Search

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25-4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor.In the present study, bla CTX-M-15 and bla KPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC 50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors., (© 2024. The Author(s).)

Imipenemase (IMP) metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactams including carbapenems and are not inhibited by any commercially available β-lactamase inhibitor. Tebipenem (TP) pivoxil is the first orally available carbapenem and possesses a unique bicyclic azetidine thiazole moiety located at the R2 position. TP has potent in vitro activity against Enterobacterales producing extended-spectrum and/or AmpC β-lactamases. Thus far, the activity of TP against IMP-producing strains is understudied. To address this knowledge gap, we explored the structure activity relationships of IMP MBLs by investigating whether IMP-6, IMP-10, IMP-25, and IMP-78 [MBLs with expanded hydrolytic activity against meropenem (MEM)] would demonstrate enhanced activity against TP. Most of the Escherichia coli DH10B strains expressing IMP-1 variants displayed a ≥twofold MIC difference between TP and MEM, while those expressing VIM or NDM variants demonstrated comparable MICs. Catalytic efficiency ( k cat / K M ) values for the TP hydrolysis by IMP-1, IMP-6, IMP-10, IMP-25, and IMP-78 were significantly lower than those obtained for MEM. Molecular dynamic simulations reveal that V67F and S262G substitutions (found in IMP-78) reposition active site loop 3, ASL-3, to better accommodate the bicyclic azetidine thiazole side chain, allowing microbiological/catalytic activity to approach that of comparison MBLs used in this study. These findings suggest that modifying the R2 side chain of carbapenems can significantly impact hydrolytic stability. Furthermore, changes in conformational dynamics due to single amino acid substitutions should be used to inform drug design of novel carbapenems., Competing Interests: The authors declare no conflict of interest.

Aims: The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021., Methods and Results: Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n = 642) and AmpC (n = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 gene groups by PCR. Genes belonging to blaCTX-M-1 and blaCTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No blaCTX-M-2 group's gene was found. A subset of strains (n = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were blaCTX-M (n = 21), with blaCTX-M-15 mostly represented (n = 15), as well as blaTEM (n = 11), blaOXA (n = 7), blaSHV (n = 9), and carbapenemase (CP) genes were observed in several strains-blaKPC-3 (n = 19), blaNDM-1 (n = 1), blaVIM-1 (n = 2), and blaOXA-244 (n = 2)-even from freshwaters., Conclusions: ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

We report a fatal case of New Delhi metallo-β-lactamase (NDM)-producing Escherichia coli in a bacteremic patient with sequential failure of aztreonam plus ceftazidime-avibactam followed by cefiderocol. Acquired resistance was documented phenotypically and mediated through preexisting and acquired mutations. This case highlights the need to rethink optimal treatment for NDM-producing organisms., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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

In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D β-lactamase genes bla OXA-1176 and bla OXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli gene encodes carbapenemase. In contrast, bla OXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that bla OXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing bla OXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of bla OXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCE Ralstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including β-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel bla OXA-1176 and bla OXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings., Competing Interests: The authors declare no conflict of interest.

Aims: Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144., Methods and Results: The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin., Conclusions: Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

In response to the increasing number of carbapenem-resistant Enterobacterales (CRE), we investigated carbapenemase-producing Klebsiella pneumoniae and non-K. pneumoniae epidemiology and genetics. We collected 76 clinical Enterobacterales and 4 stool surveillance Escherichia coli isolates resistant to ertapenem or imipenem. Using polymerase chain reaction (PCR) and DNA sequencing, we assessed carbapenemases, extended-spectrum β-lactamases, and AmpC β-lactamases. Molecular typing via pulsed-field gel electrophoresis (PFGE) and conjugation experiments were conducted to examine resistance gene transfer. Among the 80 isolates, 96.2% harbored at least one carbapenemase gene, with bla OXA-48 in 87.5%. KPC-2 and IMP-8 carbapenemases were found in 15.0 and 22.5% of the isolates, respectively, with 27.5% having 2 or more carbapenemase genes. The PFGE analysis revealed the presence of diverse genotypes. PCR-based plasmid replicon typing identified IncA/C as the most prevalent type among K. pneumoniae isolates (26/29), and IncF and IncFIB among E. coli isolates (22/28). Conjugal transfer was successful for plasmids encoding OXA-48, CTX-M-3, CTX-M-14, CMY-2, and other β-lactamases, except the KPC-2 gene. In conclusion, our study highlights high carbapenemase prevalence in CRE, primarily OXA-48. Multiple carbapenemases within strains were common, and PFGE showed diverse patterns in these carbapenem-resistant isolates.

Objectives: Since its discovery, bla NDM-5 has spread widely amongst Escherichia coli strains in clinical patients, causing carbapenem resistance. Here we report the complete genome sequence of an NDM-5-producing E. coli strain isolated from the faecal sample of a healthy individual in Hangzhou, China., Methods: The whole-genome sequence of E. coli CREC8 was obtained utilising both the Nanopore sequencer and the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes, multilocus sequence typing, and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between CREC8 and other E. coli strains was investigated using the core genome multilocus sequence typing (cgMLST) strategy., Results: The complete genome sequence of E. coli CREC8 consists of one chromosome and 7 plasmids. CREC8 belongs to ST167 according to the MLST scheme. Seven ARGs were identified, including carbapenem resistance gene bla NDM-5 which was located in an IncFIA/IncFII type plasmid. A total of 164 E. coli ST167 strains related to 25 countries across four continents can be retrieved from the NCBI database, 95 of them carrying the bla NDM gene with bla NDM-5 the most (N = 79). Phylogenetic analysis revealed a worldwide distribution of E. coli ST167 strains, with China having the highest prevalence (37%, 61/165)., Conclusion: In summary, we reported a bla NDM-5 -carrying E. coli ST167 strain isolated from a healthy individual in China. Such strains are more commonly isolated from hospitalised patients but are rarely isolated from healthy individuals. This indicates a further epidemic of carbapenem-resistant E. coli strains in the healthy population which needs our attention., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The emergence of carbapenem resistance is a major public health threat in sub-Saharan Africa but remains poorly understood, particularly at the human-animal-environment interface. This study provides the first One Health-based study on the epidemiology of Carbapenemase-Producing Gram-Negative Bacteria (CP-GNB) in Djibouti City, Djibouti, East Africa. In total, 800 community urine samples and 500 hospital specimens from humans, 270 livestock fecal samples, 60 fish samples, and 20 water samples were collected and tested for carbapenem resistance. The overall estimated CP-GNB prevalence was 1.9 % (32/1650 samples) and specifically concerned 0.3 % of community urine samples, 2.8 % of clinical specimens, 2.6 % of livestock fecal samples, 11.7 % of fish samples, and 10 % of water samples. The 32 CP-GNB included 19 Escherichia coli, seven Acinetobacter baumannii, five Klebsiella pneumoniae, and one Proteus mirabilis isolate. Short-read (Illumina) and long-read (Nanopore) genome sequencing revealed that carbapenem resistance was mainly associated with chromosomal carriage of bla NDM-1 , bla OXA-23 , bla OXA-48 , bla OXA-66 , and bla OXA-69 in A. baumannii, and with plasmid carriage in Enterobacterales (bla NDM-1 and bla OXA-181 in E. coli, bla NDM-1 , bla NDM-5 and bla OXA-48 in K. pneumoniae, and bla NDM-1 in P. mirabilis). Moreover, 17/32 CP-GNB isolates belonged to three epidemic clones: (1) A. baumannii sequence type (ST) 1697,2535 that showed a distribution pattern consistent with intra- and inter-hospital dissemination; (2) E. coli ST10 that circulated at the human-animal-environment interface; and (3) K. pneumoniae ST147 that circulated at the human-environment interface. Horizontal exchanges probably contributed to carbapenem resistance dissemination in the city, especially the bla OXA-181 -carrying ColKP3-IncX3 hybrid plasmid that was found in E. coli isolates belonging to different STs. Our study highlights that despite a relatively low CP-GNB prevalence in Djibouti City, plasmids harboring carbapenem resistance circulate in humans, animals and environment. Our findings stress the need to implement preventive and control measures for reducing the circulation of this potentially emerging public health threat., Competing Interests: Declaration of competing interest The authors declare they do not have known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Enterobacterales carrying bla NDM represent an emerging challenge in treating infectious diseases. In this study, we aimed to investigate the characteristics of bla NDM -producing Enterobacterales from three hospitals in southern Taiwan., Methods: Enterobacterales strains that were nonsusceptible to more than one carbapenem (ertapenem, meropenem, imipenem, or doripenem) were collected from hospitalized patients. Molecular typing for New Delhi metallo-β-lactamase (NDM) and antibiotic susceptibility tests were performed, followed by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and plasmid analysis by PCR-based replicon typing., Results: A total of 1311 carbapenem-nonsusceptible Enterobacterales were isolated from 2017 to 2021. bla NDM -encoding genes were detected in 108 isolates, with 53 (49.1%) harboring bla NDM-1 and 55 (50.9%) harboring bla NDM-5 . The rate of bla NDM-1 detection among isolates decreased to 2% in 2021. However, the rate of E. coli harboring bla NDM-5 increased from 1% to 12% of total isolates during the study period. Of 47 NDM-5-positive E. coli isolates, 44 (93.6%) were ST8346 with high genetic relatedness. E. coli ST8346 isolates showed high-level resistance to both carbapenems and aminoglycosides. Most (38 out of 47, 80.9%) bla NDM-5 -harboring E. coli isolates co-harbored bla OXA-181 . We analyzed the regions harboring bla NDM-5 in E. coli ST8346 via PCR amplification. bla NDM-5 and bla OXA-181 were located on two separate plasmids, IncF and IncX3, respectively., Conclusion: The dissemination of E. coli ST8346 caused an increase in bla NDM-5 and bla OXA-181 co-harboring Enterobacterales in southern Taiwan, which show high-level resistance to both carbapenems and aminoglycosides. We identified a distinct IncF plasmid encoding bla NDM-5 that has the potential for rapid spread and needs further surveillance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Antimicrobial resistance (AR) mechanisms encoded on plasmids can affect other phenotypic traits in bacteria, including biofilm formation. These effects may be important contributors to the spread of AR and the evolutionary success of plasmids, but it is not yet clear how common such effects are for clinical plasmids/bacteria, and how they vary among different plasmids and host strains. Here, we used a combinatorial approach to test the effects of clinical AR plasmids on biofilm formation and population growth in clinical and laboratory Escherichia coli strains. In most of the 25 plasmid-bacterium combinations tested, we observed no significant change in biofilm formation upon plasmid introduction, contrary to the notion that plasmids frequently alter biofilm formation. In a few cases we detected altered biofilm formation, and these effects were specific to particular plasmid-bacterium combinations. By contrast, we found a relatively strong effect of a chromosomal streptomycin-resistance mutation (in rpsL) on biofilm formation. Further supporting weak and host-strain-dependent effects of clinical plasmids on bacterial phenotypes in the combinations we tested, we found growth costs associated with plasmid carriage (measured in the absence of antibiotics) were moderate and varied among bacterial strains. These findings suggest some key clinical resistance plasmids cause only mild phenotypic disruption to their host bacteria, which may contribute to the persistence of plasmids in the absence of antibiotics., Competing Interests: Declaration of Competing Interest We have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The circulation of carbapenemase-producing Escherichia coli (CPEC) in our society is a serious concern for vulnerable patients in nosocomial environments. However, the genomic epidemiology of the circulation of CPEC bacteria among companion animals remains largely unknown. In this study, epidemiological analysis was conducted using complete genome identification of CPEC ST410 isolates obtained from companion animals. To estimate the genomic distance and relatedness of the isolates, a total of 37 whole-genome datasets of E. coli ST410 strains were downloaded and comparatively analysed. As a result of the analysis, the genomic structure of the chromosomes and plasmids was identified, revealing the genomic positions of multiple resistance and virulence genes. The isolates in this study were grouped into the subclade H24/RxC, with fimH24, and substituted quinolone resistance-determining regions (QRDRs) and multiple beta-lactamases, including extended-spectrum β-lactamase (ESBL) and carbapenemase. In addition, the in silico comparison of the whole-genome datasets revealed unidentified ST410 H24/Rx subgroups, including either high pathogenicity islands (HPIs) or H21 serotypes. Considering the genetic variations and resistance gene dissemination of the isolates carried by companion animals, future approaches for preventive measurement must include the "One Health" perspective for public health in our society., (© 2023. L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE).)

Objectives: A higher diversity of species, clones and genes have been increasingly implicated in carbapenemases spread, though the mobile genetic elements responsible for their acquisition and dispersion at local and global levels are less explored, particularly in species other than Klebsiella pneumoniae or Escherichia coli. We aim to explain the emergence of NDM-1 and KPC-3 carbapenemases in a Kluyvera cryocrescens isolate, and to shed light on the heterogeneity of genetic platforms and acquisition routes of bla NDM-1 in diverse Enterobacterales species in Portugal., Methods: A KPC-3 and NDM-1-producing K. cryocrescens colonizing a hospitalized patient in 2019 was characterized by whole-genome sequencing and antibiotic resistance profiling following standard methods. Conjugative transfer of carbapenemases genes was assessed by filter mating. Plasmids were reconstructed with in silico and in vitro approaches. bla NDM-1 genetic context was compared with that of diverse NDM-1-producing Enterobacterales species, previously described in Portugal., Results: K. cryocrescens K629 showed a multidrug resistance profile. Resistance gene bla KPC-3 was harboured by a Tn4401d transposon within a worldwide-spread IncN-ST15 plasmid (pKLU-KPC3), whereas bla NDM-1 was located in a Tn3000 within a non-typeable mosaic plasmid (pKLU-NDM1). The heterogeneous bla NDM-1 genetic platforms and variable plasmid backbones identified in various Enterobacterales species suggested multiple introductions of bla NDM-1 in Portugal, mediated by variable insertion sequences., Conclusions: We report the convergence of KPC-3 and NDM-1 in K. cryocrescens and the variable dissemination modes of these carbapenemases in different Enterobacterales species, underlining the need to track down genetic platforms responsible for carbapenemases diffusion., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages., Methods: In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240)., Results: Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates bla VIM-1 replaced bla OXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum β-lactamases decreased over time., Conclusions: This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Infections with OXA-244-carbapenemase-producing Escherichia coli with sequence type (ST)38 have recently increased in Europe. Due to its low-level activity against carbapenems, OXA-244 can be difficult to detect. Previous assessments have not revealed a clear source and route of transmission for OXA-244-producing E. coli , but there are indications of non-healthcare related sources and community spread. Here we report a hospital-associated outbreak of OXA-244-producing E. coli ST38 involving three hospitals in Western Norway in 2020. The outbreak occurred over a 5-month period and included 12 cases identified through clinical (n = 6) and screening (n = 6) samples. The transmission chain was unclear; cases were identified in several wards and there was no clear overlap of patient stay. However, all patients had been admitted to the same tertiary hospital in the region, where screening revealed an outbreak in one ward (one clinical case and five screening cases). Outbreak control measures were instigated including contact tracing, isolation, and screening; no further cases were identified in 2021. This outbreak adds another dimension to the spread of OXA-244-producing E. coli ST38, illustrating this clone's ability to establish itself in the healthcare setting. Awareness of challenges concerning OXA-244-producing E. coli diagnostic is important to prevent further spread.

Epidemiological surveys have shown that carbapenem resistance is mainly transmitted across species by carbapenemase genes located on conjugative plasmids. As chromosomal integration of carbapenemase genes has rarely been identified, only a few studies have investigated their advantages to the carbapenem-resistant bacterial community. Here, we confirmed the increased stability of bla IMP-6 isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying Escherichia coli isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying bla IMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant E. coli (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.bla IMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.

During a 2020 routine epidemiological investigation of carbapenem-resistant Enterobacterales at a local food market in Guangzhou, China, two Escherichia coli ST410 isolates coproducing NDM-5 and OXA-181 were obtained from environmental samples. Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation assays were applied to identify their resistance phenotypes, phylogenetic relatedness, and genetic characteristics. Phylogenetic analysis showed that the two isolates were clonally related with only one core-genome single-nucleotide polymorphism (SNP) difference and clustered into a branch with 87 E. coli ST410 isolates deposited in GenBank. These 89 ST410 isolates were closely related (≤51 SNPs), and most were from humans in Southeast Asian countries ( n  = 47). A Vietnamese clinical isolate collected in 2017 showed the strongest epidemiological link (seven SNPs) to the two ST410 isolates detected in this study. Complete-genome analysis revealed that the carbapenem resistance determinants bla NDM-5 and bla OXA-181 were located on an IncF1:A1:B49-IncQ1 plasmid and IncX3 plasmid, respectively. Conjugation experiments confirmed that the IncX3 plasmid was self-transmissible while the IncF1:A1:B49-IncQ1 plasmid was nonconjugative. BLASTn analysis indicated that the two plasmids showed high similarity to other bla NDM-5 This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones.bla OXA-181 -bearing IncX3 plasmids from other countries. Altogether, the high similarity of the core genomes and plasmids between the ST410 isolates found in this study and those human source isolates from foreign countries suggested the clonal spread of E. coli ST410 strains and horizontal transmission of bla OXA-181 -bearing IncX3 plasmids across Southeast Asian countries. Stringent sanitary management of food markets is important to prevent the dissemination of high-risk clones to the public. IMPORTANCE This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones., Competing Interests: The authors declare no conflict of interest.

Carbapenem-resistant Enterobacteriaceae infections are among the most serious threats to human and animal health worldwide. Of the 1013 strains of Escherichia coli isolated and identified in 14 regions of China from 2007 to 2018, seven strains were resistant to meropenem and all were positive for bla NDM . The seven New Delhi metallo-β-lactamase (NDM)-positive strains belonged to five different sequence types, indicating that most of the NDM-positive strains were nonclonal. An IncHI2 plasmid carrying the bla NDM-1 element was identified in the C1147 strain from a goose source and reported for the first time, showing a specific structure. Conjugation experiments revealed that the IncHI2 plasmid was conjugatable, and the horizontal propagation of the plasmid led to the rapid propagation of NDM in the same and different strains. This study revealed that waterfowl, as a potential transmission factor for carbapenem-resistant bla NDM-1 , poses a threat to human health.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 10 4 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 10 3 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (10 5 cell equivalents/mL) and the highest levels of the bla KPC gene (up to 10 -1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, bla CTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, bla KPC-2 and bla NDM-1 were the most frequently detected CP genes, followed by bla OXA-48 . Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (bla CTX-M-15 /bla TEM-116 ) and ST541 (bla KPC-2 ), K. pneumoniae ST101 (bla OXA-48 /bla NDM-1 ), and Enterobacter cloacae complex ST277 (bla KPC-2 /bla NDM-1 ) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the bla CTX-M , bla OXA-48 , and bla KPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Objectives: Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation., Methods: Three plasmids, IncX3-type plasmid with bla NDM-1 , IncN-type plasmid with bla KPC-2 , and ColE-type plasmid with bla OXA-232 , were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants., Results: Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin., Conclusion: In this study, we showed that a small ColE-type plasmid bearing bla OXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase., Competing Interests: Competing interests All authors report no conflicts of interest relevant to this article., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

BackgroundCarbapenemase-producing Enterobacterales (CPE) are rapidly increasing worldwide, also in Europe. Although prevalence of CPE in Germany is comparatively low, the National Reference Centre for Multidrug-resistant Gram-negative Bacteria noted annually increasing numbers of NDM-5-producing Escherichia coli isolates.AimAs part of our ongoing surveillance programme, we characterised NDM-5-producing E. coli isolates received between 2013 and 2019 using whole genome sequencing (WGS).MethodsFrom 329 identified NDM-5-producing E. coli , 224 isolates from known geographical locations were subjected to Illumina WGS. Analyses of 222 sequenced isolates included multilocus sequence typing (MLST), core genome (cg)MLST and single-nucleotide polymorphism (SNP)-based analyses.ResultsResults of cgMLST revealed genetically distinct clusters for many of the 43 detected sequence types (ST), of which ST167, ST410, ST405 and ST361 predominated. The SNP-based phylogenetic analyses combined with geographical information identified sporadic cases of nosocomial transmission on a small spatial scale. However, we identified large clusters corresponding to clonal dissemination of ST167, ST410, ST405 and ST361 strains in consecutive years in different regions in Germany.ConclusionOccurrence of NDM-5-producing E. coli rose in Germany, which was to a large extent due to the increased prevalence of isolates belonging to the international high-risk clones ST167, ST410, ST405 and ST361. Of particular concern is the supra-regional dissemination of these epidemic clones. Available information suggest community spread of NDM-5-producing E. coli in Germany, highlighting the importance of epidemiological investigation and an integrated surveillance system in the One Health framework.

Purpose: In this study, we aimed to assess the geographic distribution and molecular characteristics of β-lactamases among Enterobacterales isolates causing intra-abdominal infections (IAIs) from 2015 to 2018 in the Asia-Pacific region., Method: Isolates were investigated for extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases using multiplex PCR assays and full-gene DNA sequencing., Result: A total of 832 Enterobacterales isolates from 8 different countries with β-lactamase genes were analysed. Plasmid-mediated ESBLs and AmpC β-lactamases were encoded in 598 (71.9 %) and 314 (37.7 %) isolates, respectively. In 710 (85.3 %) carbapenemase-negative isolates, positivity for both AmpC β-lactamases and ESBLs was identified in 51 (8.5 %) Escherichia coli and 24 (3.4 %) Klebsiella pneumoniae isolates. The most prevalent countries were Taiwan and Vietnam, and the co-occurrence of CMY/CTX-M in E. coli and DHA-1/ESBLs in K. pneumoniae was predominant. All isolates showed high susceptibility to colistin, but susceptibility to carbapenems varied among different resistance mechanism combinations. Among 122 (14.7 %) isolates encoding carbapenemase, NDM (n = 67, including 64.2 % NDM-1) was the most common, followed by the OXA-48-type (n = 49), KPC (n = 24) and IMP (n = 4). The most prevalent country was Thailand (n = 44), followed by Vietnam (n = 35) and the Philippines (n = 21). Twenty-two isolates were found to encode multiple carbapenemases, 16 of which were collected from Thailand and harbored NDM-1, OXA-232 and CTX-M-15. Despite high susceptibility to amikacin, susceptibility to colistin was only 56 %., Conclusion: The emergence of carbapenem-non-susceptible AmpC/ESBL co-occurring Enterobacterales and colistin non-susceptible carbapenemases co-occurring K. pneumoniae highlights potential therapeutic challenges in the Asia-Pacific region., Competing Interests: Declaration of competing interest All authors have no conflicts of interest to disclose., (Copyright © 2021. Published by Elsevier B.V.)

Objective: The increasing trend of Escherichia coli that produce extended-spectrum beta-lactamases and carbapenemases is a global public health concern. Sequence type (ST)131, a high-risk clone, is most likely responsible for the global distribution of E. coli that carry the carbapenemase gene such as bla . In this study, carbapenemase-producing KPC . In this study, carbapenemase-producing E. coli (CPEC) isolates were recovered from the stool of patients in a tertiary care hospital in 2019 and 2020., Methods: We randomly selected nine isolates among CPEC isolates carrying KPC, NDM and OXA-type carbapenemase genes for whole genome sequencing (WGS). WGS was performed using the Illumina system (Illumina, Inc., San Diego, CA, USA) at Macrogen (Seoul, Korea). De novo assembly and assembly validation were performed at Macrogen. The whole genome of isolates was analyzed using the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org/)., Results: Seven sequence types were detected: ST131, ST410, ST68, ST216, ST405, ST224 and ST409 variant. Six isolates harbored CTX-M-type genes such as bla CTX-M-15 , bla CTX-M-14 and bla CTX-M-27 . Plasmid replicons such as IncFIA, IncFIB, IncFII and IncX3 types were detected., Conclusions: The present study showed the diversity of the molecular characteristics of CPEC by analysis of the whole genome of bacteria. WGS is a useful tool that provides much information in the clinical microbiology., (© 2022 by the Association of Clinical Scientists, Inc.)

The natural environment represents a complex reservoir of antibiotic-resistant bacteria as a consequence of different wastewater discharges including anthropogenic and agricultural. Therefore, the aim of this study was to examine sewage and waters across Ireland for the presence of antibiotic-resistant Enterobacterales. Samples were collected from the West, East and South of Ireland. Two periods of sampling took place between July 2019 and November 2020, during which 118 water (30 L) and 36 sewage samples (200 mL) were collected. Waters were filtered using the CapE method, followed by enrichment and culturing. Sewage samples were directly cultured on selective agars. Isolates were identified by MALDI-TOF and antibiotic susceptibility testing was performed in accordance with EUCAST criteria. Selected isolates were examined for bla CTX-M , bla VIM , bla IMP , bla OXA-48 , bla NDM , and bla KPC by real time PCR and whole genome sequencing (n = 146). A total of 419 Enterobacterales (348 water, 71 sewage) were isolated from all samples. Hospital sewage isolates displayed the highest percentage resistance to many beta-lactam and aminoglycoside antibiotics. Extended-spectrum beta-lactamase-producers were identified in 78% of water and 50% of sewage samples. One or more carbapenemase-producing Enterobacterales were identified at 23 individual sampling sites (18 water, 5 sewage). This included the detection of bla OXA-48 (n = 18), bla NDM (n = 14), bla KPC (n = 4) and bla OXA-484 (n = 1). All NDM-producing isolates harbored the ble-MBL bleomycin resistance gene. Commonly detected sequence types included Klebsiella ST323, ST17, and ST405 as well as E. coli ST131, ST38 and ST10. Core genome MLST comparisons detected identical E. coli isolates from wastewater treatment plant (WWTP) influent and nursing home sewage, and the surrounding waters. Similarly, one Klebsiella pneumoniae isolated from WWTP influent and the surrounding estuarine water were identical. These results highlight the need for regular monitoring of the aquatic environment for the presence of antibiotic-resistant organisms to adequately inform public health policies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

We characterized plasmids carrying bla NDM-5 detected in Escherichia coli isolated from the infection site and stool sample of a Japanese patient, with no international travel history, by whole-genome sequencing (WGS). WGS was performed using MiSeq and MinlON sequencer followed by hybrid de novo assembly. bla NDM-5 was detected on IncX3 ( bla NDM-5 /IncX3) plasmids; pMTY18530-4_IncX3 in E. coli TUM18530 isolated from a wound above the pubis; pMTY18780-5_IncX3 and pMTY18781-1_IncX3 in E. coli TUM18780 and TUM18781, respectively, isolated from stool. These three plasmids resembled each other and pGSH8M-2-4, previously detected in E. coli isolated from a Tokyo Bay water sample. E. coli TUM18530 and TUM18780 belonged to sequence type (ST) 1011 and had only two single nucleotide polymorphisms on the core-genome, whereas TUM18781 belonged to ST2040. Three bla NDM-5 /IncX3 plasmids (pMTY18530-4_IncX3, pMTY18780-5_IncX3, and pMTY18781-1_IncX3) exhibited conjugative transfer in vitro at an average frequency of 1.71 × 10 -3 per donor cell. The transconjugant was resistant to only β-lactams, including carbapenem, except aztreonam. Similarity of the bla NDM-5 /IncX3 plasmids isolated from our patient compared with that isolated from the Tokyo bay water sample suggested that the plasmids may have already spread throughout the Japanese community. The bla NDM-5 /IncX3 plasmid exhibited potential for easy transmission to different strains in the patient's intestine.

Hospitals are one of the main reservoirs of multi-resistant Enterobacterales (MRE). As MRE are resistant to the most frequently used antibiotics, therapy for patients with MRE infections is challenging. It has been previously described that MRE from hospital wastewater can pass into municipal wastewater and even surface water. In this study, we investigated the diversity and epidemiology of MRE in the wastewater of a large tertiary care hospital. Wastewater samples were collected for a four-day period and tested for the presence of Enterobacterales resistant to 3rd gen. cephalosporins. Representative isolates were further characterized by whole genome sequencing. In 120 β-glucuronidase-producing isolates, 68 Escherichia coli and, interestingly, also 52 Citrobacter freundii were identified. In 120 β-glucosidase-producing isolates 45 Serratia marcescens, 34 Klebsiella oxytoca, 32 Enterobacter cloacae and 9 Klebsiella pneumoniae were observed. For all species various MLST sequence types and different clusters of resistance genes were determined, showing a great diversity within the different Enterobacterales, further corroborated by clonal analysis performed by cgMLST. The most prominent clone was wastewater associated E. coli ST635, which accounted for 47.1% of all E. coli isolates. Interestingly, 45.6% of E. coli, 88.5% of C. freundii, 95.6% of S. marcescens, 91.2% of K. oxytoca, 96.9% of E. cloacae and 88.9% of K. pneumoniae isolates carried a carbapenemase gene, indicating a high burden with carbapenemase-producing Enterobacterales. Comparison with clinical isolates from the same hospital displayed few clonal matches. One wastewater isolate of K. pneumoniae was identified to be closely related compared to a clone that had been introduced into the hospital during an outbreak four years earlier. One E. coli isolate was identified as identical to an isolate from a patient, with inpatient stay during the sampling period. The data obtained in this study highlight the problem of antibiotic resistance of Enterobacterales in hospital wastewater. In particular, the clustered occurrence of carbapenemase genes is of great concern and underscores the problem of increasingly scarce antibiotic options against these bacteria., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Emergency department areas were repurposed as intensive care units (ICUs) for patients with acute respiratory distress syndrome during the initial months of the coronavirus disease 2019 (COVID-19) pandemic. We describe an outbreak of New Delhi metallo-β-lactamase 1 (NDM-1)-producing Escherichia coli infections in critically ill COVID-19 patients admitted to one of the repurposed units. Seven patients developed infections (6 ventilator-associated pneumonia [VAP] and 1 urinary tract infection [UTI]) due to carbapenem-resistant E. coli, and only two survived. Five of the affected patients and four additional patients had rectal carriage of carbapenem-resistant E. coli. The E. coli strain from the affected patients corresponded to a single sequence type. Rectal screening identified isolates of two other sequence types bearing bla NDM-1 . Isolates of all three sequence types harbored an IncFII plasmid. The plasmid was confirmed to carry bla NDM-1 through conjugation. An outbreak of clonal NDM-1-producing E. coli isolates and subsequent dissemination of NDM-1 through mobile elements to other E. coli strains occurred after hospital conversion during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This emphasizes the need for infection control practices in surge scenarios. IMPORTANCE The SARS-CoV-2 pandemic has resulted in a surge of critically ill patients. Hospitals have had to adapt to the demand by repurposing areas as intensive care units. This has resulted in high workload and disruption of usual hospital workflows. Surge capacity guidelines and pandemic response plans do not contemplate how to limit collateral damage from issues like hospital-acquired infections. It is vital to ensure quality of care in surge scenarios.

Fecal carriage of multidrug-resistant Escherichia coli , particularly sequence type 131 (ST131), is becoming a global concern. This study aimed at determining the prevalence rate and molecular epidemiology of extended-spectrum β -lactamase-producing E. coli (ESBL-Ec), carbapenemase-producing E. coli (CPEc), ceftazidime/avibactam (CAZ/AVI)-resistant E. coli , and ST131 isolates in healthy fecal carriers in Tehran, Iran. Among 540 samples studied, 233 (43.1%) carried ESBL-Ec, with the majority (93.9%) harboring the bla CTX-M. The carriage rate of CPEc was 2.5% ( n gene was the predominant carbapenemase gene. Most CPEc isolates ( bla NDM gene was the predominant carbapenemase gene. Most CPEc isolates ( n  = 11/14) was shown to be resistant to CAZ/AVI. Among ESBL-Ec/CPEc, 7.3% ( n  = 17/233) belonged to E. coli ST131 clone, which was identified by polymerase chain reaction and confirmed by multilocus sequence typing. The ST131 isolates genetically typed by pulsed-field gel electrophoresis were heterogeneous and four different plasmids were detected by plasmid typing, with the IncFIA/FIB being the major type. Our findings disclose that the presence of carbapenem-resistant ST131 isolates, which are also resistant to CAZ/AVI, contributes to the spread of resistant strains in the community. Therefore, screening and monitoring of such resistant clone in healthy people is necessary.

Carbapenemase-producing Escherichia coli sequence type (ST) 648 strains were isolated from two international visitors without previous medical exposure from Southeast Asian countries in a hospital in Japan. One isolate, FUJ80154, carried bla NDM-5 in a complex class 1 integron on an IncFIB/FII plasmid; the other isolate, FUJ80155, carried two copies of bla OXA-48 on the chromosome flanked by IS 1R on both sides. The core-genome based-phylogenetic analysis with publicly available genome data of E. coli ST648 carrying bla Although patients with history of international hospitalization are often subject to screening for multidrug-resistant organisms, it is unclear whether patients who reside in countries where carbapenemase-producing NDM-5 (CPE) is endemic but have no history of local hospitalization contribute to the transmission of CPE. In this study, NDM-5-producing and OXA-48-producing Escherichia coli sequence type (ST) 648, a recently recognized high-risk, multidrug-resistant clone, were detected from two overseas visitors without previous medical exposure. The findings of this study suggest that active surveillance culture on admission to hospital may be considered for travelers from countries with endemicity of carbapenem-resistant organisms even without history of local hospitalization and underscore the need to monitor cross-border transmission of high-risk clones, such as carbapenemase-producing E. coli ST648.bla OXA-48-like demonstrated high genetic similarity between FUJ80154 and NDM-5-prooducing E. coli ST648 strains isolated in South and Southeast Asian countries. On the other hand, no closely related isolates of FUJ80155 were identified. In the absence of prior hospitalization overseas, neither patient had qualified for routine screening of multidrug-resistant organisms, and the isolates were incidentally identified in cultures ordered at the discretion of the treating physician. IMPORTANCE Although patients with history of international hospitalization are often subject to screening for multidrug-resistant organisms, it is unclear whether patients who reside in countries where carbapenemase-producing Enterobacterales (CPE) is endemic but have no history of local hospitalization contribute to the transmission of CPE. In this study, NDM-5-producing and OXA-48-producing Escherichia coli sequence type (ST) 648, a recently recognized high-risk, multidrug-resistant clone, were detected from two overseas visitors without previous medical exposure. The findings of this study suggest that active surveillance culture on admission to hospital may be considered for travelers from countries with endemicity of carbapenem-resistant organisms even without history of local hospitalization and underscore the need to monitor cross-border transmission of high-risk clones, such as carbapenemase-producing E. coli ST648.

Despite the advances in β-lactamase inhibitor development, limited options exist for the class D carbapenemase known as OXA-48. OXA-48 is one of the most prevalent carbapenemases in carbapenem-resistant Enterobacteriaceae infections and is not susceptible to most available β-lactamase inhibitors. Here, we screened various low-molecular-weight compounds (fragments) against OXA-48 to identify functional scaffolds for inhibitor development. Several biphenyl-, naphthalene-, fluorene-, anthraquinone-, and azobenzene-based compounds were found to inhibit OXA-48 with low micromolar potency despite their small size. Co-crystal structures of OXA-48 with several of these compounds revealed key interactions with the carboxylate-binding pocket, Arg214, and various hydrophobic residues of β-lactamase that can be exploited in future inhibitor development. A number of the low-micromolar-potency inhibitors, across different scaffolds, synergize with ampicillin to kill Escherichia coli expressing OXA-48, albeit at high concentrations of the respective inhibitors. Additionally, several compounds demonstrated micromolar potency toward the OXA-24 and OXA-58 class D carbapenemases that are prevalent in Acinetobacter baumannii . This work provides foundational information on a variety of chemical scaffolds that can guide the design of effective OXA-48 inhibitors that maintain efficacy as well as potency toward other major class D carbapenemases.

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l -1 cefotaxime and TBX+1 mg l -1 imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla VIM and bla IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6')-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla VIM and bla IMP genes in livestock in Tunisia., (© 2021 The Society for Applied Microbiology.)

The rapid increase in carbapenemase-producing Enterobacterales is a global health concern. During 2017-2020, a total of 44 Escherichia coli isolates co-harbouring bla NDM-5 and bla OXA-181 were collected from patients at 17 hospitals in Seoul and characterized based on antimicrobial susceptibility, resistance genes and plasmid replicons detected using polymerase chain reaction (PCR). Clonal relatedness was estimated using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates had an identical multidrug resistance profile, including resistance to carbapenems, cephalosporins, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole, and susceptibility to amikacin, colistin, and tigecycline. Resistance genes (bla CTX-M-15 , bla CMY-2 , bla TEM-1B , bla OXA-1 , aac(6')-Ib-cr, and qnrS) and plasmid replicons (IncFIA, IncFIB, and IncX3) was observed in almost all isolates. All isolates belonged to ST410 and were genetically similar (>88% similarity), with some PFGE types shared among isolates from different hospitals. Analysis of the whole genome revealed that the isolates clustered together with other strains of the international high-risk clone ST410 B4/H24RxC from other countries. These findings underline the ongoing spread of the high-risk clone of NDM-5- and OXA-181-producing E. coli ST410 B4/H24RxC among hospitals in Seoul. Continuous monitoring and implementation of infection control measures are crucial to track and prevent further spread of these resistant strains., (Copyright © 2021 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S 70 , K 73 , S 118 , and K 208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T 209 was found to be important for hydrolysis of imipenem, whereas R 250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W 105 and L 158 , revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.

Carbapenemase-producing Enterobacterales (CPE) bacteria are a critical global health concern; New Delhi metallo-β-lactamase (NDM) enzymes account for >25% of all CPE found in Switzerland. We characterized NDM-positive CPE submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance during a 2-year period (January 2019-December 2020) phenotypically and by using whole-genome sequencing. Most isolates were either Klebsiella pneumoniae (59/141) or Escherichia coli (52/141), and >50% were obtained from screening swabs. Among the 108 sequenced isolates, NDM-1 was the most prevalent variant, occurring in 56 isolates, mostly K. pneumoniae (34/56); the next most prevalent was NDM-5, which occurred in 49 isolates, mostly E. coli (40/49). Fourteen isolates coproduced a second carbapenemase, predominantly an OXA-48-like enzyme, and almost one third of isolates produced a 16S rRNA methylase conferring panresistance to aminoglycosides. We identified successful plasmids and global lineages as major factors contributing to the increasing prevalence of NDMs in Switzerland.

Background: The molecular characteristics of carbapenem-resistant Escherichia coli (CREco) remain unclear., Methods: We conducted a multi-center bacterial resistance monitoring project from 2015 to 2017.The minimum inhibitory concentrations ofCREco were determined bybroth microdilution method. The genome sequencing of CREcoisolates was performed, and single-nucleotide polymorphism (SNP) was analyzed., Results: A total of 144CREcoisolatescollected from 10 cities in China were involved in this study. ST167 (n = 43) is the most popular type, followed by ST410(n = 14), ST131(n = 9). There were 102 (70.83%) CREco isolates that produced various NDMs, including NDM-1 (n = 16), NDM-4(n = 1), NDM-5(n = 79), NDM-6(n = 2) and NDM-9(n = 4). In addition, 15 isolates produced KPC-2, three isolates wereIMP-4 positive, and three isolates produced OXA-48. Genetic relatedness and phylogenetic analysis showed that isolates with the same ST had a high degree of homology. Some STs (including ST167, ST410, ST131, ST46, ST405 and ST617) exhibited a trend of outbreak., Conclusions: The majority of CREco belonged to ST167, followed by ST410 and ST131, and most of them carried various NDM-coding genes. The spread of high-risk clones of CREco has occurred in different regions of China., (© 2021. The Author(s).)

Background: Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), commonly used for microorganism identification, can also be applied for the detection of carbapenemase-producing bacteria by the evaluation of carbapenem hydrolysis. Since KPC- and NDM-producing bacteria are related to high mortality rates, diagnostic assays for its detection are essential. The aim of this study was to develop and evaluate a method to establish a quantitative measure (hydrolysis index - HI) to detect meropenem hydrolysis by MLADI-TOF MS., Methods: bla KPC and bla NDM positive and negative Klebsiella pneumoniae isolates and Escherichia coli ATCC 25922 (control) were incubated in a meropenem solution for 2 h. Protein extraction from these suspensions were submitted to MALDI-TOF MS analysis. The intensity of peaks at 384 m/z and 379 m/z of each isolate were used to establish the HI as follows: HI = (Peak intensity 384 Test / Peak intensity 379 Test ) / (Peak intensity 384 Control / Peak intensity 379 Control ). Receiver Operating Characteristic curve was used to determine a cutoff value to differentiate carbapenemase-producing from carbapenemase non-producing bacteria., Results: As all carbapenemase-producing K. pneumoniae presented HI ≤0.55 and all carbapenemase non-producing isolates presented a HI ≥0.57, the index of 0.56 was established as a cutoff value to differentiate carbapenemase (KPC and NDM) producing and non-producing bacteria., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Water bodies worldwide have proven to be vast reservoirs of clinically significant antibiotic resistant organisms. Contamination of waters by anthropogenic discharges is a significant contributor to the widespread dissemination of antibiotic resistance. The aim of this research was to investigate multiple different anthropogenic sources on a national scale for the role they play in the environmental propagation of antibiotic resistance. A total of 39 water and 25 sewage samples were collected across four local authority areas in the West, East and South of Ireland. In total, 211 Enterobacterales were isolated (139 water, 72 sewage) and characterised. A subset of isolates (n=60) were chosen for whole genome sequencing. Direct comparisons of the water versus sewage isolate collections revealed a higher percentage of sewage isolates displayed resistance to cefoxitin (46%) and ertapenem (32%), while a higher percentage of water isolates displayed resistance to tetracycline (55%) and ciprofloxacin (71%). Half of all isolates displayed extended spectrum beta-lactamase (ESBL) production phenotypically (n = 105/211; 50%), with bla CTX-M detected in 99/105 isolates by PCR. Carbapenemase genes were identified in 11 isolates (6 sewage, 5 water). The most common variant was bla OXA-48 (n=6), followed by bla NDM-5 (n=2) and bla KPC-2 (n=2). Whole genome sequencing analysis revealed numerous different sequence types in circulation in both waters and sewage including E. coli ST131 (n=15), ST38 (n=8), ST10 (n=4) along with Klebsiella ST405 (n=3) and ST11 (n=2). Core genome MLST (cgMLST) comparisons uncovered three highly similar Klebsiella isolates originating from hospital sewage and two nearby waters. The Klebsiella isolates from an estuary and seawater displayed 99.1% and 98.8% cgMLST identity to the hospital sewage isolate respectively. In addition, three pairs of E. coli isolates from different waters also revealed cgMLST similarities, indicating widespread dissemination and persistence of certain strains in the aquatic environment. These findings highlight the need for routine monitoring of water bodies used for recreational and drinking purposes for the presence of multi-drug resistant organisms., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: The global success of carbapenem-resistant pathogens has been attributed to large plasmids carrying bla KPC genes circulating among high-risk clones. In this study, we sequenced the genome of a carbapenem-resistant Escherichia coli strain (Ec351) isolated from a human infection. Phylogenomic analysis based on single nucleotide polymorphisms (SNPs) as well as the comparative resistome and plasmidome of globally disseminated bla KPC-2 -positive E. coli strains with identical sequence type (ST) were further investigated., Methods: Total DNA was sequenced using an Illumina NextSeq 500 platform and was assembled using Unicycler. Genomic data were evaluated through bioinformatics tools available from the Center of Genomic Epidemiology and by in silico analysis., Results: Genomic analysis revealed the convergence of a wide resistome and virulome in E. coli ST648, showing a high-level phylogenetic relationship with a KPC-2-positive ST648 cluster identified in the USA and association with international clade 2. Additionally, the emergence of an IncQ1 small plasmid (pEc351) carrying bla KPC-2 (on an NTE KPC -IId element), aph(3')-VIa, and plasmid regulatory and replication genes in the pandemic clone ST648 is reported., Conclusion: Identification of a bla KPC-2 -positive IncQ1 plasmid in a high-risk E. coli clone represents rapid adaptation and expansion of these small plasmids encoding carbapenemases to novel bacterial hosts with global distribution, which deserves continued monitoring., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates., Methodology: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumβ-lactamase (ESBL), carbapenemase and metallo β-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method., Results: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene., Conclusions: The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Sohni Naeem, Hazrat Bilal, Hafsah Muhammad, Muhammad Asif Khan, Fareeha Hameed, Sher Bahadur, Tayyab Ur Rehman.)

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary β-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing bla OXA-48 , on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and β5-β6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of β-lactams can drive the evolution of β-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance. IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of β-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low β-lactam concentrations on the widespread β-lactamase OXA-48. Our data indicate that the exposure to β-lactams at very low concentrations enhances β-lactamase diversity and drives the evolution of β-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases., (Copyright © 2021 Fröhlich et al.)

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 bla OXA-244 inside the entire or truncated Tn51098.

Objective: To analyze the incidence and resistance of microorganisms to antibacterial drugs isolated in urine cultures of patients with urinary tract infections from 2012 to 2019., Materials and Methods: In the Pirogov City Clinical Hospital No1 and in the Bauman City Clinical Hospital No 29 analyzed the results of 15083 urine cultures in 12554 patients from 2012 to 2019., Results: Enterococcus faecalis (41%), Escherichia coli (36.4%), Klebsiella pneumonia (23.4%) and Proteus mirabilis (7.6%) predominate in the occurrence of complicated UTIs. the number of strains resistant to certain groups of antibacterial drugs increased: mesitillin-resistant staphylococci (+ 4%), producers of -lactamases (+ 19.8% (for E. coli) and + 34.7% (for Klebsiella pneumoniae)), vancomycin-resistant enterococci (+ 1.89%), carbapenemase producers (+ 32.9%). A high level of resistance among Enterococcus faecalis strains to ciprofloxacin (23.1%) and gentamicin (38.4%) was revealed. Among strains of Escherichia coli, an increase in resistance to ampicillin (85.7%), ceftazidime (66.7%), ciprofloxacin (54.1%) and nitrofurantoin (42.9%) was noted. The appearance of carbapenem-resistant strains is noted. Among the strains of Klebsiella pneumonia, there is a significant increase in resistance to all antibacterial drugs used. Separately, one can note a sharp (27.1%) increase in resistance to carbapenems. A high level of resistance was found in Proteus mirabilis to ciprofloxacin (66.7%), ampicillin (75%). There is an increase in resistance of Pseudomonas aeruginosa to ciprofloxacin (66.7%), meropenem (50%). There is a high level of resistance for Acinetobacter baumannii to amikacin (94.9%), imipenem (92.3%), ceftazadime (100%), ciprofloxacin (100%), tigecycline (53.6%). Relatively high sensitivity remains to polymyxin E (88.9%)., Conclusions: The results of our analysis demonstrate a rapid increase in resistance to most antibacterial drugs among community-acquired and hospital-acquired strains.

Objectives: The aim of this study was to screen for the presence of β-lactamase-producing Gram-negative bacteria (GNB) from Algerian currency collected from food vendors in Batna city, Algeria., Methods: During two periods (May 2018 and March-April 2019), a total of 408 coins and currency notes of different denominations of Algerian Dinar were randomly recovered from several food vendors. Samples were subjected to selective isolation of extended-spectrum cephalosporin- and carbapenem-resistant GNB. Bacterial species identification was performed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was performed by the disk diffusion method. Carbapenemase and extended-spectrum β-lactamase (ESBL) genes were searched for by real-time PCR, standard PCR and sequencing. The clonal relationship of carbapenemase-producing isolates was investigated by multilocus sequence typing (MLST). The transferability of the detected carbapenemase-encoding gene was verified by conjugation experiments., Results: Twelve cefotaxime- and/or carbapenem-resistant strains were isolated in this study and were identified as Enterobacter cloacae, Raoultella ornithinolytica, Citrobacter freundii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas libanensis and Pseudomonas stutzeri. The bla OXA-48 gene was detected in only one E. cloacae strain belonging to sequence type 108 (ST108), whilst the two R. ornithinolytica isolates harboured bla CTX-M-27 and one E. coli strain carried bla CTX-M-14 . The detected bla OXA-48 gene was transferable by conjugation., Conclusions: We report for the first time the detection of an OXA-48-producing E. cloacae isolate from money. This calls for consciousness development on the potential risks associated with poor handling of currency., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n = 57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using bla NDM -positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20 kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the bla KPC , bla NDM , bla SHV and bla TEM genes, respectively. The bla KPC-2 gene was observed in one E. coli isolate. The bla NDM-1 gene was detected in Stenotrophomonas maltophilia (n = 2), Acinetobacter baumannii (n = 1) and Ochrobactrum anthropi (n = 1) isolates. These isolates co-harboured the bla TEM and bla SHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of bla NDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks., (© 2020 The Society for Applied Microbiology.)

Objectives: The aim of this study was to explore the genomic content of a bla KPC-2 - and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance., Methods: Antimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing., Results: Escherichia coli strain QE11-421 was an mcr-1-positive colistin-resistant isolate that co-harboured the bla KPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11-421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The bla KPC-2 gene was located on a Tn3-Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon., Conclusions: Horizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of bla KPC-2 -IncN and mcr-1-IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

OXA-244 is a derivative of OXA-48 showing weaker carbapenemase activity, compromising the detection of corresponding producers in clinical laboratories. Since 2017, the Swiss National Reference Center for Emerging Antibiotic Resistance noticed an increased identification of OXA-244-producing Escherichia coli (n=15) within the country. Different methods (biochemical and immunoassay tests, screening culture media) were tested for the detection of OXA-244 producers. Whole genome sequencing was used to investigate the genetic relatedness between the isolates and the genetic structures at the origin of the acquisition of the bla OXA-244 gene. The mSuperCARBA® medium and the NG-Test CARBA5 assay were found to be suitable tools for detecting all OXA-244-producing isolates. Other selective media did not perform optimally. Among the fifteen strains, five sequence types were identified, with ST38 being predominant. The bla OXA-244 gene was located on the chromosome for all isolates. Overall, detection of OXA-244 producers is challenging and specific guidelines must be followed., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)