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Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that bla TEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of bla TEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of bl a TEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and bla TEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of bla TEM gene in P/T-R isolates, generated by three different genetic events: (1) IS 26 -mediated duplication of the bla TEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying bla TEM , and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms. • IS26-mediated duplication of the bla TEM gene is involved in the acquisition of P/T-R by E. coli. • Generation of a small multicopy plasmid carrying bla TEM is involved in the acquisition of P/T-R by E. coli. • Adaptive evolution via plasmid size reduction, leading to a higher plasmid copy number is involved in the P/T-R by E. coli. • Reduction of OmpC expression is associated with P/T-R in E. coli. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/S1473-3099(09)70054-4 Byline: Patrice Nordmann (a), Gaelle Cuzon (a), Thierry Naas (a) Abstract: From early this decade, Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases (KPC) were reported in the USA and subsequently worldwide. These KPC-producing bacteria are predominantly involved in nosocomial and systemic infections; although they are mostly Enterobacteriaceae, they can also be, rarely, Pseudomonas aeruginosa isolates. KPC [beta] lactamases (KPC-1 to KPC-7) confer decreased susceptibility or resistance to virtually all [beta] lactams. Carbapenems (imipenem, meropenem, and ertapenem) may thus become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are, in addition, resistant to many other non-[beta]-lactam molecules, leaving few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. It is therefore crucial to implement efficient infection control measures to limit the spread of these pathogens. Author Affiliation: (a) Service de Bacteriologie-Virologie-Hygiene, INSERM U914 'Emerging Resistance to Antibiotics', Hopital de Bicetre, Assistance Publique/Hopitaux de Paris, Faculte de Medecine and Universite Paris Sud, K -Bicetre, France

The high frequency autonomous retrotransposition of the human long interspersed nuclear elements (L1) L1.2 and LRE2in a cultured human cell line is demonstrated. The retrotransposed products resemble endogenous L1 insertions. Point mutations in conserved domains of L1.2-encoded proteins decreased retrotransposition by 100 to 1,000 times. L1.2 also retrotransposed in a mouse cell line, suggesting that L1-based vectors play a significant part in random insertional mutagenesis.

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]

New resistance problems have emerged recently among hospital and community-acquired pathogens such as in Staphylococcus aureus, Enterococcus faecium and Pseudomonas aeruginosa. Hospital-acquired and now community-acquired methicillin-resistant S. aureus are emerging worldwide whereas vancomycin-resistant S. aureus remain extremely rare. Hospital-acquired outbreaks of vancomycin-resistant enterococci and multidrug resistant Pseudomonas aeruginosa infections are increasingly reported worldwide. Whereas novel molecules are being developed for treating Gram-positive infections, difficult to non possible-to-treat pandrug-resistant P. aeruginosa infections may become a therapeutic challenge soon. [Copyright &y& Elsevier]

The recently released CHROMagar™ ESBL/CHROMagar™ mSuperCARBA bi-plate medium was evaluated for the detection of ESBL- and carbapenemase-producing Enterobacteriaceae. Spiked stools were used to mimic in vivo stool colonization. Two-hundred enterobacterial isolates were tested. Respective sensitivities of 93.9% and 97.8% were obtained for the detection of ESBL and carbapenemase producers. [ABSTRACT FROM AUTHOR]

In low- and middle-income countries (LMICs), data related to antimicrobial resistance (AMR) are often inconsistently collected. Humanitarian, private and non-governmental medical organizations (NGOs), working with or in parallel to public medical systems, are sometimes present in these contexts. Yet, what is the role of NGOs in the fight against AMR, and how can they contribute to AMR data collection in contexts where reporting is scarce? How can context-adapted, high-quality clinical bacteriology be implemented in remote, challenging and underserved areas of the world? The aim was to provide an overview of AMR data collection challenges in LMICs and describe one initiative, the Mini-Lab project developed by Médecins Sans Frontières (MSF), that attempts to partially address them. We conducted a literature review using PubMed and Google scholar databases to identify peer-reviewed research and grey literature from publicly available reports and websites. We address the necessity of and difficulties related to obtaining AMR data in LMICs, as well as the role that actors outside of public medical systems can play in the collection of this information. We then describe how the Mini-Lab can provide simplified bacteriological diagnosis and AMR surveillance in challenging settings. NGOs are responsible for a large amount of healthcare provision in some very low-resourced contexts. As a result, they also have a role in AMR control, including bacteriological diagnosis and the collection of AMR-related data. Actors outside the public medical system can actively contribute to implementing and adapting clinical bacteriology in LMICs and can help improve AMR surveillance and data collection. [ABSTRACT FROM AUTHOR]

Background: New antibiotics are needed for the treatment of patients with life-threatening carbapenem-resistant Gram-negative infections. We assessed the efficacy and safety of cefiderocol versus best available therapy in adults with serious carbapenem-resistant Gram-negative infections.Methods: We did a randomised, open-label, multicentre, parallel-group, pathogen-focused, descriptive, phase 3 study in 95 hospitals in 16 countries in North America, South America, Europe, and Asia. We enrolled patients aged 18 years or older admitted to hospital with nosocomial pneumonia, bloodstream infections or sepsis, or complicated urinary tract infections (UTI), and evidence of a carbapenem-resistant Gram-negative pathogen. Participants were randomly assigned (2:1 by interactive web or voice response system) to receive either a 3-h intravenous infusion of cefiderocol 2 g every 8 h or best available therapy (pre-specified by the investigator before randomisation and comprised of a maximum of three drugs) for 7-14 days. For patients with pneumonia or bloodstream infection or sepsis, cefiderocol treatment could be combined with one adjunctive antibiotic (excluding polymyxins, cephalosporins, and carbapenems). The primary endpoint for patients with nosocomial pneumonia or bloodstream infection or sepsis was clinical cure at test of cure (7 days [plus or minus 2] after the end of treatment) in the carbapenem-resistant microbiological intention-to-treat population (ITT; ie, patients with a confirmed carbapenem-resistant Gram-negative pathogen receiving at least one dose of study drug). For patients with complicated UTI, the primary endpoint was microbiological eradication at test of cure in the carbapenem-resistant microbiological ITT population. Safety was evaluated in the safety population, consisting of all patients who received at least one dose of study drug. Mortality was reported through to the end of study visit (28 days [plus or minus 3] after the end of treatment). Summary statistics, including within-arm 95% CIs calculated using the Clopper-Pearson method, were collected for the primary and safety endpoints. This trial is registered with ClinicalTrials.gov (NCT02714595) and EudraCT (2015-004703-23).Findings: Between Sept 7, 2016, and April 22, 2019, we randomly assigned 152 patients to treatment, 101 to cefiderocol, 51 to best available therapy. 150 patients received treatment: 101 cefiderocol (85 [85%] received monotherapy) and 49 best available therapy (30 [61%] received combination therapy). In 118 patients in the carbapenem-resistant microbiological ITT population, the most frequent carbapenem-resistant pathogens were Acinetobacter baumannii (in 54 patients [46%]), Klebsiella pneumoniae (in 39 patients [33%]), and Pseudomonas aeruginosa (in 22 patients [19%]). In the same population, for patients with nosocomial pneumonia, clinical cure was achieved by 20 (50%, 95% CI 33·8-66·2) of 40 patients in the cefiderocol group and ten (53%, 28·9-75·6) of 19 patients in the best available therapy group; for patients with bloodstream infection or sepsis, clinical cure was achieved by ten (43%, 23·2-65·5) of 23 patients in the cefiderocol group and six (43%, 17·7-71·1) of 14 patients in the best available therapy group. For patients with complicated UTIs, microbiological eradication was achieved by nine (53%, 27·8-77·0) of 17 patients in the cefiderocol group and one (20%, 0·5-71·6) of five patients in the best available therapy group. In the safety population, treatment-emergent adverse events were noted for 91% (92 patients of 101) of the cefiderocol group and 96% (47 patients of 49) of the best available therapy group. 34 (34%) of 101 patients receiving cefiderocol and nine (18%) of 49 patients receiving best available therapy died by the end of the study; one of these deaths (in the best available therapy group) was considered to be related to the study drug.Interpretation: Cefiderocol had similar clinical and microbiological efficacy to best available therapy in this heterogeneous patient population with infections caused by carbapenem-resistant Gram-negative bacteria. Numerically more deaths occurred in the cefiderocol group, primarily in the patient subset with Acinetobacter spp infections. Collectively, the findings from this study support cefiderocol as an option for the treatment of carbapenem-resistant infections in patients with limited treatment options.Funding: Shionogi. [ABSTRACT FROM AUTHOR]

• Culture-based screening of CPE using enrichment broth is sensitive but causes delay • Molecular-based screening of CPE is rapid but might miss low-level carriage • Molecular biology on enriched broth might have both sensitivity and rapidity Early detection of patients colonized with carbapenemase-producing Enterobacterales is crucial to limit their spread. Molecular biology tests are rapid but might miss low-level carriage. Culture-based methods using enrichment are cheap and detect low-level carriage but cause delay. Two clinical cases are reported, which demonstrated that molecular biology (Xpert® Carb-R) on enriched broth cumulates advantages of both strategies. In both cases, this strategy enabled early detection of patients colonized at low-level with carbapenemase-producing Enterobacterales because it saved 24 hours in detection time. [ABSTRACT FROM AUTHOR]

Escherichia coli ST410 (Ec -ST410) is an emerging, multidrug-resistant clone. Recent investigations of its global epidemiology and evolution have been based almost exclusively on isolates from Europe and North America. It is unclear whether Southeast Asian-origin Ec- ST410 (SEA- Ec -ST410) belong to these same clones or represent regionally disseminated variants. Antimicrobial resistance mechanisms among SEA- Ec -ST410 were characterised, and whether they belonged to regional variants was investigated by contextualising them within a global collection. Seven Ec -ST410 were identified among a recent collection of expanded-spectrum cephalosporin-resistant E. coli recovered from 91 healthy women (stool) and 26 infected patients (blood and urine) living in Phnom Penh, Cambodia. Nine additional Ec -ST410 genomes were identified from Thailand (n = 7) and Vietnam (n = 2) through EnteroBase and PubMed searches. The assembled genomes were characterised and a SNP-based phylogenetic tree was created comparing these 16 SEA -Ec -ST410 with a previously published Ec -ST410 collection, primarily sourced from Europe (97/128) and North America (24/128). SEA -Ec -ST410 belonged to several distinct branches within previously described clonal clades. SEA -Ec -ST410 within the B3/ H 24Rx sublineage encoded bla CTX-M-55 (8/12) and F18:A-:B1 plasmid replicons (6/12), neither of which were detected among other Ec -ST410 belonging to this clade. Three of four SEA -Ec -ST410 within the B4/ H 24RxC sublineage lacked both bla OXA-181 and an IncX3 plasmid replicon that were harboured by 97% and 100% of all other Ec -ST410 in this sublineage (n = 64), respectively. In conclusion, Ec -ST410 are present in Southeast Asia following multiple introductions. The unique pattern of antimicrobial resistance elements harboured by SEA -Ec -ST410 suggests independent circulation in the region. [ABSTRACT FROM AUTHOR]

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

• Eleven bla PER-2 -harboring plasmids from Enterobacterales were fully sequenced. • The bla PER-2 gene was encoded on a variety of plasmid types (IncA, C, FIB, HI1B, N2). • IS Pa12 -composite transposons are suggested to be involved in bla PER-2 origin recruitment and dissemination. • A novel IS Pa12 -composite transposon, Tn 7390 , carrying the bla PER-2 gene was identified. • IS Kox2 -like elements seem to play a role in bla PER-2 gene dissemination. The production of PER-like extended-spectrum β-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring bla PER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with bla PER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the bla PER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the bla PER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of bla PER genes), indicates a role of IS Pa12 in bla PER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the bla PER-2 gene was carried by a novel IS Pa12 -composite transposon, Tn 7390. In addition, its association with IS Kox2 -like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of bla PER-2 genes. [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]

• 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]

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]

We describe the case of NDM-1-producing Leclercia adecarboxylata recovered from the clinical sample of a patient hospitalized for a trauma-related injury to his foot. The isolate was resistant to all beta-lactams, quinolones, trimetroprim-sulfametoxazol, gentamicin and tobramicyn. The bla NDM-1 gene was located in a conjugative plasmid that also contained the bla SHV-12 gene and was preceded by a disrupted insertion sequence of IS Aba125. The plasmid belongs to the incompatibility group X3, which is known to be an important vector for NDM-1 dissemination in China. This is the first reported case of NDM-1 L. adecarboxylata in our country and evidences that species of uncertain clinical relevance can act as hidden sources of clinically important resistance determinants. [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]

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. [Display omitted] • Forty azetidinimines synthesized and evaluated for their activity against carbapenemases. • Several compounds are submicromolar inhibitors of both MBLs (NDM-1) and SBLs of class A (KPC-2) and D (OXA-48). • One compound is also active against 4 other MBLs (NDM-4, NDM-7, NDM-9 and VIM-1), CTX-M-15 (class A) and CMY-2 (class C). • The same compound can repotentiate imipenem against a NDM-1-producing clinical isolate of E. coli. [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]

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

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