Europe, involving 12 isolates of an ST90 Escherichia coli clone and nine isolates of an ST147 Klebsiella pneumoniae clone. All isolates co-produced the cephalosporinase CMY-4, and 60% of them co-produced the extended-spectrum beta-lactamase CTX-M-15. The bla OXA-204 gene was located on a 150-kb IncA/C plasmid, isolated from various enterobacterial species in the same patient, indicating a high conjugative ability of this genetic vehicle.

Objectives: To describe a novel plasmid-borne class D carbapenemase (CHDL) named OXA-427 identified in several Enterobacteriaceae clinical isolates from nine patients in one Belgian hospital., Methods: OXA-427-producing isolates were analysed by an electrochemical imipenem hydrolysis method (BYG Carba test), Carba NP test, conventional phenotypic assays and by molecular methods (PCR, whole sequencing of the OXA-427-encoding plasmid and cloning). The antimicrobial resistance profile of OXA-427 was analysed by expression of the cloned gene in Escherichia coli DH10B and J53., Results: Eleven OXA-427-producing Enterobacteriaceae isolates of various species were identified from clinical specimens of nine patients between March 2012 and June 2014. OXA-427 shares only 22%-29% amino acid identity with OXA-48-like enzymes and other acquired CHDL (e.g. OXA-23, -24/40 and -58 of Acinetobacter spp.). Conversely, it appeared closely related to the chromosomal class D β-lactamase of Aeromonas media, Aeromonas hydrophila and Aeromonas sobria (99%, 89% and 77% of identity, respectively). When expressed in E. coli, OXA-427 hydrolysed imipenem and conferred resistance to extended-spectrum cephalosporins (mostly ceftazidime), penicillins including temocillin, and reduced susceptibility to carbapenems. The blaOXA-427 gene was located in a 45 kb resistance island on a 177 kb IncA/C plasmid., Conclusions: OXA-427 is a novel CHDL most closely related to chromosomal class D β-lactamase of A. media WS. It confers resistance to penicillins, ceftazidime and aztreonam and in some instances to carbapenems. OXA-427, which is not detectable by classical molecular tests, caused a protracted outbreak in one university hospital over a 2 year period., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We describe the case of NDM-1-producing Leclercia adecarboxylata recovered from the clinical sample of a patient hospitalized for a trauma-related injury to his foot. The isolate was resistant to all beta-lactams, quinolones, trimetroprim-sulfametoxazol, gentamicin and tobramicyn. The bla NDM-1 gene was located in a conjugative plasmid that also contained the bla SHV-12 gene and was preceded by a disrupted insertion sequence of ISAba125. The plasmid belongs to the incompatibility group X3, which is known to be an important vector for NDM-1 dissemination in China. This is the first reported case of NDM-1L. adecarboxylata in our country and evidences that species of uncertain clinical relevance can act as hidden sources of clinically important resistance determinants., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we have evaluated a novel colorimetric test (the β-CARBA™ test; Bio-Rad) to detect carbapenemase-producing Gram-negative bacilli from cultured colonies., Methods: The performance of the β-CARBA™ test was compared with that of the Carba NP test (or the CarbAcineto NP test) and RAPIDEC ® CARBA NP (bioMérieux) using a collection of 290 isolates with characterized β-lactamase content. This collection included 199 carbapenemase producers (121 Enterobacteriaceae, 36 Pseudomonas and 42 Acinetobacter baumannii ) and 91 non-carbapenemase producers (55 Enterobacteriaceae, 20 Pseudomonas and 16 A. baumannii )., Results: The β-CARBA™ test correctly detected 84.9% of the carbapenemase producers, including all KPC and IMP, 96.4% of VIM, 85.3% of NDM, 80.5% of OXA-48-like and 91.2% of A. baumannii -related OXA carbapenemases (OXA-23, OXA-40, OXA-58, OXA-143 and overexpressed OXA-51). All rare metallo-β-lactamases (SPM, AIM, GIM, DIM and SIM) were detected. Importantly, all non-KPC Ambler class A carbapenemases were not detected, including GES variants with carbapenemase activity ( n  = 6), IMI ( n  = 3), NMC-A ( n  = 1), SME ( n  = 2), FRI-1 ( n  = 1) and BIC-1 ( n  = 1). All non-carbapenemase producers gave a negative result except with OXA-163-, OXA-405- and one TEM-3-producing Citrobacter freundii . The overall sensitivity and specificity of the β-CARBA™ test were 84.9% and 95.6%, respectively. This test is easy to perform and to interpret by non-specialized staff members., Conclusions: Despite lack of specificity towards non-KPC Ambler class A and OXA-48-like carbapenemases, the β-CARBA™ test could complete the existing panel of tests available for the confirmation of carbapenemases in Gram-negatives., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: There is an urgent need for accurate and rapid diagnostic tests to identify carbapenemase producing enterobacteria (CPE). Here, we have evaluated the Carbapenem Inactivation Method (CIM) test to detect CPEs from cultured colonies., Methods: A total of 256 enterobacterial isolates were used to evaluate the performance of the CIM in comparison to Carba NP test and molecular detection used a reference method. Ninety three well-characterized isolates (including 29 non-CPE and 63 CPEs of worldwide origin) with decreased susceptibility to at least one carbapenem were used to (i) evaluate the efficacy of CIM test and (ii) to compare it to the Carba NP test. We also tested different carbapenems to determine the best substrate for this test. Finally, the CIM test was then evaluated prospectively against 164 isolates referred to the French National Reference Center (NRC) for Antimicrobial Resistance from may 2016 to july 2016., Results: Based on the results of this retrospective study, sensitivity and specificity of the CIM and the Carba NP test were 92.1% and 100%, respectively. We demonstrated that the meropenem was the best substrate to perform the CIM test since sensitivity and specificity were 81.1% and 100% using ertapenem disk, and 100% and 65,6% using imipenem disk, and respectively. Taking in account the results of retrospective and prospective studies, CIM and Carba NP tests have similar sensitivity, specificity, positive predictive value and negative predictive values being 96.3%, 98.9%, 99.0% and 98.4% for the CIM test versus 96.9%, 100%, 100% and 100% for the Carba NP test., Conclusions: Our results confirm that the CIM test may be a useful tool for the reliable confirmation of carbapenemase-activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources, no trained personnel, and no specialized equipment., Competing Interests: Competing Interests: LD is co-inventor of the Carba NP Test, which patent (WO2012175637 A1) has been licenced to bioMérieux (La Balmes les Grottes, France). This does not interfere with the adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors.

Multidrug resistant bacteria and emerging antibiotic resistance traits. Since the 1990s, the dissemination of extended spectrum β-lactamase producing-Enterobacteriaceae that are resistant to a wide variety of common antimicrobials is serious global health concern. Carbapenems have rapidly become the treatment of choice of severe infections due to ESBL-producing Enterobacteriaceae, but carbapenem- resistant Enterobacteriaceae have been increasingly reported in France and worldwide. These bacteria are mainly found in feacal carriage but the number of clinically-relevant infections is increasing. In that context, we observe a renewed interest for old drugs like temocillin, tigecyclin or polymyxins. When susceptible, aminoglycosides are also often used to treat infections due to multiresistant bacteria. Unfortunately, in addition to significant risks of toxicity, the use of these last resort drugs can be compromised due to the emergence of new mechanisms of resistance., Competing Interests: Les auteurs déclarent n’avoir aucun lien d’intérêts.

The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array 'Check-MDR CT103 XL' (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs (blaTEM, blaSHV, blaCTX-M, blaBEL, blaPER, blaGES and blaVEB), pAmpCs (blaCMY-2-like, blaDHA, blaFOX, blaACC-1, blaACT/MIR and blaCMY-1-like/MOX) and carbapenemases (blaKPC, blaOXA-48, blaVIM, blaIMP, blaNDM, blaGIM, blaSPM and blaOXA-23, -24 and -58) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for blaIMP. The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents., (Copyright © 2016. Published by Elsevier B.V.)

Five GES-producing Enterobacteriaceae isolates that displayed an extended-spectrum β-lactamase (ESBL) phenotype harbored two GES variants: GES-7 ESBL and GES-6 carbapenemase. In all isolates, the two GES alleles were located on the same integron that was inserted into an 80-kb IncM1 self-conjugative plasmid. Whole-genome sequencing suggested in vivo horizontal gene transfer of the plasmid along with clonal diffusion of Enterobacter cloacae To our knowledge, this is the first description in Europe of clustered Enterobacteriaceae isolates carrying two GES β-lactamases, of which one has extended activity toward carbapenems., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify MDR bacteria. Here, we evaluated an immunochromatographic assay (the OXA-48 K-SeT assay) to detect OXA-48-like carbapenemase-producing Enterobacteriaceae from culture colonies., Methods: One hundred and sixty-one collection isolates with characterized β-lactamase content and 185 non-duplicate consecutive clinical isolates referred to the Associated French National Reference Center between 15 February and 15 March 2015 were used to evaluate the OXA-48 K-SeT assay. Among these 346 isolates, 100 were OXA-48-like carbapenemase producers, 3 were OXA-48-like producers lacking carbapenemase activity and 243 were ESBL, AmpC, oxacillinase and/or non-OXA-48 carbapenemase producers., Results: All 100 OXA-48-like carbapenemase producers were correctly detected by the OXA-48 K-SeT assay, including OXA-48 (n = 73), OXA-181 (n = 18), OXA-162 (n = 1), OXA-204 (n = 4), OXA-232 (n = 2) and OXA-244 (n = 2) variants. The three OXA-48 variants lacking carbapenemase activity, OXA-163 (n = 2) and OXA-405 (n = 1), were not detected. All non-OXA-48 producers gave a negative result with the OXA-48 K-SeT assay. No cross-reaction was evidenced with the carbapenemases (VIM, IMP, NDM and KPC), ESBLs (TEM, SHV and CTX-M), AmpCs (CMY-2, DHA-2 and ACC-1) and oxacillinases (OXA-1, -2, -9 and -10). Overall, the sensitivity and specificity of the assay were 100% for OXA-48-like carbapenemase detection., Conclusions: The OXA-48 K-SeT assay was efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of OXA-48-like carbapenemase-producing Enterobacteriaceae. It could complete the existing panel of tests available for the confirmation of OXA-48-like carbapenemases, especially in countries with high OXA-48 prevalence., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of blaOXA-181 and blaOXA-232 genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceae epidemiology of many countries worldwide., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: The objective of this study was to assess the performance of an algorithm based on the disc diffusion method for the screening of carbapenemase-producing Enterobacteriaceae (CPE) referred to the French National Reference Centre for Antibiotic Resistance., Methods: From April to June 2014, all isolates of Enterobacteriaceae referred to the French National Reference Centre for Antibiotic Resistance were included. The inhibition zone diameters of imipenem, ticarcillin/clavulanate and temocillin using EUCAST disc diffusion methodology were recorded. All isolates were subjected to the algorithm proposed by the Antibiogram Committee of the French Society of Microbiology (CA-SFM) for the screening of carbapenemase producers. Phenotypic, biochemical and molecular detection of carbapenemases was performed for all isolates., Results: A total of 621 consecutive enterobacterial isolates with decreased susceptibility to carbapenems were tested. They included 213 CPE [OXA-48-like (n = 183), NDM (n = 22), VIM (n = 3), KPC (n = 3) and OXA-48-like + NDM (n = 2)] and 408 non-carbapenemase producers. The CA-SFM algorithm combining cut-off values of 15 mm, 15 mm and 22 mm for ticarcillin/clavulanate, temocillin and imipenem, respectively, perfectly detected 204 isolates (32.8%) as non-carbapenemase producers, leading to a negative predictive value of 100% for this algorithm., Conclusions: Implementation of the CA-SFM algorithm in clinical microbiology laboratories may avoid additional testing for CPE in one-third of the enterobacterial isolates with decreased susceptibility to carbapenems., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The objective of this study was the evaluation of the performance of two commercially available biochemical tests for the rapid detection of carbapenemase-producing Enterobacteriaceae compared with a home-made technique., Methods: A collection of 150 enterobacterial isolates, including 132 isolates with decreased susceptibility to at least one carbapenem molecule, were tested for carbapenemase activity using the RAPIDEC(®) CARBA NP (bioMérieux), the Rapid CARB Screen(®) (Rosco Diagnostica) and the home-made Carba NP test. This strain collection included 55 non-carbapenemase producers, 21 KPC producers, 21 NDM producers, 17 VIM producers, 11 IMP producers, 16 OXA-48 producers and 9 OXA-48-like producers (OXA-162, OXA-181, OXA-204, OXA-232 and OXA-244)., Results: The RAPIDEC(®) CARBA NP detected all carbapenemase producers except a single OXA-244 producer. Using the Rapid CARB Screen(®), one KPC-2, two NDM-1, one OXA-48 and five OXA-48 variant producers gave equivocal results and one OXA-244 producer was not detected. Using the Carba NP test, the same OXA-244 producer was not detected and one OXA-181 producer and one OXA-244 producer gave equivocal results. Sensitivity and specificity were 99% (95% CI 94.3%-99.8%) and 100% (95% CI 93.5%-100%), respectively, for the RAPIDEC(®) CARBA NP test, 89.5% (95% CI 81.7%-94.2%) and 70.9% (95% CI 57.9%-81.2%) for the Rapid CARB Screen(®) and 96.8% (95% CI 91.1%-98.9%) and 100% (95% CI 93.5%-100%) for the Carba NP test. The impact of the use of an adequate bacterial inoculum for obtaining the optimal performance with the RAPIDEC(®) CARBA NP was noted., Conclusions: The RAPIDEC(®) CARBA NP possesses the best performance for rapid and efficient detection of carbapenemase-producing Enterobacteriaceae., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Fifteen carbapenemase-producing Enterobacteriaceae isolates and 12 carbapenemase-producing Pseudomonas aeruginosa isolates were recovered from patients hospitalized between August 2011 and March 2013 at the Hospital of Infectious Disease, Cluj-Napoca, Romania. One KPC-, nine NDM-1-, four OXA-48-, and one VIM-4-producing Enterobacteriaceae isolates along with 11 VIM-2-producing and one IMP-13-producing P. aeruginosa isolates were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were associated with other resistance determinants, including extended-spectrum β-lactamases and RmtC methylases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The recognition of carbapenemase-producing Enterobacteriaceae (CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum β-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of ≥0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Klebsiella pneumoniae carbapenemase (KPC)-type enzymes have largely disseminated worldwide among K. pneumoniae isolates. In this study, 11 non-K. pneumoniae KPC-producing enterobacterial isolates from four hospitals located in different Colombian cities were genetically investigated. All isolates were multidrug-resistant and harboured the bla(KPC-2) gene along with several other acquired β-lactamase genes. The bla(KPC-2) gene was associated with transposon Tn4401b inserted in different loci of plasmids varying in size and replicon type. The presence of KPC-2 in different enterobacterial species from different cities within Colombia underlines the spread of KPC beyond K. pneumoniae., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Carbapenemases increasingly have been reported in Enterobacteriaceae in the past 10 years. Klebsiella pneumoniae carbapenemases have been reported in the United States and then worldwide, with a marked endemicity at least in the United States and Greece. Metallo-enzymes (Verona integron-encoded metallo-β-lactamase, IMP) also have been reported worldwide, with a higher prevalence in southern Europe and Asia. Carbapenemases of the oxacillinase-48 type have been identified mostly in Mediterranean and European countries and in India. Recent identification of New Delhi metallo-β-lactamase-1 producers, originally in the United Kingdom, India, and Pakistan and now worldwide, is worrisome. Detection of infected patients and carriers with carbapenemase producers is necessary for prevention of their spread. Identification of the carbapenemase genes relies mostly on molecular techniques, whereas detection of carriers is possible by using screening culture media. This strategy may help prevent development of nosocomial outbreaks caused by carbapenemase producers, particularly K. pneumoniae.

Objectives: CTX-M extended-spectrum beta-lactamases (ESBLs) are emerging worldwide. Fast and reliable detection techniques may become mandatory for implementing proper treatment and infection control measures. Here, a bla(CTX-M)-specific LightCycler real-time PCR (LC-PCR) assay based on hybridization probes was developed., Methods: Urine samples positive for Gram-negative bacilli as revealed by Gram staining were collected over a 3 month period at Bicêtre hospital, France. Aliquots of these urine samples were frozen for subsequent molecular analysis, and the bacteria were cultured and identified by standard bacteriological techniques (biochemical tests, disc diffusion antibiogram and synergy testing). LC-PCR and standard PCR followed by sequencing was performed on all ESBL-positive and on 70 randomly chosen ESBL-negative urine samples., Results: Over the study period, 810 urine samples were collected from 655 patients. Thirty-six ESBL-producing Enterobacteriaceae, mostly Escherichia coli (77%), were identified from 29 patients, of which half were outpatients. Twenty-five urine samples (19 patients) were found to be positive for bla(CTX-M) genes using the LC-PCR assay. The bla(CTX-M) genes belonged to the bla(CTX-M-1), bla(CTX-M-9) and bla(CTX-M-2) groups (68%, 24% and 8%, respectively). Standard PCR and sequencing of the entire bla(CTX-M) genes confirmed the LC-PCR results; 17 CTX-M-15, 6 CTX-M-9 and 2 CTX-M-2. Among the remaining ESBLs, eight were of the TEM type and three of the SHV type., Conclusions: The LC-PCR assay represents a powerful tool for rapid identification of CTX-M producers in urine samples.

The chromogenic agar medium chromID ESBL (bioMérieux) was compared with BLSE agar medium (AES) for selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from clinical samples. A total of 765 samples (468 rectal swabs, 255 urine samples and 42 pulmonary aspirations) obtained from 547 patients was processed. All bacterial strains isolated on either medium were further characterized using biochemical tests, and ESBL producers were confirmed by synergy testing. Genetic characterization of ESBL genes was determined by PCR. A total of 33 ESBL-producing Enterobacteriaceae strains [Escherichia coli (n=16), Klebsiella pneumoniae (n=8), Enterobacter spp. (n=3), Citrobacter spp. (n=5) and Proteus mirabilis (n=1)] was recovered. The sensitivity after 24 h incubation was 88 % for chromID ESBL and 85 % for BLSE agar. At 48 h, the sensitivity of chromID ESBL increased to 94 % and was higher than that obtained with BLSE agar. The positive predictive value at 24 h for chromID ESBL was 38.7 % [95 % confidence interval (95 % CI) 28.3 -50.2 %)], which was significantly higher than that for BLSE agar [15.4 %, 95 % CI 10.1 -21.5 %]. On both media, false-positive results were mostly due to Pseudomonas aeruginosa and to Enterobacteriaceae overproducing chromosomal cephalosporinase (Enterobacter spp.) or a chromosomal penicillinase (Klebsiella oxytoca). This study showed that chromID ESBL, a ready-to-use chromogenic selective medium, is sensitive and specific for rapid, presumptive identification of ESBL-producing Enterobacteriaceae. Its chromogenic properties and its selectivity are particularly useful in specimens containing resident associated flora.

IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla(VEB-1) and bla(OXA-48). 5' Rapid amplification of cDNA ends experiments revealed that expression of these beta-lactamase genes was driven by the outward-directed promoter, P(out), located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

Since 2016, OXA-244-producing Escherichia coli has been increasingly isolated in France. We sequenced 97 OXA-244-producing E. coli isolates and found a wide diversity of sequence types and a high prevalence of sequence type 38. Long-read sequencing demonstrated the chromosomal location of blaOXA-244 inside the entire or truncated Tn51098. [ABSTRACT FROM AUTHOR]

In Enterobacterales, the most common carbapenemases are Ambler's class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996. [ABSTRACT FROM AUTHOR]

Objectives: Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood.Methods: Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates.Results: We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure.Conclusions: This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs. [ABSTRACT FROM AUTHOR]

Background: SME carbapenemases are increasingly reported, especially from North and South America. Here, we describe an SME-4-producing Serratia marcescens (SME-Sm) clinical isolate from Argentina and compare its genome with other SME-Sm and Sm isolates recovered from public databases.Methods: Sm isolates were characterized by WGS using Illumina technology, susceptibility testing and MIC determination. Carbapenemase activity was revealed by biochemical tests based on imipenem hydrolysis. A whole-genome phylogeny was estimated for all the Sm isolates retrieved from public databases with kSNP3 and a whole-genome phylogenetic analysis based on non-recombinant core SNPs was inferred for Sm complete genomes and for those encoding any blaSME variants.Results: Sm163 was resistant to amoxicillin, temocillin, aztreonam and carbapenems, remaining susceptible to extended-spectrum cephalosporins. WGS analysis of Sm163 revealed a genome of 5139329 bp and a chromosomally encoded blaSME-4 carbapenemase gene located on a genomic island closely related to SmarGI1-1 of Sm N11-02820. Comparison of the Sm genomes revealed that the 14 SME-Sm isolates possess this genomic island inserted at the same loci, that 13/14 belong to clade 1 and that 11/14 form a well-defined subcluster of cluster I of Sm clade 1, while Sm163 belongs to clade 2, suggesting that an SME-encoding genomic island may have been transferred between isolates from different clades.Conclusions: To the best of our knowledge this is the first report of an SME-4-encoding Sm from Argentina. The blaSME-4 gene is located on a SmarGI1-1-like genomic island. The genome of Sm163 belongs to clade 2, unlike all the other SME-Sm isolates, which belong to clade 1. [ABSTRACT FROM AUTHOR]

Objectives: Available commercial tools (molecular methods or immunochromatographic assays) usually allow the detection of the five most prevalent carbapenemases (KPC, NDM, VIM, IMP and OXA-48-like), but miss minor carbapenemases. Here, we characterize two enterobacterial isolates with reduced susceptibility to carbapenems and negative for the most commonly encountered carbapenemase genes.Methods: Enterobacter hormaechei and Citrobacter freundii isolates were recovered from a bile sample and rectal screening, respectively. Both isolates were investigated by WGS. Resistance genes were detected using ResFinder. The blaTMB-1-harbouring plasmid was reconstructed using CLC genomic workbench 10.0 and was annotated using the RAST tool. Transfer frequency was determined by conjugation experiments using the laboratory strain Escherichia coli J53.Results: The two isolates were resistant to broad-spectrum cephalosporins and carbapenems. WGS revealed the presence of blaTMB-1, which has previously only been described in non-fermenters. blaTMB-1 was located within an ISKpn19-based composite class 1 transposon. Comparative genomics revealed that this structure was carried on a conjugative IncN-type plasmid within an integration hotspot. Conjugation experiments revealed high transfer frequencies of ∼1 × 10-3.Conclusions: To the best of our knowledge, this study corresponds to the first report of Tripoli MBL 1-producing Enterobacteriaceae. Despite always being described as likely to be chromosomally located in non-fermenters, the blaTMB-1 gene is now found to be carried by a conjugative plasmid among Enterobacteriaceae, raising concern about the possible dissemination of this carbapenemase. The blaTMB-1 gene should now be suspected when PCRs targeting the main carbapenemases remain negative. [ABSTRACT FROM AUTHOR]

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here, we have evaluated three MALDI-TOF-based techniques to detect carbapenemase-producing Enterobacteriaceae (CPE) from cultured colonies.Methods: The performance of three MALDI-TOF-based techniques, including the commercialized MBT STAR®-Carba IVD Kit (Bruker Daltonics) and two in-house protocols performed on the Microflex LT Biotyper (Bruker Daltonics) and the VITEK® MS Plus (bioMérieux), were compared with those of the RAPIDEC® CARBA NP (bioMérieux). A collection of 175 isolates including 120 carbapenemase producers and 55 non-carbapenemase producers was tested. Samples were tested blind in the three participating centres. The repeatability of the MBT STAR®-Carba IVD Kit was also evaluated.Results: The three MALDI-TOF techniques possess sensitivities ranging from 95% to 100% and specificities from 98.2% to 100% compared with 99.2% and 100%, respectively, for the RAPIDEC® CARBA NP. The MBT STAR®-Carba IVD Kit gave highly reproducible results and is the only technique able to provide a concomitant identification of the bacterial isolate. The three MALDI-TOF techniques possess a fast turnaround time (less than 1.5 h).Conclusions: Overall, MALDI-TOF is a reliable technique for the rapid detection of CPE from cultured colonies. MBT STAR®-Carba IVD Kit, the only commercially available assay, could easily be implemented in a clinical microbiology laboratory if it is already equipped with a Microflex LT Biotyper mass spectrometer. [ABSTRACT FROM AUTHOR]

Objectives: Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection.Methods: The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017.Results and discussion: The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media.Conclusions: The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories. [ABSTRACT FROM AUTHOR]

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like).Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5.Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity.Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapene- mases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like). Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5. Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non- targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity. Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratoryfor confirmation of the five main carbapenemases encountered in Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Objectives: There is an urgent need for accurate and fast diagnostic tests capable of identifying carbapenemase producers. Here, we assessed the performance of a new multiplex lateral flow assay (OKN K -SeT) for the rapid detection of OXA-48-like, KPC and NDM carbapenemase-producing Enterobacteriaceae from culture colonies.Methods: Two hundred collection isolates with characterized β-lactamase content and 183 non-duplicate consecutive isolates referred to two National Reference Centres over a 2 month period in 2016 were used to evaluate the OKN K -SeT assay.Results: The assay correctly detected all 42 OXA-48-like-, 27 KPC- and 30 NDM-producing isolates from the collection panel, including 7 isolates that co-produced NDM and OXA-181 carbapenemases. No cross-reactivity was observed with non-targeted carbapenemases ( n  =   41) or with non-carbapenemase producers ( n  =   60). Prospectively, all OXA-48-like ( n  =   69), KPC ( n  =   9) and NDM ( n  =   19) carbapenemase-producing Enterobacteriaceae isolates were correctly detected, while 11 carbapenemase producers not targeted by the assay went undetected [VIM ( n  =   8) and OXA-23/OXA-58-like ( n  =   3)]. Overall, the sensitivity and specificity of the assay were 100%.Conclusions: The OKN assay is efficient, rapid and easy to implement in the workflow of a clinical microbiology laboratory for the confirmation of OXA-48, NDM and KPC carbapenemases. This test represents a powerful diagnostic tool as it enables the rapid detection of the most clinically important carbapenemases without the need for more costly and less frequently available molecular assays. [ABSTRACT FROM AUTHOR]

Objectives KPC-producing Pseudomonas aeruginosa are increasingly isolated in the Americas and in the Caribbean islands. Here, we determined the whole-plasmid sequence of two plasmids carrying the blaKPC-2 gene from multidrug-resistant P. aeruginosa clinical isolates from Colombia. Methods The two plasmids, pCOL-1 and pPA-2, were transferred to Escherichia coli recipient strain TOP10 and completely sequenced using high-throughput pyrosequencing for pCOL-1 and classical Sanger sequencing for pPA-2. Results Both plasmids could be transferred to E. coli by transformation and displayed no other resistance marker besides KPC. Plasmid pCOL-1 was 31 529 bp in size, contained 31 open reading frames (ORFs) and belonged to the IncP-6 replicon group. It exhibited genes involved in replication, mobilization and partitioning, but none involved in conjugation. Plasmid pPA-2 was 7995 bp in size and contained seven ORFs. It exhibited a replicase gene of IncU, but was lacking genes involved in mobilization, partitioning and conjugation. Only 2072 bp matched Tn4401, including the blaKPC-2 gene, part of ISKpn6 and a 73 bp segment located upstream of the blaKPC-2 gene, containing the P1 promoter. Sequence identity was interrupted by a Tn3 transposon, itself interrupted by an IS26 element inserted within the β-lactamase blaTEM-1 gene. Conclusions Here we present the genetic features of the very first plasmids carrying the blaKPC-2 gene from P. aeruginosa. The emergence of the blaKPC-2 gene on unrelated plasmids, differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes in P. aeruginosa isolates suggests that this resistance trait may follow a dissemination scheme in P. aeruginosa similar to that seen in Enterobacteriaceae. [ABSTRACT FROM PUBLISHER]

The structure of the β-lactamase SME-1 from Serratia marcescens, a class A enzyme characterized by its significant activity against imipenem, has been determined to 2.13 Å resolution. The overall structure of SME-1 is similar to that of other class A β-lactamases. In the active-site cavity, most of the residues found in SME-1 are conserved among class A β-lactamases, except at positions 104, 105 and 237, where a tyrosine, a histidine and a serine are found, respectively, and at position 238, which is occupied by a cysteine forming a disulfide bridge with the other cysteine residue located at position 69. The crucial role played by this disulfide bridge in SME-1 was confirmed by site-directed mutagenesis of Cys69 to Ala, which resulted in a mutant unable to confer resistance to imipenem and all other β-lactam antibiotics tested. Another striking structural feature found in SME-1 was the short distance separating the side chains of the active serine residue at position 70 and the strictly conserved glutamate at position 166, which is up to 1.4 A shorter in SME4 compared with other class A β-lactamases. Consequently, the SME-1 structure cannot accommodate the essential catalytic water molecule found between Ser70 and Glu166 in the other class A β-lactamases described so far, suggesting that a significant conformational change may be necessary in SME-1 to properly position the hydrolytic water molecule involved in the hydrolysis of the acyl-enzyme intermediate. [ABSTRACT FROM AUTHOR]

A clinical isolate of Proteus mirabilis Lil-1 was obtained from a Vietnamese patient hospitalized in Paris, France. This isolate was resistant to cephalosporins, and there was marked synergy between cephalosporins and clavulanic acid together with unusual synergy between cefoxitin and cefuroxime. PCR analysis revealed the presence of blaVEB-1, an integron-located gene coding for an extended-spectrum β-lactamase (ESBL) identified previously in an Escherichia coli isolate MG-1 from Vietnam. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1-containing integron along with flanking sequences were amplified from P. mirabilis Lil-1 whole-cell DNA. A novel class 1 integron, In55, was identified that contained, in addition to intI1, qacEΔ1, sul1 and Orf5 genes, an 8 kb variable region. This region was comparable in size to that found previously in E. coli MG-1, but different from those previously identified in two Pseudomonas aeruginosa isolates from Thailand. In55 was located on a 190 kb self-transferable plasmid, which was different in size and structure from that found in E. coli MG-1. The finding of blaVEB-1 on different plasmids and integrons in enterobacterial isolates underlines the interspecies spread of this novel ESBL gene. [ABSTRACT FROM PUBLISHER]

Summary: From early this decade, Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases (KPC) were reported in the USA and subsequently worldwide. These KPC-producing bacteria are predominantly involved in nosocomial and systemic infections; although they are mostly Enterobacteriaceae, they can also be, rarely, Pseudomonas aeruginosa isolates. KPC β lactamases (KPC-1 to KPC-7) confer decreased susceptibility or resistance to virtually all β lactams. Carbapenems (imipenem, meropenem, and ertapenem) may thus become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are, in addition, resistant to many other non-β-lactam molecules, leaving few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. It is therefore crucial to implement efficient infection control measures to limit the spread of these pathogens. [Copyright &y& Elsevier]

As carbapenemase-producing Enterobacteriaceae (CPE) and extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The ELITe InGenius is a fully automated sample-to-result system designed for the extraction and detection by multiplex real-time polymerase chain reaction of carbapenemases KPC, NDM, VIM, IMP, and OXA-48–like variants and CTX-M group 1 and 9-producers from diverse sample matrices such as colonies, positive blood cultures, and rectal swabs. CRE and ESBL ELITe MGB® kits were evaluated on 153 cultured colonies of enterobacterial isolates with characterized β-lactamase content, on 30 spiked blood cultures, and the CRE kit was also evaluated on 53 clinical rectal swabs collected prospectively during a 3-month period and 10 spiked rectal swabs. CRE ELITe MGB® kit’s performances reached 100% sensitivity and 100% specificity, while for the ESBL ELITe kit, 100% sensitivity and 96.6% specificity were observed, with a sample to result of less than 3 h and a total percentage of agreement with expected results of 99.6% (255/256). [ABSTRACT FROM AUTHOR]

Highlights • First outbreak of Enterobacteriaceae producing IMI-1 carbapenemase. • IMI-1-producers can be underdetected in routine because of their unusual antimicrobial resistance. • bla IMI-1 gene is borne by the new integrative mobile element EcloIMEX-8. ABSTRACT The spread of carbapenemase-producing Enterobacteriaceae in the Southwest Indian Ocean islands is poorly known. Here we describe an outbreak of colistin-resistant Enterobacter cloacae harbouring bla IMI-1 in the French overseas department of Mayotte. Between October 2015 and January 2017, all isolates of imipenem-non-susceptible E. cloacae at Mayotte Medical Center and University Hospital of Reunion Island were screened for carbapenemase production. Positive isolates were typed by pulsed-field gel electrophoresis and whole-genome sequencing (WGS)-based multilocus sequence typing (MLST), and all β-lactamase genes were identified by PCR and sequencing. Resistance profiles were determined by agar diffusion and Etest. Genetic support of the bla IMI-1 gene was determined by WGS. A total of 18 E. cloacae isolates harbouring bla IMI-1 were detected in 17 patients from Mayotte. Pulsed-field gel electrophoresis (PFGE) analysis showed 16 of the 18 strains to be clonally related and belonging to ST820. Based on clinical data, this outbreak most likely had a community origin. The bla IMI-1 gene in the 18 isolates was carried by a new variant of an integrative mobile element involving the Xer recombinases, called EcloIMEX-8. The mcr-1 – mcr-5 genes were absent from the collection. The isolates belonged to E. cloacae cluster XI, known to be colistin heteroresistant. Here we report the first outbreak of IMI-1-producing Enterobacteriaceae. IMI-1-producers may be underdetected in microbiology laboratories because of their unusual antimicrobial resistance profile (resistant to imipenem but with intermediate resistance to ertapenem and susceptible to extended-spectrum cephalosporins) and the absence of bla IMI-1 in the panel of genes targeted by molecular diagnostic kits. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the performance of the Carbapenemase Detection Set™ (CDS; Mast Diagnostics) in association with i) the EUCAST meropenem screening cut-off and ii) the faropenem–temocillin algorithm (FTa) for the screening of carbapenemase-producing Enterobacteriaceae (CPE). A total of 200 well-characterized enterobacterial isolates with reduced susceptibility to at least one carbapenem including 63 non-CPEs and 137 CPEs belonging to different Ambler classes were initially screened for CPEs using i) the EUCAST meropenem cut-off (diameter <25 mm) and ii) the FTa. Highly suspected CPEs underwent further testing using the CDS, which is based on the inhibition zone diameters determination of combined disks (A: meropenem, B: meropenem + dipicolinic acid, C: meropenem + cloxacillin, and D: meropenem + boronic acid). With the FTa, 66.7% of the non-CPE isolates were correctly identified. Most OXA-48-like producers (90.5%) were detected with 98.6% specificity. The FTa discriminates CPE from non-CPE with 100% sensitivity, but complementary tests were still needed for 59 % (118/200) of the strains. The EUCAST cut-off led to 3 false-negative results (2 OXA-181 and 1 NMC-A producer) resulting in a sensitivity of 97.8% for the discrimination between CPE and non-CPE, and 75.5% (151/200) of the strains still required complementary test. The CDS reduced the number of isolates requiring additional tests from 59% to 22%, and from 75.5% to 38% for FTa and EUCAST cut-off, respectively. FTa possesses very good specificities for the detection and classification of Ambler class A and most class B carbapenemase-producers, except for IMP producers, which were almost not detected (10/11). In conclusion, the association of the CDS with the FTa presented only 22% of inconclusive results, while this number was 38% with the EUCAST meropenem CPE screening cut-off. [ABSTRACT FROM AUTHOR]

Early detection of patients colonised with carbapenemase-producing Enterobacteriaceae (CPE) is crucial for implementing proper infection control measures. Here we evaluated the biological performance of the Xpert ® Carba-R v2 (Cepheid) in the daily workflow of a hygiene unit in a country with a low CPE prevalence. Patients repatriated from countries known for high CPE prevalence or contact patients of a known CPE carrier were targeted as being ‘high-risk patients’ for CPE carriage. Between September 2015 and March 2016, 241 ‘high-risk patients’ for CPE carriage were screened using the Xpert ® Carba-R v2 and by plating on chromID ® CARBA Smart medium (bioMérieux) with and without ertapenem-containing enrichment culture for 24 h. Of these patients, 81.7% were previously hospitalised abroad and 18.3% were contact patients of known CPE carriers. The Xpert ® Carba-R v2 was able to detect 12 OXA-48-like, 1 KPC and 1 OXA-48-like/NDM carriers. For 2 of the 14 Xpert ® Carba-R v2-positive samples, cultures remained negative even on two additional screenings (performed at Days 4 and 7). The Xpert ® Carba-R v2 presents 100% sensitivity, 99.13% specificity, 85.71% positive predictive value and 100% negative predictive value. This study demonstrated that the Xpert ® Carba-R v2 kit is well adapted for rapid screening of high-risk patients even in low prevalence regions (in <1 h versus 24/48 h for culture). This assay may guide infection control programmes to limit the spread of CPE. [ABSTRACT FROM AUTHOR]

Eleven carbapenem-resistant Klebsiella pneumoniae isolates were recovered from the military hospital of Tunis, Tunisia. Three of these isolates carried the carbapenemase bla OXA-232 gene recently identified in France. These isolates were clonally related and co-expressed the extended-spectrum ß-lactamase, CTX-M-15. This work identified the occurrence of OXA-232 producers in North-African countries. [ABSTRACT FROM AUTHOR]

We investigated the presence of carbapenemases in carbapenem-resistant Pseudomonas aeruginosa isolates, which were collected over a 14-month period in a Turkish hospital, with in-depth molecular characterization of carbapenemase-producing isolates. Among 45 study isolates, 2 isolates were identified as carbapenemase producers by both Carba NP and Carbapenem Inactivation Method tests, and only 1 of them gave a positive result in polymerase chain reaction tests for a carbapenemase gene (bla VIM). Whole genome sequencing of the 2 isolates revealed the presence of bla VIM-5 gene in an ST308 isolate, while the other one expressed IMP-7 in an ST357 isolate; both STs are considered high-risk clones. The 2 carbapenemase-producing isolates were multidrug resistant, as they harbored other resistance determinants, including a variant of the recently described plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. We report for the first time P. aeruginosa high-risk clones carrying VIM-5– and IMP-7–type carbapenemases with multiple resistance determinants in Turkey. • Twenty percent carbapenem-resistant Pseudomonas aeruginosa isolates over a 14-month period in a Turkish hospital. • Among carbapenem-resistant P. aeruginosa isolates, 5% were carbapenemase producers. • Carbapenemase-producing P. aeruginosa high-risk clones from Turkey. • VIM-5–producing ST308 and IMP-7–producing ST357. • Novel plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. [ABSTRACT FROM AUTHOR]

An OXA-181 producing Escherichia coli isolate that went recurrently undetected directly from rectal swab sample using Xpert® Carba-R, was successfully detected using ertapenem supplemented broth enrichment followed by culture-based method. Our data suggest that implementation of culture-based methods plus enrichment might be crucial for the efficient screening of patients considered to be at "high-risk" of colonization with carbapenemase producers and who are colonized at low level. [ABSTRACT FROM AUTHOR]

This prospective study evaluated the ability of the qPCR Amplidiag® CarbaR+VRE assay to detect Carbapenemase-producing Gram-negative bacilli (CP-GNB) directly on 1830 rectal swabs extracted using the fully automated platform Amplidiag® Easy instrument. The Amplidiag® CarbaR+VRE assay gave a positive signal for 94 rectal swabs, whereas only 70 grew with CP-GNB on chromogenic media including 4 VIM-producing P. aeruginosa , 8 OXA-23-producing A. baumannii and 58 carbapenemase-producing Enterobacteriaceae. All the CP-GNB culture positive were detected by the Amplidiag® CarbaR+VRE assay. Twenty-four qPCR-positive and culture-negative samples were further investigated using targeted PCRs and subsequent DNA sequencing. Seventeen and 7 of these were positive and negative with PCR/DNA sequencing, respectively. Taken together, the Amplidiag® CarbaR+VRE could detect carbapenemases directly from rectal swabs in 3 h 30 using a fully automated platform and showed high biological performances (sensitivity, specificity, and negative and positive predictive values were 100%, 98.6%, 100%, and 74.5%, respectively). [ABSTRACT FROM AUTHOR]