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OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests. [ABSTRACT FROM AUTHOR]

This article discusses a study on antimicrobial resistance (AMR) in Lebanon, specifically focusing on the presence of the blaNDM-5 gene in E. coli isolates. The study found that blaNDM-5-harbouring E. coli ST617 was present in Syrian refugees, their animals, and the camp environment, posing a health risk to both the refugees and the hosting community. The study emphasizes the importance of integrated surveillance programs and One Health studies to address AMR in vulnerable populations and highlights the role of war, mass migration, and displacement in the spread of AMR. Funding for the study was provided by various organizations, and ethical considerations were taken into account. [Extracted from the article]

This article discusses the discovery of the OXA-198 carbapenemase gene cassette in the genus Candidatus Accumulibacter. The Candidatus Accumulibacter genus is known for its role in enhanced biological phosphate removal in wastewater treatment. The OXA-198 gene was first identified in clinical Pseudomonas aeruginosa isolates and later in Enterobacterales. The study found that the blaOXA-198-like gene was present in three different regions of Denmark, but unfortunately, the isolates were not cultivated. The genetic contexts of the blaOXA-198-like genes were analyzed, and it was determined that they were not acquired from other sources. The study suggests that Candidatus Accumulibacter may be the progenitor of OXA-198-like carbapenemase-encoding genes. This finding is significant as it provides insight into the origins of carbapenemase genes and may aid in future research and prevention efforts. [Extracted from the article]

Cefiderocol resistance is increasingly reported in New Delhi metallo-β-lactamase–producing Enterobacterales. Genomic and phenotypic analysis of Escherichia coli sequence type 361, a primary clone causing carbapenemase spread in France, revealed mutations leading to cefiderocol resistance. Continued genomic surveillance of carbapenem-resistant Enterobacterales could clarify prevalence of cefiderocol-resistant E. coli in Europe. [ABSTRACT FROM AUTHOR]

Background As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. Objectives Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type β-lactamases. Methods The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various β-lactamases grown on Mueller–Hinton (MH) agar, chromogenic, and chromogenic/selective media. Results The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type β-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. Conclusions The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type β-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters. [ABSTRACT FROM AUTHOR]

During 2013-2021, increased prevalence of oxacillinase 232-producing Enterobacterales was observed in France, mostly driven by its emergence in Klebsiella pneumoniae. Whole-genome sequencing identified that oxacillinase 232-producing K. pneumoniae belonged to 14 sequence types (STs), among which 2 polyclonal high-risk clones, ST-231 and ST-2096, were overrepresented. [ABSTRACT FROM AUTHOR]

Carbapenem resistance (CR) is an emerging health issue. Epidemiological surveys on carbapenem-resistant Gram-negative bacilli (CR-GNB) in Lebanon remain scarce. In this study, we determined the prevalence of CR-GNB isolated between 2015 to 2019 in three hospitals in northern Lebanon: 311 CR-Enterobacterales (out of 11210; 2.8%), 155 CR-Pseudomonas (out of 1034; 15%) and 106 CR- Acinetobacter (out of 184; 57.6%) were identified. CR mechanisms were determined for 146 randomly chosen isolates: the Carba NP test revealed an enzymatic resistance to carbapenems in 109 isolates (out of 146, 74.7%). Produced carbapenemases were evaluated by the NG-Test Carba5, NG-Test OXA-23 immunochromatographic assays and PCR. Carbapenemase-producing (CP) Enterobacterales expressed blaOXA-48-like, blaNDM-like and blaVIM-like genes and CP-Pseudomonas expressed blaIMP-like and blaVIM-like genes, whereas CP-Acinetobacter expressed blaOXA-23-like genes. The NG-Test Carba5 results were confirmed by PCR sequencing and revealed several variants, such as NDM-19, VIM-62 and OXA-162, never described so far in Lebanon. Isolates with discordant results were sequenced by WGS and highlighted novel variants of the natural oxacillinases of Pseudomonas aeruginosa: blaOXA-50-like genes. Their role in carbapenem resistance should be further studied. Overall, our findings highlight an alarming situation and encourage health care centers to establish performant registration systems that could help in limiting resistance spread. [ABSTRACT FROM AUTHOR]

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The bla VIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the bla VIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding bla VIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the bla VIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype. [ABSTRACT FROM AUTHOR]

While there are numerous variants of CIM that have been proposed [[1]], the "rCIM-A" name has already been published almost a year ago to define a rapid CIM (rCIM) protocol that optimizes results for chromosomal cephalosporinase (AmpC) hyper-producing Enterobacterales [[3]]. The Carbapenem Inactivation Method became "rapid" with the work by Muntean et al. [[6]] with final test results in less than 3 h, a time-to-result similar to those of other rapid CPE confirmation methods (colorimetric, spectrometry, etc.), thus changing the paradigm of CIM testing. As stated by Gauthier et al. interpretation of growth of I E. coli i ATCC 25922 on Mueller-Hinton agar in relation to the antibiotic is not always interpretable at 6 h [[5]]. References 1 Mitteregger D., Wessely J., Barisi'c I., Bedeni'c B., Kosak D., Kundi M. A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter Baumannii Group with Shortened Time-to-Result: RCIM-A. Pathogens. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

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

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

The worldwide spread of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non-Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non-KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone. [ABSTRACT FROM AUTHOR]

Proteus spp. are commensal Enterobacterales of the human digestive tract. At the same time, P. mirabilis is commonly involved in urinary tract infections (UTI). P. mirabilis is naturally resistant to several antibiotics including colistin and shows reduced susceptibility to imipenem. However higher levels of resistance to imipenem commonly occur in P. mirabilis isolates consecutively to the loss of porins, reduced expression of penicillin binding proteins (PBPs) PBP1a, PBP2, or acquisition of several antibiotic resistance genes, including carbapenemase genes. In addition, resistance to non-β-lactams is also frequently reported including molecules used for treating UTI infections (e.g., fluoroquinolones, nitrofurans). Emergence and spread of multidrug resistant P. mirabilis isolates, including those producing ESBLs, AmpC cephalosporinases and carbapenemases, are being more and more frequently reported. This review covers Proteus spp. with a focus on the different genetic mechanisms involved in the acquisition of resistance genes to multiple antibiotic classes turning P. mirabilis into a dreadful pandrug resistant bacteria and resulting in difficult to treat infections. [ABSTRACT FROM AUTHOR]

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

Highlights • Genomic analysis of NDM-producing Acinetobacter baumannii from Tunisia. • The clonal relationship revealed that these isolates were highly related. • This clone corresponds to a clone recovered from France 5 years ago. • Conjugation and electroporation experiments suggested that the bla NDM-1 gene is likely to be chromosomally located. ABSTRACT 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 1st June 2013 and 31st 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 Tn 125 transposon (ΔTn 125). The bla OXA-23 gene was located within transposon Tn 2008. 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. [ABSTRACT FROM AUTHOR]

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

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

Objectives: 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 theCIM and Carba NPtests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion: The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions: The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an opportunistic pathogen in healthcare facilities responsible for nosocomial infections mostly in immunocompromised patients. Colistin resistance is increasingly reported worldwide in A. baumannii . Here we describe the in vivo selection of colistin and rifampicin resistance in carbapenem-resistant A. baumannii . Antimicrobial susceptibility testing, plasmid analysis and whole-genome sequencing (WGS) were performed to fully characterise the resistome of two clinical isolates ( Ab S1 and Ab S2) selected during treatment. Clinical isolate Ab S1 remained susceptible to colistin, rifampicin and tigecycline, whilst Ab S2 was susceptible only to tigecycline. PCR analysis revealed the presence of a bla OXA-23-like carbapenemase gene. Kieser extraction revealed an ca. 74 kb plasmid harbouring bla OXA-23 . WGS revealed genomes of 3.8 Mbp in size with a G + C content of 38.9%, and both belonged to ST281 according to the Oxford MLST scheme and ST641 according to the Institut Pasteur scheme. The resistome was also composed of naturally occurring β-lactamases, i.e. ADC-25 cephalosporinase and OXA-82 oxacillinase, aminoglycoside resistance genes [ aac(3)-Ia , aadA1 and aph(3')-VIa ( aphA6 )], and mutations in DNA gyrases explaining fluoroquinolone resistance. Single nucleotide polymorphism analysis revealed that both isolates were identical except for a 30-nucleotide duplication within the pmrB gene and a point mutation in the rpoB gene resulting in colistin and rifampicin resistance, respectively. This study highlights the genomic plasticity of A. baumannii under antibiotic pressure. The 10-amino acid duplication in PmrB affects colistin susceptibility by regulating lipopolysaccharide modification through the PmrAB two-component system. These findings provide further information on the molecular mechanisms leading to colistin resistance in A. baumannii . [ABSTRACT FROM AUTHOR]

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

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

Objectives: KPC-producing pathogens exhibit variable carbapenem susceptibility levels, which is probably the result of the genetic environment of the bla KPC genes. Here we determined the transcriptional start sites (TSSs) and the expression of the bla KPC-2 gene in various genetic contexts and in different hosts ( Escherichia coli , Pseudomonas aeruginosa and Acinetobacter baumannii ).Methods: The bla KPC-2 genes along with the upstream sequences derived from Tn 4401b (structure A), Tn 4401b interrupted by Tn 3 /IS 26 (structure B) and Tn 4401b interrupted by Tn 5563 (structure C) were cloned in two E. coli shuttle vectors (pBBR1MCS.3 for expression studies in P. aeruginosa and pIM-arr2 for expression studies in A. baumannii ). MICs were determined by Etests. 5' RACE (where RACE stands for rapid amplification of cDNA ends) and quantitative RT-PCR experiments were performed to determine TSSs and transcription levels, respectively.Results: Depending on the bacterial host, different promoters were used for bla KPC-2 gene expression. The highest transcriptional level was obtained in P. aeruginosa with structure C, described only in P. aeruginosa . Tn 4401b (structure A), harbouring two promoters (P1 and P2), was the most efficient in E. coli and A. baumannii . This structure was also efficient in P. aeruginosa , although the same deduced promoter was not used (P1, instead of P2 used by E. coli and A. baumannii ). Two novel TSSs and putative promoters (P2b and P3b) were identified in structure B. In this structure, P2b and P3b were preferably used in E. coli and in P. aeruginosa , respectively, whereas P1 was used in A. baumannii .Conclusions: We determined the preferred TSSs of the bla KPC gene in each species and described two novel deduced promoters in structure B. [ABSTRACT FROM AUTHOR]

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

Since the first description of OXA-48, more than forty variants have been recovered from Enterobacterales isolates. Whereas some OXA-48-related enzymes have been reported as conferring similar resistance patterns, namely, the hydrolysis of carbapenems and penicillins with very weak or almost no activity against expanded-spectrum cephalosporins, some have reduced carbapenem and temocillin hydrolysis, and others hydrolyze expanded-spectrum cephalosporins and carbapenems only marginally. With such drastic differences in the hydrolytic profile, especially of carbapenems, it becomes urgent to establish hydrolytic cutoffs in order to determine when an OXA-48-like enzyme may be considered as a carbapenemase or not. With this aim, the coefficient of activity for imipenem (kcat/Km) was determined for a total of 30 enzymes, including OXA-48, OXA-48-like natural variants, and OXA-48 synthetic mutants. In addition, six different methods for the detection of carbapenemase-producers were performed. The coefficients of activity for imipenem for all the different enzymes went from 550 mM−1·s−1 to 0.02 mM−1·s−1. In order to match the coefficient of activity results with the biochemical confirmatory tests, we suggest the value of 0.27 mM−1·s−1 as the cutoff above which an OXA-48 variant may be considered a carbapenem-hydrolyzing enzyme. [ABSTRACT FROM AUTHOR]

OXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the β5–β6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the β5–β6 loop, which is shorter for OXA-405, suggesting that the length of the β5–β6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors. [ABSTRACT FROM AUTHOR]

Objectives: This study evaluated the performance of the Novodiag® CarbaR+ an automated qualitative nucleic acid-based diagnostic assay detecting the blaVIM, blaNDM, blaIMP, blaOXA-48, blaKPC, blaOXA-23, blaOXA-58, blaOXA-24, and ISAba1 associated blaOXA-51 carbapenemase genes and colistin resistance mcr-1 gene from clinical isolates or directly from rectal swabs. Materials and Methods: CarbaR+ was evaluated on 201 clinical isolates and on 100 rectal swabs (80 selected swabs from patients that were evaluated by culture method and/or Xpert Carba-R assay and 20 spiked samples). PCR-sequencing on colonies was considered as the gold standard. Results: The CarbaR+ detected all the variants of the targeted resistance genes (39 blaVIM-, 30 blaNDM-, 20 blaIMP-, 19 blaOXA-48-, 15 blaKPC-, 19 blaOXA-23-, 13 blaOXA-58-, 4 blaOXA-24-, 8 ISAba1-blaOXA-51-, and 3 mcr-1-like genes) with sensitivity and specificity of 98.2% and 99.7%, respectively. On the 80 rectal swabs, 71 CarbaR+ results were fully concordant with the results on selective culture media (66 positive samples with 1 to 3 carbapenemases and 5 negative samples). In eight rectal swabs, CarbaR+ identified additional carbapenemase genes. One false negative result with an Escherichia coli producing-OXA-181 was observed and one CarbaR+ result for OXA-48 was in agreement with Xpert Carba-R assay, without growth on culture media. A concordance of 100% was observed on spiked samples. Conclusions: Novodiag CarbaR+ is a random-access fully automated system that achieves excellent performances for the detection of carbapenemase and/or colistin resistance determinants either from cultured clinical isolates or directly from rectal swabs in 80 minutes. [ABSTRACT FROM AUTHOR]

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

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

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

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

• Susceptibility of a large collection of OXA-48-like-producing Enterobacterales to relebactam and vaborbactam. • Susceptibility rate was 75.7% to imipenem whereas it was 81.3% for imipenem/relebactam combination. • 95.1% of total isolates were susceptible to meropenem/vaborbactam. • Susceptibility was 63.9% for meropenem at standard dosage but increased to 94% at high meropenem exposure. • Ceftazidime/avibactam remains the best therapeutic option with a susceptibility rate of 99.5%. Relebactam and vaborbactam are among the newest β-lactamase inhibitors marketed. They were originally designed to inhibit the Ambler class A carbapenemase KPC. In this study, susceptibility to imipenem/relebactam and meropenem/vaborbactam was determined against a collection of OXA-48-like-producing Enterobacterales (n = 407). The clonality and resistomes of the isolates were determined by whole-genome sequencing. Comparison was performed with other relevant antibiotics such as carbapenems alone, ceftazidime/avibactam and ceftolozane/tazobactam. Addition of relebactam and vaborbactam did not significantly modify the MIC 50 and MIC 90 values obtained for imipenem and meropenem alone. In contrast, addition of avibactam strongly restored ceftazidime susceptibility. According to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, MIC 50 /MIC 90 values were at 2/4, 2/4, 2/8, 2/8, 32/>32 and 0.5/2 mg/L for imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, ceftazidime and ceftazidime/avibactam, respectively. No differences were observed depending on the species. This study highlights the lack of benefit in vitro for carbapenem/inhibitor combination compared with carbapenem alone against OXA-48-producing isolates as well as the difficulties in comparing molecules since carbapenem/inhibitor combinations were not developed with the same dosage of carbapenem. [ABSTRACT FROM AUTHOR]

The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array ‘Check-MDR CT103 XL’ (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs ( bla TEM , bla SHV , bla CTX-M , bla BEL , bla PER , bla GES and bla VEB ), pAmpCs ( bla CMY-2-like , bla DHA , bla FOX , bla ACC-1 , bla ACT/MIR and bla CMY-1-like/MOX ) and carbapenemases ( bla KPC , bla OXA-48 , bla VIM , bla IMP , bla NDM , bla GIM , bla SPM and bla OXA-23 , -24 and -58 ) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for bla IMP . The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents. [ABSTRACT FROM AUTHOR]

Cefiderocol is a catechol-substituted cephalosporin dedicated to the treatment of infections caused by multidrug resistant gram-negative rods. Cefiderocol susceptibility testing might be complex. We compared cefiderocol susceptibility testing methods on a relevant collection of carbapenem-resistant Enterobacterales. CE-IVD (European CE marking required for all in vitro diagnostic (IVD)) broth microdilution (BMD) plate (ThermoFisher, Waltham, MA, USA) using regular Mueller-Hinton broth, MIC test strip (Liofilchem, Teramo, Italy), and disk diffusion (Liofilchem) were compared to a frozen BMD plate prepared with iron depleted Mueller-Hinton broth. First, a collection of 100 entirely sequenced carbapenem-resistant Enterobacterales was used to compare these methods. Then, a prospective comparison of disk diffusion and CE-IVD BMD was performed on 827 consecutive carbapenem non-susceptible Enterobacterales including 634 carbapenemase-producers. Compared to reference method, CE-IVD BMD plate gave 95.0% (95% CI, 88.8–97.9) categorisation agreement (CA), 2.8% (95% CI, 0.4–14.2) very major errors (VME), and 1.6% (95% CI, 0.3–8.7) major errors (ME) with high reproducibility. MIC strip gave only 63% (95% CI, 53.2–71.8) of CA and 94.9% (95% CI, 83.1–98.6) of VME due to critical underestimation of the MICs. Disk diffusion gave 77% (95% CI, 67.9–84.2) CA with additional 8% of the isolates within the area of technical uncertainty of 18–22 mm. Prospectively, disk diffusion gave 81.7% (95% CI, 79.0–84.2) CA, 23.3% (95% CI, 15.1–34.2%)VME, and 4.9% (95% CI, 3.6–6.7) ME. Additionally, 21.3% (95% CI, 18.6–24.2) of CRE were within the area of technical uncertainty. Commercial CE-IVD BMD (ThermoFisher) is accurate for cefiderocol MIC determination in difficult-to-treat Enterobacterales whereas MIC test strip (Liofilchem), that was formulated for Pseudomonas aeruginosa only, should be avoided. Disk diffusion might be useful for screening, but many of these CRE have to be re-tested using BMD to assess definitive categorization. [ABSTRACT FROM AUTHOR]

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

• First description of VIM-2-producing Enterobacter hormaechei subsp. steigerwaltii. • First description of Tn 1332 in Enterobacterales. • Isolates exhibited different resistance levels explained notably by deletion of 117-kb on the chromosome including ompD. • A mutant was obtained possessing an increase of bla VIM-2 copy number as well as a mutation in WecA. 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 Tn 1332 and was embedded in a remnant of a class 1 integron. Tn 1332 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 Tn 1332 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 Tn 1332 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 Tn 1332 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 Tn 1332 in Enterobacterales and the phenotypic diversity of VIM-2-producing E. hormaechei. [ABSTRACT FROM AUTHOR]

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

• Second description worldwide of LMB-1, a novel subclass B3 MBL in Citrobacter freundii 164 from Argentina. • LMB-1 confers reduced susceptibility to carbapenems and gives inconsistent results with carbapenemase confirmatory tests. • The bla LMB-1 gene was located on a 176-kb IncA/C2 plasmid. • The genetic environment of bla LMB-1 indicates an IS CR -like mobilization from the chromosome of Rheinheimera pacifica. • C. freundii 164 expressed CMY-150, a novel CMY-2 variant that increases ceftazidime and aztreonam MICs when expressed in E. coli. Carbapenemase-producing Enterobacterales expressing OXA-48, KPC, NDM, VIM or IMP enzymes are increasingly reported worldwide. We have characterized LMB-1, a novel metallo-β-lactamase (MBL) of Ambler class B3 from Citrobacter freundii 164 (Cf 164) clinical isolate from Buenos Aires, Argentina. Cf 164 displayed reduced susceptibility to carbapenems but gave inconsistent results with carbapenemase confirmatory tests, indicating the presence of a weak carbapenemase. Analysis of whole-genome sequencing (WGS) of Cf 164 using Resfinder revealed four β-lactamase genes coding for CTX-M-8, PER-2, TEM-1 and CMY-150, a novel chromosomally-encoded CMY variant. Kinetic parameters of purified CMY-150 did not reveal any carbapenemase activity. However, CMY-150 conferred higher minimum inhibitory concentrations (MICs) to E. coli for ceftazidime and aztreonam compared with CMY-2. The in-house-developed β-lactamase search software (ResMiner) in WGS data revealed a novel subclass B3 MBL named LMB-1. LMB-1 conferred resistance to penicillins and expanded-spectrum cephalosporins and reduced susceptibility to carbapenems in E. coli. The bla LMB-1 gene was located on a 176-kb IncA/C2 plasmid. LMB-1 shared 99% amino acid sequence identity with the MBL encoded in the chromosome of Rheinheimera pacifica, it's likely progenitor. Despite repeated attempts, LMB-1 could not be purified, thus only specific activities could indicate hydrolysis of carbapenems. Here we report on CMY-150, a novel CMY-2 variant that confers increased ceftazidime and aztreonam MICs to E. coli and the first description of LMB-1 in Argentina. This work underlines the need for several carbapenemase-producing Enterobacteriaceae (CPE) confirmatory tests, as this novel enzyme might have been missed using only one. Image, graphical abstract [ABSTRACT FROM AUTHOR]

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