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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.)

We characterized a collection of IMI-like-producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1-producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria.

Background: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp., Methods: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A., Findings: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-β-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A., Interpretation: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons., Funding: None., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase bla OXA-48 gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion., (© 2024. The Author(s).)

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.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories., Importance: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment., Competing Interests: H.V. has filed on behalf of CEA a patent for the SPID platform. M.S.P. and L.F. are employees of NG Biotech and were involved in the BL-DetecTool manufacturing but not in the data analysis. All the other authors declare no competing interests.

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: The fecal carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) is a major driver of the global spread of these antibiotic resistance determinants. Here we determined the rate of fecal ESBL-PE carriage in pediatric hospitals and community-serving healthcare centers serving adults and children in the Gaza Strip, Palestine., Methods: A total of 373 fecal and rectal samples were collected from different hospitals and clinics in Gaza. The antibiotic susceptibility was determined using the disk diffusion method and interpreted according to CLSI guidelines. The bacterial isolates were tested for ESBL production using phenotypic methods (double disk synergy test and growth on selective chromogenic media). Bla CTX-M , bla SHV , and bla TEM genes were sought by PCR., Results: Out of the 373 isolates tested, 138 (37%) were considered ESBL positive as revealed by phenotypic tests. The prevalence of ESBLs among hospitalized patients was 39.1% (hospital setting) whereas, among outpatients attending community healthcare centers, it was 35.1% (community setting). ESBL production among Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Klebsiella aerogenes isolates was 52.8%, 39.1%, 26.7%, 2.8%, and 2.1% respectively. Meropenem and amikacin were the most effective antibiotics against ESBL producers (68.9% and 73.6% susceptibility, respectively), while only 15.2%, 22.5%, and 24.6% remained susceptible to ceftazidime, cefotaxime, and ceftriaxone, respectively. Out of 138 phenotypically ESBL-positive isolates, 98 randomly chosen were screened for bla CTX-M , bla TEM , and bla SHV genes. The prevalence rate of bla CTX-M was 45.9%, while bla TEM and bla SHV genes were detected in 16.8% and 5.2% of CTX-M-negative isolates (corresponding mostly for K. pneumoniae isolates in the case of SHV-PCR), respectively., Conclusions: The study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing Enterobacterales among hospitalized children but also in the community of the Gaza Strip. In addition, 30% of ESBL-producers were already resistant to carbapenems, the treatment of choice of infections with ESBL-producers., (© 2023. The Author(s).)

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: "What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?" From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

Early detection of expanded-spectrum cephalosporin (ESC) resistance is essential not only for an effective therapy but also for the prompt implementation of infection control measures to prevent dissemination in the hospital. We have developed and validated a lateral flow immunoassay (LFIA), called LFIA-CTX test, for the detection of ESC (cefotaxime) hydrolytic activity based on structural discrimination between the intact antibiotic and its hydrolysed product. A single bacterial colony was suspended in an extraction buffer containing cefotaxime. After a 30-min incubation, the solution is loaded on the LFIA for reading within 10 min. A total of 348 well-characterized Gram-negative isolates were tested. Among them, the 38 isolates that did not express any cefotaxime-hydrolysing β-lactamase gave negative results. Of the 310 isolates expressing at least one cefotaxime-hydrolysing β-lactamase, all were tested positive, except three OXA-48-like producers, which were repeatedly detected negative. Therefore, the sensitivity was 99.1% and the specificity was 100%. The LFIA-CTX test is efficient, fast, low-cost and easy to implement in the workflow of a routine microbiology laboratory., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.)

Carbapenemase-producing Enterobacterales (CPE) is a major public-health concern. Here we describe the occurrence of bla VIM-2 in three isolates of Enterobacter hormaechei subsp. steigerwaltii. The bla VIM-2 gene was part of a class II transposon Tn1332 and was embedded in a remnant of a class 1 integron. Tn1332 was carried by a large, conjugative, non-typeable plasmid. The three isolates belonged to sequence type 90 (ST90). Two isolates (90H2 and 90H3) were highly related [<10 single nucleotide polymorphisms (SNPs)], whereas isolate 104D2 exhibited more than 50 SNPs and Tn1332 was inserted in a different place in the plasmid. Another IncHI-type plasmid carrying the extended-spectrum β-lactamase (ESBL) gene bla CTX-M-15 was identified in 90H2 and 90H3. Among the three isolates, isolate 104D2 was negative for detection of carbapenemase activity using the biochemical Carba NP test, despite the presence of Tn1332 on the same plasmid. Mutants of 104D2 with higher minimum inhibitory concentrations (MICs) for carbapenems were obtained and one mutant (m104D2) was analysed. In contrast to 104D2, mutant m104D2 gave a positive Carba NP test. The mutant possessed two copies of Tn1332 per cell and a nonsense mutation in WecA, an enzyme involved in enterobacterial common antigen and peptidoglycan intermediate biosynthesis. This study describes the first occurrence of Tn1332 in Enterobacterales and the phenotypic diversity of VIM-2-producing E. hormaechei., (Copyright © 2021 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Klebsiella pneumoniae is a leading cause of intractable hospital-acquired multidrug-resistant infections and carbapenemase-producing K. pneumoniae (CPKp) are particularly feared. Most of the clinical isolates produce capsule as a major virulence factor. Recombination events at the capsule locus are frequent and responsible for capsule diversity within Klebsiella spp. Capsule diversity may also occur within clonal bacterial populations generating differences in colony aspect. However, little is known about this phenomenon of phenotypic variation in CPKp and its consequences., Results: Here, we explored the genetic causes of in vitro switching from capsulated, mucoid to non-mucoid, non-capsulated phenotype in eight clinical CPKp isolates. We compared capsulated, mucoid colony variants with one of their non-capsulated, non-mucoid isogenic variant. The two colony variants were distinguished by their appearance on solid medium. Whole genome comparison was used to infer mutations causing phenotypic differences. The frequency of phenotypic switch was strain-dependent and increased along with colony development on plate. We observed, for 72 non-capsulated variants that the loss of the mucoid phenotype correlates with capsule deficiency and diverse genetic events, including transposition of insertion sequences or point mutations, affecting genes belonging to the capsule operon. Reduced or loss of capsular production was associated with various in vitro phenotypic changes, affecting susceptibility to carbapenem but not to colistin, in vitro biofilm formation and autoaggregation., Conclusions: The different impact of the phenotypic variation among the eight isolates in terms of capsule content, biofilm production and carbapenem susceptibility suggested heterogeneous selective advantage for capsular loss according to the strain and the mutation. Based on our results, we believe that attention should be paid in the phenotypic characterization of CPKp clinical isolates, particularly of traits related to virulence and carbapenem resistance.

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 bla OXA-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 bla OXA-23 gene in that isolate. By contrast to the bla OXA-23 genes, the bla OXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the bla OXA-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.

OXA-48 carbapenemase has rapidly spread in many countries worldwide with several OXA-48-variants being described, differing by a few amino acid (AA) substitutions or deletions, mostly in the β5-β6 loop. While single AA substitutions have only a minor impact on OXA-48 hydrolytic profiles, others with 4 AA deletions result in loss of carbapenem hydrolysis and gain of expanded-spectrum cephalosporin (ESC) hydrolysis. We have replaced the β5-β6 loop of OXA-48 with that of OXA-18, a clavulanic-acid inhibited oxacillinase capable of hydrolyzing ESCs but not carbapenems. The hybrid enzyme OXA-48Loop18 was able to hydrolyze ESCs and carbapenems (although with a lower k cat ), even though the β5-β6 loop was longer and its sequence quite different from that of OXA-48. The kinetic parameters of OXA-48Loop18 were in agreement with the MIC values. X-ray crystallography and molecular modeling suggest that the conformation of the grafted loop allows the binding of bulkier substrates, unlike that of the native loop, expanding the hydrolytic profile. This seems to be due not only to differences in AA sequence, but also to the backbone conformation the loop can adopt. Finally, our results provide further experimental evidence for the role of the β5-β6 loop in substrate selectivity of OXA-48-like enzymes and additional details on the structure-function relationship of β-lactamases, demonstrating how localized changes in these proteins can alter or expand their function, highlighting their plasticity.

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 Revogene Carba C assay (formerly GenePOC Carba assay) is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-producing Enterobacterales (CPE) from cultured colonies. This assay was evaluated directly on colonies of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 multidrug-resistant (MDR) Pseudomonas aeruginosa and 40 MDR Acinetobacter baumannii isolates. The Revogene Carba C assay's performance was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). In Enterobacterales , sensitivity and specificity were 100%. When extrapolating the results to the French CPE epidemiology between 2012 and 2018, this assay would have detected 99.28% of the 9,624 CPE isolates sent to the French NRC, missing 69 CPE isolates (2 GES-5, 10 OXA-23, 2 TMB-1, 1 SME-4, 53 IMI, and 1 FRI). The overall sensitivity and specificity for CP P. aeruginosa were 93.7 and 100%, respectively, as two rare IMP variants (IMP-31 and -46) were not detected. Extrapolating these results to the French epidemiology of CP P. aeruginosa in 2017, 93.3% would have been identified, missing only 1 DIM and 10 GES variants. The Revogene Carba C assay accurately identified the targeted carbapenemase genes in A. baumannii , but when extrapolating these results to the French CP A. baumannii epidemiology of 2017, only 12.50% of them could be detected, as OXA-23 is the most prevalent carbapenemase in CP A. baumannii The Revogene Carba C assay showed excellent sensitivity and specificity for the five most common carbapenemases regardless of the bacterial host. It is well adapted to the CPE and CP P. aeruginosa epidemiology of many countries worldwide, which makes it suitable for use in the routine microbiology laboratory, with a time to result of ca. 85 min for eight isolates simultaneously., (Copyright © 2020 Girlich et al.)

A carbapenem-resistant Citrobacter sp. was recovered from routine screening of multidrug-resistant bacteria. This isolate coproduced OXA-48 and OXA-198. OXA-48 was carried by the prototypical IncL plasmid, whereas OXA-198 was carried by a peculiar IncHI-type plasmid. This carbapenemase gene was inserted within a class 1 integron located on a conjugative plasmid. This report describes the first occurrence of OXA-198 in Enterobacterales ., (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.)

Background: Carbapenem-resistant Enterobacteriaceae are considered by WHO as "critical" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes., Methods: We first analyzed the whole-genome sequence and the evolution of the E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing., Results: In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination., Conclusions: Lineages of CP-Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.

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.)

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 3h 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)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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

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., (© 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.)

Metallo-β-lactamase (MBL)-producing Gram-negative bacteria are often extremely resistant, leading to a real therapeutic dead end. Here, we evaluated the in vitro and in vivo efficacy of aztreonam in combination with ceftazidime-avibactam, ceftolozane-tazobactam, or amoxicillin-clavulanate for the treatment of infections caused by MBL-producing Enterobacteriaceae , MBL-producing Pseudomonas aeruginosa , and extremely drug-resistant Stenotrophomonas maltophilia First, we report two clinical cases, namely, a urinary tract infection caused by an NDM-5-producing Escherichia coli isolate and a pulmonary infection caused by a S. maltophilia isolate efficiently treated with the association of aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate, respectively. Then, a total of 50 MBL-producing Enterobacteriaceae isolates, 3 MBL-producing P. aeruginosa isolates, and 5 extremely drug-resistant S. maltophilia isolates were used to test aztreonam susceptibility in combination with ceftolozane-tazobactam, ceftazidime-avibactam, or amoxicillin-clavulanate. The Etest strip superposition method was used to determine the MICs of the aztreonam/inhibitor combinations. According to CLSI breakpoints, aztreonam susceptibility was fully restored for 86%, 20%, and 50% of the MBL-producing Enterobacteriaceae isolates when combined with ceftazidime-avibactam, ceftolozane-tazobactam, and amoxicillin-clavulanate, respectively. In P. aeruginosa , the aztreonam-ceftazidime-avibactam combination was the most potent, even though the reduction in MICs was at most 2-fold. With the 5 S. maltophilia isolates, aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate were found to be equal (100% susceptibility). Overall, aztreonam-ceftazidime-avibactam was the most potent combination to treat infections caused by MBL producers compared with aztreonam-amoxicillin-clavulanate and aztreonam-ceftolozane-tazobactam. However, in many cases aztreonam-amoxicillin-clavulanate was found to be as efficient as aztreonam-ceftazidime-avibactam, offering the main advantage to be markedly cheaper. We also confirmed the validity of Etest superpositions as a very simple method to determine MICs of aztreonam combinations., (Copyright © 2019 American Society for Microbiology.)

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.)

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.)

With the widespread use and abuse of antibiotics for the past decades, antimicrobial resistance poses a serious threat to public health nowadays. β-Lactams are the most used antibiotics, and β-lactamases are the most widespread resistance mechanism. Class C β-lactamases, also known as cephalosporinases, usually do not hydrolyze the latest and most potent β-lactams, expanded spectrum cephalosporins and carbapenems. However, the recent emergence of extended-spectrum AmpC cephalosporinases, their resistance to inhibition by classic β-lactamase inhibitors, and the fact that they can contribute to carbapenem resistance when paired with impermeability mechanisms, means that these enzymes may still prove worrisome in the future. Here we report and characterize the CMY-136 β-lactamase, a Y221H point mutant derivative of CMY-2. CMY-136 confers an increased level of resistance to ticarcillin, cefuroxime, cefotaxime, and ceftolozane/tazobactam. It is also capable of hydrolyzing ticarcillin and cloxacillin, which act as inhibitors of CMY-2. X-ray crystallography and modeling experiments suggest that the hydrolytic profile alterations seem to be the result of an increased flexibility and altered conformation of the Ω-loop, caused by the Y221H mutation.

We compared extended-spectrum β-lactamase-producing Escherichia coli isolates from meat and fish, gut-colonized women, and infected patients in Cambodia. Nearly half of isolates from women were phylogenetically related to food-origin isolates; a subset had identical multilocus sequence types, extended-spectrum β-lactamase types, and antimicrobial resistance patterns. Eating sun-dried poultry may be an exposure route.

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.

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.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A multidrug-resistant Klebsiella pneumoniae 1210 isolate with reduced carbapenem susceptibility revealed the presence of a novel plasmid-encoded bla OXA-48-like gene, named bla OXA-519 The 60.7-kb plasmid (pOXA-519) was similar to the IncL-OXA-48 prototypical plasmid except for a ca. 2-kb deletion due to an IS 1R insertion. OXA-519 differed from OXA-48 by a Val120Leu substitution, which resulted in an overall reduced β-lactam-hydrolysis profile, except those for ertapenem and meropenem, which were increased. Thus, detection of OXA-519 producers using biochemical tests that monitor imipenem hydrolysis will be difficult., (Copyright © 2018 American Society for Microbiology.)

With the dissemination of carbapenemase-producing Enterobacteriaceae (CPE) strains worldwide, carbapenem-hydrolyzing enzymes are increasingly reported among isolates of Escherichia coli , the first hospital and community-acquired opportunistic pathogen. Here, we have performed an epidemiological survey of carbapenemase-producing E. coli (CP- Ec ) isolates received at the French National Reference Centre (F-NRC) in 2012 and 2013. Antimicrobial susceptibilities for last-resort antibiotics and antimicrobial compounds commonly used to treat urinary tract infections were determined by broth microdilution. Clonal relationship was assessed using repetitive sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). From this collection of 140 carbapenemase-producing E. coli isolates, 74% produced an OXA-48-like carbapenemase and 21% produced an NDM carbapenemase. A link with a foreign country was suspected for 37% of infected/colonized patients. Most of the isolates were from screening (56%) and from urine samples (26%). Colistin, fosfomycin, and nitrofurantoin possessed the most consistent activity, with 100%, 95%, and 96% isolates susceptible, respectively. A wide diversity of carbapenemase-producing E. coli isolates has been found (50 different sequence types [STs]). The most prevalent clones were (i) E. coli sequence type 38 (ST38) producing OXA-48 ( n = 21), a clone linked to Turkey and North African countries, (ii) E. coli ST-90 producing OXA-204 ( n = 9), which was responsible for an outbreak related to a contaminated duodenoscope, and (iii) E. coli ST-410 producing OXA-181 ( n = 5), which was recovered from patients of different geographical origins. These specific clones might be considered high-risk clones for the dissemination of carbapenemases in E. coli The wide diversity of STs, combined with the increasing number of CP- Ec isolates received by the F-NRC, suggests a likely dissemination of CP- Ec isolates in the community., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2018. Published by Elsevier Inc.)

Carbapenemase-producing Pseudomonadaceae have increasingly been reported worldwide, with an ever-increasing heterogeneity of carbapenem resistance mechanisms, depending on the bacterial species and the geographical location. OXA-198 is a plasmid-encoded class D β-lactamase involved in carbapenem resistance in one Pseudomonas aeruginosa isolate from Belgium. In the setting of a multicenter survey of carbapenem resistance in P. aeruginosa strains in Belgian hospitals in 2013, three additional OXA-198-producing P. aeruginosa isolates originating from patients hospitalized in one hospital were detected. To reveal the molecular mechanism underlying the reduced susceptibility to carbapenems, MIC determinations, whole-genome sequencing, and PCR analyses to confirm the genetic organization were performed. The plasmid harboring the bla OXA-198 gene was characterized, along with the genetic relatedness of the four P. aeruginosa isolates. The bla OXA-198 gene was harbored on a class 1 integron carried by an ∼49-kb IncP-type plasmid proposed as IncP-11. The same plasmid was present in all four P. aeruginosa isolates. Multilocus sequence typing revealed that the isolates all belonged to sequence type 446, and single-nucleotide polymorphism analysis revealed only a few differences between the isolates. This report describes the structure of a 49-kb plasmid harboring the bla OXA-198 gene and presents the first description of OXA-198-producing P. aeruginosa isolates associated with a hospital-associated cluster episode., (Copyright © 2018 American Society for Microbiology.)

Integrons are genetic elements that can acquire and rearrange gene cassettes. The bla BEL-1 gene encodes an extended-spectrum β-lactamase, BEL-1, that is present at the second position of the variable region of class 1 integrons identified in Pseudomonas aeruginosa The mobility of the bel-1 gene cassette was analyzed under physiological conditions and with the integrase gene being overexpressed. Cassette mobility in Escherichia coli was detected by excision/integration into the recipient integron In 3 on the conjugative plasmid R388 with the overproduced integrase. Despite several antibiotic pressures, the bel-1 cassette remained at the second position in the integron, highlighting its stability in P. aeruginosa Overexpression of the integrase gene in E. coli induced bel-1 cassette recombination. However, cassettes containing two genes ( bla BEL-1 and smr2 or bla BEL-1 and aacA4 ) were excised, suggesting that the bel-1 cassette attC site was defective. We show that bel-1 is a stable gene cassette under physiological growth conditions, irrespective of the selective antibiotic pressure, that may be mobilized upon overexpression of the integrase gene., (Copyright © 2018 American Society for Microbiology.)

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.

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.