Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli.

The emergence of drug-resistant Enterobacteriaceae carrying plasmid-mediated ß-lactamase genes has become a significant threat to public health. Organisms in the Enterobacteriaceae family containing New Delhi metallo-ß-lactamase­1 (NDM-1) and its variants, which are capable of hydrolyzing nearly all ß-lactam antibacterial agents, including carbapenems, are referred to as superbugs and distributed worldwide. Despite efforts over the past decade, the discovery of an NDM-1 inhibitor that can reach the clinic remains a challenge. Here, we identified oxidized glutathione (GSSG) as a metabolic biomarker for blaNDM-1 using a non-targeted metabolomics approach and demonstrated that GSSG supplementation could restore carbapenem susceptibility in Escherichia coli carrying blaNDM-1 in vitro and in vivo. We showed that exogenous GSSG promotes the bactericidal effects of carbapenems by interfering with intracellular redox homeostasis and inhibiting the expression of NDM-1 in drug-resistant E. coli. This study establishes a metabolomics-based strategy to potentiate metabolism-dependent antibiotic efficacy for the treatment of antibiotic-resistant bacteria.

A systematic review and meta-analysis of carbapenem resistance and its possible treatment options with focus on clinical Enterobacteriaceae: Thirty years of development in Pakistan.

Background: Carbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO 'watch group' with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options. Methods: In this systematic review and meta-analysis, we investigated the pooled prevalence (PPr) of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae by organizing available data, from Web of Science and PubMed by April 2, 2020, in various groups and subgroups including species, years, provinces, extended spectrum ß-lactamase production, clinical presentation, carbapenemase and metallo-ß-lactamase production, and New Delhi metallo-ß-lactamase (NDM) prevalence. Literature review was updated for the studies publisehd by December 07, 2023. Moreover, we descriptively reviewed the molecular epidemiology of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae in Pakistan. Lastly, we statistically explored different treatment options available for carbapenem resistant infections. We used R package 'metafor' for performing meta-analysis and influence diagnostics and determining treatment options. Results: From two academic databases Web of Science and PubMed we identified 343 studies. Eighty-eight studies were selected for the systematic review and meta-analysis. Seventy-four studies were selected for phenotypic analysis, 36 for genotypic analysis, and 31 for available treatment options. PPr-ID of 12% [0.12 (0.07, 0.16)] was observed for phenotypic carbapenem resistance in Enterobacteriaceae with more prevalence recorded in Klebsiella pneumoniae 24% [0.24 (0.05, 0.44)] followed by 9% [0.09 (-0.03, 0.20)] in Escherichia coli. During the last two decades we observed a striking increase in carbapenem resistance PPr i.e., from 0% [0.00 (-0.02, 0.03)] to 36% [0.36 (0.17, 0.56)]. blaNDM with PPr 15% [0.15 (0.06, 0.23)] in naive isolates was found to be the fundamental genetic determinant for carbapenem resistance in Enterobacteriaceae in Pakistan. Polymyxin B, colistin, tigecycline, and fosfomycin were identified as the suggested treatment options available for multidrug resistant infections not responding to carbapenems. Various studies reported carbapenem resistance from human, animal, and environment sources. Conclusion: In conclusion, we found that NDM-1 producing carbapenem resistant Enterobacteriaceae are increasing in Pakistan. Meta-analysis showed that metallo-ß-lactamases producing E. coli ST405 and K. pneumoniae sequence type11 are the major resistant clones. Number of reported studies in various subgroups and inconsistency in following CLSI guidelines are the potential limitations of this meta-analysis. A National antimicrobial resistance (AMR) surveillance strategy based on One Health is urgently needed to check any future AMR crisis in Pakistan.

Draft genome sequence data of multidrug-resistant Pseudomonas aeruginosa WO7 from a hospital wastewater treatment plant in Thailand.

Multidrug-resistant Pseudomonas aeruginosa WO7 was isolated from an untreated water sample from a hospital wastewater treatment plant in Thailand. This report presents the draft genome sequence data of P. aeruginosa WO7. Genomic DNA was obtained from a pure culture of P. aeruginosa WO7, and paired-end reads were generated using an Illumina MiSeq sequencer. The draft genome consisted of 111 contigs with a total size of 6,784,206 base pairs, an N50 of 209,424 base pairs, and a GC content of 65.85%. The dDDH value between WO7 and Pseudomonas aeruginosa DSM 50071T was determined to be 90.7%, indicating that the strain is Pseudomonas aeruginosa. The data presented indicate the potential for bacterial classification, comparative genomics, comprehensive analysis of antimicrobial resistance, and assessment of bacterial virulence factors in P. aeruginosa. The draft genome sequence data have been deposited at the NCBI under Bioproject accession number PRJNA550309.

Epidemiology and clinical significance of carbapenemases in Australia: a narrative review.

Carbapenemase-producing gram-negative bacteria (CP-GNB) infections threaten public health with high mortality, morbidity and treatment costs. Although frequencies remain low in Australia (total number of CP-GNB infections reported was 907 in 2022), blaIMP-4 has established low levels of endemicity in many states. Imipenemase metallo-ß-lactamase types alone accounted for more than half of all carbapenemases in carbapenemase-producing Enterobacterales isolates in Australia, particularly in Enterobacter cloacae complex. New Delhi metallo-ß-lactamase constitutes almost 25% of all carbapenemases in Australia and was identified predominantly in Escherichia coli. The OXA-48-like carbapenemases include almost 10% of all carbapenemases and are mainly seen in Klebsiella pneumoniae and E. coli. Although K. pneumoniae carbapenemase-type carbapenemases are rare in Australia, some local outbreaks have occurred. Most carbapenem-resistant (CR) Pseudomonas aeruginosa strains in Australia do not produce carbapenemases. Finally, OXA-23-like carbapenemases are overwhelmingly positive in CR-Acinetobacter baumannii strains in Australia. Treatment of CR-GNB infections challenges physicians. Of 10 new antibiotics active against at least some CR-GNB infections that are approved by the US Food and Drug Administration, just three are approved for use in Australia. In this context, there is still an unmet need for novel antibacterials that can be used for the treatment of CR-GNB infections in Australia, as well as a pressing requirement for new mechanisms to 'de-link' antibiotic sales from their availability. In this narrative review, we aim to overview the epidemiology and clinical significance of carbapenem resistance in Australia as it pertains to Enterobacterales, P. aeruginosa and A. baumannii.

Camel-Derived Nanobodies as Potent Inhibitors of New Delhi Metallo-ß-Lactamase-1 Enzyme.

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of ß-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting ß-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation.

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-ß-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to ß-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs.

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-ß-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a "one health approach".

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

BACKGROUND: blaNDM gene was prevalent among children, and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%) and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%), and was dominant in blaNDM-K. penumoniae (79.8%), blaNDM-E. coli (58.2%) and blaNDM-E. cloacae (61.0%), respectively. Majority (79.0%) of clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, p<0.01). CONCLUSION: blaNDM is highly prevalent in CREs among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.

Evaluation of the in vitro susceptibility of clinical isolates of NDM-producing Klebsiella pneumoniae to new antibiotics included in a treatment regimen for infections.

Background: Due to the growing resistance to routinely used antibiotics, the search for new antibiotics or their combinations with effective inhibitors against multidrug-resistant microorganisms is ongoing. In our study, we assessed the in vitro drug susceptibility of Klebsiella pneumoniae strains producing New Delhi metallo-ß-lactamases (NDM) to antibiotics included in the Infectious Diseases Society of America (IDSA) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommendations. Methods: A total of 60 strains of NDM-producing K. pneumoniae were obtained from different patients hospitalized at the 4th Military Hospital in Wroclaw between 2019 and 2022 and subjected to drug susceptibility to selected antibiotics, including the effects of drug combinations. Results: Among the tested antibiotics, the highest sensitivity (100%) was observed for cefiderocol, eravacycline (interpreted according to the European Committee on Antimicrobial Susceptibility Testing [EUCAST]), and tigecycline. Sensitivity to intravenous fosfomycin varied depending on the method used. Using the "strip stacking" method, determining cumulative sensitivity to ceftazidime/avibactam and aztreonam demonstrated 100% in vitro sensitivity to this combination among the tested strains. Conclusion: The in vitro susceptibility assessment demonstrated that, the best therapeutic option for treating infections caused by carbapenemase-producing strains seems to be a combination of ceftazidime/avibactam with aztreonam. Due to the safety of using both drugs, cost effectiveness, and the broadest indications for use among the tested antibiotics, this therapy should be the first-line treatment for carbapenemase-producing Enterobacterales infections. Nevertheless, a comprehensive evaluation of the efficacy of treating infections caused by NDM-producing K. pneumoniae strains should include not only in vitro susceptibility assessment but also an analysis of clinical cases.

Transmission of blaNDM in Enterobacteriaceae among animals, food and human.

Despite carbapenems not being used in animals, carbapenem-resistant Enterobacterales (CRE), particularly New Delhi metallo-ß-lactamase-producing CRE (NDM-CRE), are prevalent in livestock. Concurrently, the incidence of human infections caused by NDM-CRE is rising, particularly in children. Although a positive association between livestock production and human NDM-CRE infections at the national level was identified, the evidence of direct transmission of NDM originating from livestock to humans remains largely unknown. Here, we conducted a cross-sectional study in Chengdu, Sichuan Province, to examine the prevalence of NDM-CRE in chickens and pigs along the breeding-slaughtering-retail chains, in pork in cafeterias of schools, and in colonizations and infections from children's hospital and examined the correlation of NDM-CRE among animals, foods and humans. Overall, the blaNDM increases gradually along the chicken and pig breeding (4.70%/2.0%) -slaughtering (7.60%/22.40%) -retail (65.56%/34.26%) chains. The slaughterhouse has become a hotspot for cross-contamination and amplifier of blaNDM. Notably, 63.11% of pork from the school cafeteria was positive for blaNDM. The prevalence of blaNDM in intestinal and infection samples from children's hospitals was 21.68% and 19.80%, respectively. whole genome sequencing (WGS) analysis revealed the sporadic, not large-scale, clonal spread of NDM-CRE along the chicken and pig breeding-slaughtering-retail chain, with further spreading via IncX3-blaNDM plasmid within each stage of whole chains. Clonal transmission of NDM-CRE is predominant in children's hospitals. The IncX3-blaNDM plasmid was highly prevalent among animals and humans and accounted for 57.7% of Escherichia coli and 91.3% of Klebsiella pneumoniae. Attention should be directed towards the IncX3 plasmid to control the transmission of blaNDM between animals and humans.

Novel partially reversible NDM-1 inhibitors based on the naturally occurring houttuynin.

Discovering novel NDM-1 inhibitors is an urgent task for treatment of 'superbug' infectious diseases. In this study, we found that naturally occurring houttuynin and its sulfonate derivatives might be effective NDM-1 inhibitors with novel mechanism, i.e. the attribute of partially covalent inhibition of sulfonate derivatives of houttuynin against NDM-1. Primary structure-activity relationship study showed that both the long aliphatic side chain and the warhead of aldehyde group are vital for the efficiency against NDM-1. The homologs with longer chains (SNH-2 to SNH-5) displayed stronger inhibitory activities with IC50 range of 1.1-1.5 µM, while the shorter chain the weaker inhibition. Further synergistic experiments in cell level confirmed that all these 4 compounds (at 32 µg/mL) recovered the antibacterial activity of meropenem (MER) against E. coli BL21/pET15b-blaNDM-1.

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae ST307 revealed multiple introductions in Buenos Aires, Argentina.

OBJECTIVE: to describe at genomic level nine carbapenemase-producing Klebsiella pneumoniae ST307 (Kp-ST307) clinical isolates recovered in Buenos Aires during 2017-2021, investigating their resistome, virulome and phylogeny. METHODS: Antimicrobial susceptibility was determined according to CLSI. Genomic DNA was sequenced by Illumina MiSeq and analyzed using SPAdes, PROKKA and Kleborate. Phylogeny of 355 randomly selected Kp-ST307 genomes and those from nine local isolates was inferred by a maximum-likelihood approach. The tree was visualized using Microreact. RESULTS: Besides resistance to ß-lactams and fluoroquinolones, six out of nine Kp-ST307 were also resistant to ceftazidime/avibactam (CZA). This difficult-to-treat resistance phenotype was mediated by blaSHV-28 and gyrA-83I/parC-80I mutations in addition to carbapenemase coding genes. Among CZA susceptible isolates, two of them harbored blaKPC-3 while the other harbored blaKPC-2+blaCTX-M-15. Respect to CZA resistant isolates, three harbored blaKPC-3+blaNDM-1+blaCMY-6, two carried blaKPC-2+blaNDM-5+blaCTX-M-15, and blaNDM-5+blaCTX-M-15 were detected in the remaining isolate. Besides, five colistin resistant isolates presented a nonsense mutation in mgrB. Global Kp-ST307 isolates were distributed in two deep-branching lineages while local isolates set in the main clade of the phylogenetic tree. The five isolates from a same hospital, harboring blaKPC-3 or blaKPC-3+blaNDM-1+blaCMY-6, clustered in a monophyletic subclade with Italian isolates. Also, an isolate harboring blaKPC-2+blaNDM-5+blaCTX-M-15 recovered in another hospital was closed to this group. The remaining local Kp-ST307 grouped in other subclades containing isolates of diverse geographically origin. CONCLUSION: The inferred resistome was consistent with the resistant phenotype. Phylogeny suggested multiple introduction events in our region and a single major introduction in one hospital followed by local spread.

High-risk clones of carbapenem resistant Klebsiella pneumoniae recovered from pediatric patients in Southern Brazil.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) exhibit high mortality rates in pediatric patients and usually belong to international high-risk clones. This study aimed to investigate the molecular epidemiology and carbapenem resistance mechanisms of K. pneumoniae isolates recovered from pediatric patients, and correlate them with phenotypical data. Twenty-five CRKP isolates were identified, and antimicrobial susceptibility was assessed using broth microdilution. Carbapenemase production and ß-lactamase genes were detected by phenotypic and genotypic tests. Multilocus sequence typing was performed to differentiate the strains and whole-genome sequencing was assessed to characterize a new sequence type. Admission to the intensive care unit and the use of catheters were significantly positive correlates of CRKP infection, and the mortality rate was 36%. Almost all isolates showed multidrug-resistant phenotype, and most frequent resistant gene was blaKPC. We observed the dissemination of ST307 and clones belonging to CG258, which are considered high risk. In pediatric patients, these clones present with high genomic plasticity, favoring adaptation of the KPC and NDM enzymes to healthcare environments.

Identification of Potential Inhibitors of Three NDM Variants of Klebsiella Species from Natural Compounds: A Molecular Docking, Molecular Dynamics Simulation, and MM-PBSA Study.

BACKGROUND: Klebsiella species have emerged as well-known opportunistic pathogens causing nosocomial infections with ß-lactamase-mediated resistance as a prevalent antibiotic resistance mechanism. The discovery and emergence of metallo-ß-lactamases, mainly new- Delhi metallo-ß-lactamases (NDMs), have increased the threat and challenges in healthcare facilities. OBJECTIVE: A computational screening was conducted using 570 natural compounds from Dr. Duke's Phytochemical and Ethnobotanical data to discover promising inhibitors for NDM-6, NDM-9, and NDM-23 of the Klebsiella species. METHODS: Using homology modeling on the Raptor-X web server, the structures of the three NDM variants were predicted. The structures were validated using various computational tools and MD simulation for 50 ns. Lipinski - Vebers' Filter and ADMET Screening were used to screen 570 compounds, followed by docking in Biovia Discovery Studio 2019 using the CDOCKER module. GROMACS was used to simulate the compounds with the highest scores with the proteins for 50 ns. Using the MM-PBSA method and g_mmpbsa tool, binding free energies were estimated and per-residue decomposition analysis was conducted. RESULTS: The three structures predicted were found stable after the 50 ns MD Simulation run. The compounds Budmunchiamine-A and Rhamnocitrin were found to have the best binding energy towards NDM-6, NDM-9, and NDM-23, respectively. From the results of MD Simulation, MM-PBSA binding free energy calculations, and per-residue decomposition analysis, the Protein-ligand complex of NDM-6 with Budmunchiamine A and NDM-9 with Rhamnocitrin was relatively more stable than the complex of NDM-23 and Rhamnocitrin. CONCLUSION: The study suggests that Budmunchiamine-A and Rhamnocitrin are potential inhibitors of NDM-6 and NDM-9, respectively, and may pave a path for in-vivo and in-vitro studies in the future.

Phenotypic and Genotypic Characterization of Pan-Drug-Resistant Klebsiella pneumoniae Isolated in Qatar.

In secondary healthcare, carbapenem-resistant Enterobacterales (CREs), such as those observed in Klebsiella pneumoniae, are a global public health priority with significant clinical outcomes. In this study, we described the clinical, phenotypic, and genotypic characteristics of three pan-drug-resistant (PDR) isolates that demonstrated extended resistance to conventional and novel antimicrobials. All patients had risk factors for the acquisition of multidrug-resistant organisms, while microbiological susceptibility testing showed resistance to all conventional antimicrobials. Advanced susceptibility testing demonstrated resistance to broad agents, such as ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam. Nevertheless, all isolates were susceptible to cefiderocol, suggested as one of the novel antimicrobials that demonstrated potent in vitro activity against resistant Gram-negative bacteria, including CREs, pointing toward its potential therapeutic role for PDR pathogens. Expanded genomic studies revealed multiple antimicrobial-resistant genes (ARGs), including blaNMD-5 and blaOXA derivative types, as well as a mutated outer membrane porin protein (OmpK37).

High prevalence of carbapenem resistance and clonal expansion of blaNDM gene in Klebsiella pneumoniae isolates in an Iranian referral pediatric hospital.

BACKGROUND: The increasing global concern regarding antibiotic resistance necessitates in-depth studies to comprehend the phenotypic and genotypic characteristics of resistant bacterial strains. This study aimed to investigate the prevalence, antibiotic resistance profiles, and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in an Iranian referral pediatrics hospital. METHODS: In this study, we examined CRKP isolates collected from hospitalized pediatric patients across various wards. The isolates underwent antimicrobial susceptibility testing, the polymerase chain reaction (PCR) analysis for carbapenemase genes (blaNDM, blaVIM and blaIMP), and genetic relatedness assessment using pulsed-field gel electrophoresis (PFGE). RESULTS: Among 166 K. pneumoniae isolates, 54 (32.5%) exhibited resistance to carbapenems. Notably, all these resistant isolates were resistant to imipenem, with 35 (65%) displaying resistance to both imipenem and meropenem. Of the 54 CRKP isolates, 24 (44%) were metallo-ß-lactamases (MBL)-producing. The prevalence of the blaNDM gene among CKCP and MBL-producing isolates was 20% (n = 11) and 44% (n = 24), respectively. The blaVIM and blaIMP genes were not detected in any of the isolates. Twenty-six CRKP isolates (48%) were recovered from ICUs. PFGE analysis of CRKP isolates revealed 20 clusters, with cluster S being the most prevalent, comprising 24% of the total (n = 13). CONCLUSION: Our study reveals a concerning prevalence of carbapenem resistance in K. pneumoniae isolates. Specifically, the detection of the blaNDM gene in 20% of CRKP isolates, with a significant proportion (82%) observed in isolated CRKP from the ICUs and emergency departments, underscores the potential clonal expansion of these resistant strains within these critical hospital wards.

Genomic insights into the evolution and mechanisms of carbapenem-resistant hypervirulent Klebsiella pneumoniae co-harboring blaKPC and blaNDM: implications for public health threat mitigation.

BACKGROUND: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing blaKPC and blaNDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on blaKPC and blaNDM dual-positive hvKP strains. METHODS: Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. RESULTS: WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. CONCLUSION: KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable blaNDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes.

Genomic characterisation of blaNDM-5-IncX3 plasmid in an ST4 Klebsiella aerogenes clinical isolate.

OBJECTIVES: The dissemination of New Delhi metallo-ß-lactamase-5 (NDM-5) among various species of Enterobacterales has attracted serious global attention. Here, we characterise the genomic characterisation of blaNDM-5-IncX3 plasmid (pNDM-KA3) in an ST4 Klebsiella aerogenes (KA3) strain isolated from a neonate with pneumonia. METHODS: Antimicrobial susceptibility and multilocus sequence typing was performed for the KA3. The plasmid conjugation assay and plasmid stability of the KA3 (pNDM-KA3) were also analysed. The pNDM-KA3 plasmid was further analysed by whole-genome sequencing and comparative analysis to determine the genetic environment of blaNDM-5. RESULTS: The KA3 strain belongs to ST4 and shows high resistance to ß-lactam antibiotics, including carbapenems, but is susceptible to ciprofloxacin, amikacin, tigecycline, and colistin. The pNDM-KA3 was successfully transferred to the recipient E. coli J53 and showed strong stability in K. aerogenes. Genomic sequencing revealed that the pNDM-KA3 plasmid was assigned to plasmid incompatibility group X3 with 43367 bp, and a conserved structure sequence of △IS3000-△ISAba125-IS5-blaNDM-5-bleMBL- trpF-dsbC-IS26 was detected upstream and downstream of the blaNDM-5 gene. Further analysis revealed that insertion sequences mediated the dissemination of blaNDM-5 from other species of Enterobacterales. The pNDM-KA3 showed high similarity to blaNDM-5-harbouring plasmids in other species of Enterobacterales, with these plasmids carrying genes for replication (repB), partitioning (parA and parB), stability (hns), and conjugative transfer (virB and virD). CONCLUSIONS: Continued monitoring for the dissemination of blaNDM-5 among uncommon Enterobacterales species should be further reinforced.

Assessing the risk of exposure to antimicrobial resistance at public beaches: Genome-based insights into the resistomes, mobilomes and virulomes of beta-lactams resistant Enterobacteriaceae from recreational beaches in Lagos, Nigeria.

The role of recreational water use in the acquisition and transmission of antimicrobial resistance (AMR) is under-explored in low- and middle-income countries (LMICs). We used whole genome sequence analysis to provide insights into the resistomes, mobilomes and virulomes of 14 beta-lactams resistant Enterobacterales isolated from water and wet-sand at four recreational beaches in Lagos, Nigeria. Carriage of multiple beta-lactamase genes was detected in all isolates except two, including six isolates carrying blaNDM-1. Most detected antibiotic resistance genes (ARGs) were located within a diverse landscape of plasmids, insertion sequences and transposons including the presence of ISKpn14 upstream of blaNDM-1 in a first report in Africa. Virulence genes involved in adhesion and motility as well as secretion systems are particularly abundant in the genomes of the isolates. Our results confirmed the four beaches are contaminated with bacteria carrying clinically relevant ARGs associated with mobile genetic elements (MGE) which could promote the transmission of ARGs at the recreational water-human interface.

Enterobacter asburiae ST229: an emerging carbapenemases producer.

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. "Policlinico San Matteo" in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.