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Background: Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco., Materials and Methods: P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types., Results: P. aeruginosa O82J1 co-expressed two metallo-β-lactamases, blaNDM-1 and blaVIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The blaVIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the blaNDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a blaPER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB., Conclusions: The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The bla OXA-933 gene was borne on Tn 2208 , a 2,696-bp composite transposon made of two IS 1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the bla OXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the bla OXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the β5-β6 loop, but interacting with key residues of it., Competing Interests: The authors declare no conflict of interest.

Objectives: In-depth phenotypic and genomic analyses on a carbapenem-resistant Escherichia coli isolate, recovered from the faeces of a farm dog in Lebanon, focusing on its antimicrobial resistance (AMR) patterns and the underlying resistome., Methods: E. coli strain EC-106 was identified using MALDI-TOF-MS. Analyses using Carba NP, immunochromatographic assay NG Carba5, and other antimicrobial susceptibility testing were performed. Whole-genome sequencing (WGS) using the Illumina technology and different software available at the Center of Genomic Epidemiology wwere used to predict the resistome, sequence type (ST), plasmid types, and virulence genes., Results: Susceptibility testing revealed that E. coli EC-106 was multi-drug resistant, including against newer antimicrobials such as imipenem-relebactam (MIC = 16 µg/mL), meropenem-vaborbactam (MIC = 16 µg/mL), and ceftazidime-avibactam (MIC > 32 µg/mL), but remained susceptible to aztreonam (MIC = 0.12 µg/mL), aztreonam-avibactam (MIC = 0.06 µg/mL), and cefiderocol (MIC = 0.5 µg/mL). WGS analyses showed that E. coli EC-106 carried 13 acquired resistance genes associated with resistance to β-lactams (bla NDM-5 and bla TEM-1B ), aminoglycosides (aac(3)-IId, aph(3')-Ia, aadA1, and aadA2), tetracyclines (tetA), amphenicols (partial catA1), macrolides (mphA), sulphonamides (sul1 and sul3), trimethoprim (dfrA12), and quaternary ammonium compounds (partial qacE). The bla NDM-5 was located on an IncX3 plasmid. The isolate was predicted to be a human pathogen (92.9%) and belonged to ST1011., Conclusion: To our knowledge, this is the first report of the detection of an IncX3 plasmid carrying the bla NDM-5 gene in animals in Lebanon, highlighting the severe AMR challenges in the country. Taken together, our current and previous findings suggest that bla NDM-5 might be spreading in different hosts and genetic backgrounds across clinical and non-clinical settings., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties., Objectives: To determine the steady-state kinetic parameters of OXA-244., Methods: The blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed., Results: A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems., Conclusions: Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases., (© The Author(s) 2021. 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.)

The occurrence of resistances in Gram negative bacteria is steadily increasing to reach extremely worrying levels and one of the main causes of resistance is the massive spread of very efficient β-lactamases which render most β-lactam antibiotics useless. Herein, we report the development of a series of imino-analogues of β-lactams (namely azetidinimines) as efficient non-covalent inhibitors of β-lactamases. Despite the structural and mechanistic differences between serine-β-lactamases KPC-2 and OXA-48 and metallo-β-lactamase NDM-1, all three enzymes can be inhibited at a submicromolar level by compound 7dfm, which can also repotentiate imipenem against a resistant strain of Escherichia coli expressing NDM-1. We show that 7dfm can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler classes A (KPC-2), B (NDM-1) and D (OXA-48) with Ki's below 0.3 μM, but also the cephalosporinase CMY-2 (class C, 86% inhibition at 10 μM). Our results pave the way for the development of a new structurally original family of non-covalent broad-spectrum inhibitors of β-lactamases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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.)

A novel real-time multiplex PCR assay, BD MAX Check-Points CPO, was evaluated to detect carbapenemase-producing organisms in clinical settings on the BD MAX system. A total of 175 well-characterized isolates (including 123 carbapenemase producers) and 128 rectal swab specimens (including 83 positives) of patients considered at high risk for carriage of carbapenemase producers were included. Bacterial suspensions were used to spike true-negative rectal swabs to mimic a clinical sample. Sample (50 μL), containing either the spiked or the patient's sample, was processed. The BD MAX Check-Points CPO assay detected carbapenemases KPC, VIM/IMP, NDM, and OXA-48-like producers with a high sensitivity and specificity of 97.1% and 98.8%, respectively. Rare variants of the IMP type (IMP-11, IMP-13, and IMP-14) and one rare and distantly related OXA-48 variant (OXA-535) remained undetected. With patients' rectal swabs, sensitivity and specificity were 92.8% and 97.8%, respectively. Failure of detection was due to weak inoculum. The time to result was short: approximately. 2.5 hours for 12 samples (including extraction and PCR). The automated sample-in results-out platform is an efficient, quick, and easy-to-use tool for the detection of the main five carbapenemases. The lack of distinction between producers of VIM and IMP may be limiting in countries where these enzymes are widespread, as in Asia, but not in France, where IMP producers are extremely rare., (Copyright © 2020 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 American Society for Microbiology.)

Background: KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions., Objectives: To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243)., Methods: The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity., Results: Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate., Conclusions: We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection., (© The Author(s) 2019. 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.)

In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The bla ACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla ACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla ACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests., (Copyright © 2019 American Society for Microbiology.)

Background: New Delhi metallo-β-lactamase (NDM)-producing Acinetobacter baumannii have been described in several countries worldwide, and studies have suggested that Acinetobacter spp. could play the role of intermediate progenitor of the bla NDM-1 gene between environmental progenitor and Enterobacteriaceae., Materials and Methods: In total, 246 carbapenem-resistant A. baumannii isolates from a teaching hospital in Sousse, Tunisia were investigated between 1 st June 2013 and 31 st December 2015 to detect metallo-ß-lactamase (MBL) production. Polymerase chain reaction (PCR), antibiotic susceptibility testing, and genetic and whole-genome sequencing tools were used to study the underlying carbapenem resistance mechanisms., Results: PCR screening of the 246 carbapenem-resistant A. baumannii isolates revealed that 242 of 246 isolates harboured carbapenemase genes (seven of 246 positive for bla NDM-1 , four of 246 positive for bla NDM-1 and bla OXA-23 , 231 positive for bla OXA-23 ). Conjugation and electroporation experiments suggested that the bla NDM-1 gene is likely to be chromosomally located. All the NDM-1-producing A. baumannii isolates were clonally related, and belonged to ST85 according to the Pasteur Institute's multi-locus sequence typing scheme. Analysis of the immediate genetic environment of the bla NDM-1 gene revealed that the gene was located within a truncated isoform of Tn125 transposon (ΔTn125). The bla OXA-23 gene was located within transposon Tn2008., Conclusion: This study showed the dissemination of a single clone of NDM-1-producing A. baumannii in a Tunisian hospital. Countries in north Africa may constitute a significant reservoir for NDM-1-producing A. baumannii. The spread of the bla NDM-1 gene in A. baumannii was linked to clonal spread in this study., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

As carbapenemase-producing Gram-negative bacilli (CP-GNB) ( Enterobacteriaceae , Pseudomonadaceae , and Acinetobacter spp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag CarbaR+VRE assay is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from Acinetobacter baumannii (OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like β-lactamase associated with an upstream inserted IS Aba1 However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in Pseudomonas aeruginosa , or OXA-143-like in A. baumannii were not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases-NDM, VIM, IMP, KPC, and OXA-48-as well as OXA-type carbapenemases from Acinetobacter spp. that are currently emerging also among Proteus mirabilis and other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs., (Copyright © 2018 American Society for Microbiology.)

Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to-date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of β-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements constitute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online at http://bldb.eu without any registration and supports all modern browsers.

The global spread of carbapenemase-producing Enterobacteriaceae (CPE) that are often resistant to most, if not all, classes of antibiotics is a major public health concern. The NDM-1 carbapenemase is among the most worrisome carbapenemases given its rapid worldwide spread. We have developed and evaluated a lateral flow immunoassay (LFIA) (called the NDM LFIA) for the rapid and reliable detection of NDM-like carbapenemase-producing Enterobacteriaceae from culture colonies. We evaluated the NDM LFIA using 175 reference enterobacterial isolates with characterized β-lactamase gene content and 74 nonduplicate consecutive carbapenem-resistant clinical isolates referred for expertise to the French National Reference Center (NRC) for Antibiotic Resistance during a 1-week period (in June 2016). The reference collection included 55 non-carbapenemase producers and 120 carbapenemase producers, including 27 NDM producers. All 27 NDM-like carbapenemase producers of the reference collection were correctly detected in less than 15 min by the NDM LFIA, including 22 strains producing NDM-1, 2 producing NDM-4, 1 producing NDM-5, 1 producing NDM-7, and 1 producing NDM-9. All non-NDM-1 producers gave a negative result with the NDM LFIA. No cross-reaction was observed with carbapenemases (VIM, IMP, NDM, KPC, and OXA-48-like), extended-spectrum β-lactamases (ESBLs) (TEM, SHV, and CTX-M), AmpCs (CMY-2, DHA-2, and ACC-1), and oxacillinases (OXA-1, -2, -9, and -10). Similarly, among the 74 referred nonduplicate consecutive clinical isolates, all 7 NDM-like producers were identified. Overall, the sensitivity and specificity of the assay were 100% for NDM-like carbapenemase detection with strains cultured on agar. The NDM LFIA was efficient, rapid, and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of NDM-like carbapenemase-producing Enterobacteriaceae ., (Copyright © 2017 Boutal et al.)

An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found for Klebsiella pneumoniae carbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. The blaFRI-1 gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5' end of the β-lactamase gene was identified, leading to inducible expression of the blaFRI-1 gene., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type β-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like β-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Objectives: Carbapenem-hydrolysing class D β-lactamases of the OXA-48 type are increasingly reported from Enterobacteriaceae. β-Lactamase OXA-48 hydrolyses penicillins very efficiently, but carbapenems only weakly and spares broad-spectrum cephalosporins. Recently, diverse OXA-48-like β-lactamases have been identified worldwide (OXA-162, OXA-181, OXA-163, OXA-204 and OXA-232). They differ by few amino acid substitutions or by amino acid deletions., Methods: blaOXA-48, blaOXA-162, blaOXA-163, blaOXA-181, blaOXA-204 and blaOXA-232 were cloned into the same expression vector and expressed in the same Escherichia coli background. Kinetic studies were performed with enzymes purified by ion-exchange chromatography. Determination of hydrolytic activities was performed by UV spectrophotometry. MICs were determined for all recombinant strains, using as background either the WT E. coli TOP10 strain or a porin-deficient E. coli strain., Results: Kinetic studies showed that OXA-162 and OXA-204 shared the same hydrolytic properties as OXA-48. On the other hand, OXA-181 possessed a higher ability to hydrolyse carbapenems, while OXA-232 hydrolysed those substrates less efficiently. In contrast to the other OXA-48-like β-lactamases, OXA-163 hydrolysed broad-spectrum cephalosporins very efficiently, but did not possess significant carbapenemase activity. Although several of these OXA-48-like enzymes possess low activity against carbapenems, MICs of carbapenems were significantly elevated when determined for strains possessing permeability defects., Conclusions: A detailed comparative analysis of the kinetic properties of the OXA-48-like β-lactamases is provided here. It clarifies the respective features of each OXA-48-like variant and their respective impacts in terms of carbapenem resistance., (© 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.)