Corrigendum: Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center.

[This corrects the article DOI: 10.3389/fcimb.2024.1407246.].

Occurrence of blaNDM-1, blaNDM-5, blaNDM-7, and blaKPC-2 genes in clinical isolates of enterobacterales with high genetic variability, from colonization and infection in patients with or without COVID-19, from a hospital in Brazil.

AIMS: This study aimed to investigate the presence of beta-lactams resistance genes and the clonal relationship of clinical isolates of Enterobacterales obtained from patients with and without COVID-19, in a hospital in northeastern Brazil. METHODS AND RESULTS: The study analyzed 45 carbapenem-resistant clinical isolates using enterobacterial repetitive intergenic consensus (ERIC-PCR), PCR, and amplicon sequencing to detect resistance genes (blaKPC, blaGES, blaNDM, blaVIM, and blaIMP). The main species were Klebsiella pneumoniae, Serratia marcescens, and Proteus mirabilis. Detected genes included blaNDM (46.66%), blaKPC (35.55%), and both (17.79%). ERIC-PCR showed multiclonal dissemination and high genetic variability. The main resistance gene was blaNDM, including blaNDM-5 and blaNDM-7. CONCLUSIONS: The presence of Enterobacterales carrying blaKPC and blaNDM in this study, particularly K. pneumoniae, in infections and colonizations of patients with COVID-19 and non-COVID-19, highlights genetic variability and resistance to carbapenems observed in multiple species of this order.

Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center.

Introduction: In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. Methods: This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). Results: Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. Discussion: The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

Genomic Insights into the First Emergence of blaNDM-5-Carrying Carbapenem-Resistant Salmonella enterica Serovar London Strain in China.

Carbapenem-resistant Salmonella enterica (S. enterica) pose a significant threat to public health, causing gastroenteritis and invasive infections. We report the first emergence of a carbapenem-resistant S. enterica serovar London strain, A132, carrying the blaNDM-5 gene in China. Whole-genome sequencing and bioinformatics analysis assigned A132 to be ST155, a multidrug-resistant clone frequently reported in China. The strain A132 exhibited resistance to multiple antibiotics, with 20 acquired antibiotic resistance genes (ARGs) identified, predominantly located on the IncFIB plasmid (pA132-1-NDM). Notably, the blaNDM-5 gene was located within an IS26 flanked-class 1 integron-ISCR1 complex, comprising two genetic cassettes. One cassette is the class 1 integron, which may facilitate the transmission of the entire complex, while the other is the blaNDM-5-containing ISCR1-IS26-flanked cassette, carrying multiple other ARGs. Genbank database search based on the blaNDM-5-carrying cassette identified a similar genetic context found in transmissible IncFIA plasmids from Escherichia coli (p91) and Enterobacter hormaechei (p388) with a shared host range, suggesting the potential for cross-species transmission of blaNDM-5. To our knowledge, this is the first reported case of Salmonella serovar London ST155 harboring blaNDM-5 gene. Phylogenetic analysis indicated a close relationship between A132 and eight S. London ST155 strains isolated from the same province. However, A132 differed by carrying the blaNDM-5 gene and four unique ARGs. Given the high transmissibility of the F-type plasmid harboring blaNDM-5 and 18 other ARGs, it is imperative to implement vigilant surveillance and adopt appropriate infection control measures to mitigate the threat to public health.

Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic.

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-ß-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

Genomic Characterization of Escherichia coli Co-Producing KPC-2 and NDM-5 Carbapenemases Isolated from Intensive Care Unit in a Chinese Hospital.

Background: Around the world, carbapenemase-producing Escherichia coli is becoming more prevalent. The purpose of this research was to analyze the whole plasmid sequences from YL03 isolates of the E. coli strain that produce both KPC-2 and NDM-5 carbapenemases. Materials and Methods: Whole-genome sequencing (WGS) and analysis of E. coli strain YL03, which was isolated from a wound sample, was performed by Illumina Novaseq 6000 and Pacific Biosciences Sequel (PacBio, Menlo Park, CA) sequencers. Following that, the WGS results were used to predict and analyze the YL03 genome composition and function. A complete gene sequence for YL03 with the accession number CP093551 has been uploaded to GenBank. Results: The results showed that YL03 co-carried five resistance genes, which included blaKPC-2, blaNDM-5, blaTEM-1B, blaCTX-M-14, and mdf(A). Furthermore, three resistance plasmids were found in YL03: pYL03-KPC, pYL03-NDM, and pYL03-CTX. Among them, the 53 kb-long pYL03-KPC plasmid belonging to the IncP, carried the replicase gene (repA) and the carbapenemase gene (blaKPC-2). The blaKPC-2 gene was flanked by a composite transposon-like element (Tn3-[Tn3] tnpR-ISKpn27 blaKPC--ISKpn6). Conclusions: The YL03 strain co-carried blaKPC-2 and blaNDM-5 and had a unique multidrug resistance plasmid containing blaKPC-2.

Co-Occurrence of tet(X4) and blaNDM-5 in Escherichia coli Isolates of Inpatient Origin in Guangzhou, China.

Tigecycline, one of the last-resort therapeutic options for complicated infections caused by multidrug-resistant pathogens, especially carbapenem-resistant Enterobacterales and Acinetobacter in recent years. The emergence of antibiotic-resistant bacteria and antibiotic-resistant genes has threatened the effectiveness of antibiotics and public health with the excessive use of antibiotics in clinics. However, the emergence and dissemination of high-level mobile tigecycline-resistance gene tet(X) is challenging for clinical effectiveness of antimicrobial agent. This study aimed to characterize an E. coli strain T43, isolated from an inpatient in a teaching hospital in China. The E. coli T43 was resistant to almost all antimicrobials except colistin and consisted of a 4,774,080 bp chromosome and three plasmids. Plasmids pT43-1 and pT43-2 contained tigecycline-resistance gene tet(X4). Plasmid pT43-1 had a size of 152,423 bp with 51.05% GC content and harbored 151 putative open reading frames. pT43-1 was the largest plasmid in strain T43 and carried numerous resistance genes, especially tigecycline resistance gene tet(X4) and carbapenemase resistance gene blaNDM-5. The tet(X) gene was associated with IS26. Co-occurrence of numerous resistance genes in a single plasmid possibly contributed to the dissemination of these genes under antibiotics stress. It might explain the presence of clinically crucial resistance genes tet(X) and blaNDM-5 in clinics. This study suggested the applicable use of antibiotics and continued surveillance of tet(X) and blaNDM-5 in clinics are imperative.

Characterisation of a Novel Hybrid IncFIIpHN7A8:IncR:IncN Plasmid Co-Harboring blaNDM-5 and blaKPC-2 from a Clinical ST11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Purpose: We aimed to characterize a novel blaNDM-5 and blaKPC-2 co-carrying hybrid plasmid from a clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) strain. Methods: Antimicrobial susceptibility was determined by the broth microdilution method. Plasmid size and localization were estimated using S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Plasmid transfer ability was evaluated by conjugation experiments. Whole genome sequencing (WGS) was performed using Illumina NovaSeq6000 and Oxford Nanopore MinION platforms. Genomic characteristics were analyzed using bioinformatics methods. Results: Strain ZY27320 was a multidrug-resistant (MDR) clinical ST11 K. pneumoniae strain that confers high-level resistance to carbapenems (meropenem, MIC 128 mg/L; imipenem, MIC 64 mg/L) and ceftazidime/avibactam (MIC >128/4 mg/L). Both S1-PFGE-Southern blotting and whole genome sequencing revealed that the carbapenemase genes blaKPC-2 and blaNDM-5 were carried by the same IncFIIpHN7A8:IncR:IncN hybrid plasmid (pKPC2_NDM5). Conjugation experiments indicated that pKPC2_NDM5 was a non-conjugative plasmid. Conclusion: This is the first report of a hybrid plasmid carrying both carbapenemase genes blaNDM-5 and blaKPC-2 in a clinical K. pneumoniae ST11 isolate that confers resistance to both ceftazidime/avibactam and carbapenems, thereby presenting a serious threat to treatment in clinical practice.

Molecular characterization of IncFII plasmid carrying blaNDM-5 in a Salmonella enterica serovar Typhimurium ST34 clinical isolate in China.

IMPORTANCE: In this study, an IncFII plasmid pIncFII-NDM5 carrying blaNDM-5 was found in carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), which has conjugative transferability and carried blaNDM-5, bleMBL, mph(A), and blaTEM-1 four resistance genes that can mediate resistance to multiple antibiotics including cephalosporins, beta-lactamase inhibitor combinations, carbapenems, and macrolides. Phylogenetic analysis showed that 1104-65 and 1104-75 were closely related to other S. enterica serovar Typhimurium in this area. The above-mentioned S. enterica serovar Typhimurium chromosome carries blaCTX-M-55, qnrS1, and tet(A) genes, so the antibiotic resistance of isolates will be further enhanced after obtaining the pIncFII_NDM5-like plasmid. Meanwhile, we discovered a novel genetic structure of blaNDM-5 mediated by the IS26 composite transposon, which will expand our understanding of the emergence and spread of carbapenem-resistance genes. Altogether, the presence of the IncFII plasmid pIncFII-NDM5 further underscores the need for vigilant surveillance and appropriate infection control measures to mitigate the impact of carbapenem-resistant S. enterica serovar Typhimurium in clinical settings.

First Emergence of NDM-5 and OqxAB Efflux Pumps Among Multidrug-Resistant Klebsiella pneumoniae Isolated from Pediatric Patients in Assiut, Egypt.

Introduction: New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae poses a high risk, especially among Egyptian pediatric patients who consume carbapenems antibiotics very widely and without adequate diagnostic sources. In addition, presence of efflux pump genes such as OqxAB increases resistance against many groups of antimicrobials which exacerbates the problem faced for human health. This study aimed to determine NDM variants among K. pneumoniae strains isolated from pediatric patients in Egypt, analyze the presence of OqxAB genes, and molecular characterization of blaNDM-5-positive K. pneumoniae. Methods: Fifty-six K. pneumoniae isolates were recovered from pediatric patients, and tested for carbapenemase by modified carbapenem inactivation methods (mCIM) test. Minimum inhibitory concentrations of meropenem and colistin were determined by meropenem E-test strips and broth microdilution, respectively. PCR was used for the detection of the resistant genes (ESBL gene (blaCTX-M), carbapenemase genes (blaNDM, blaKPC) colistin resistant (mcr1, mcr2)) and genes for efflux pump (oqxA and oqxB). BlaNDM was sequenced. The effect of efflux pump in NDM-5-producing isolates was assessed by measuring MIC of ciprofloxacin and meropenem before and after exposure to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The horizontal gene transfer ability of blaNDM-5 was determined using liquid mating assay and PCR-based replicon typing (PBRT) was done to determine the major plasmid incompatibility group. Results: Twenty-nine isolates were positive for blaNDM-1, nine isolates were positive for blaNDM-5, and 15 isolates were positive for blaKPC. There is a significant increase of meropenem MIC of NDM-5-positive isolates compared with NDM-1-positive isolates. In addition, 38 isolates were positive for CTX-M, and 15 isolates were positive for mcr1. Both OqxA and OqxB were detected in 26 isolates and 13 isolates were positive for OqxA while 11 isolates were positive for OqxB only. All NDM-5-producing isolates except one isolate could transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). Plasmid replicon typing showed that FII was predominant in NDM-5-producing K. pneumoniae. Similar strains were found between the three isolates and similarity was also detected between the two isolates. Conclusion: The highly resistant K. pneumoniae producing blaNDM-5 type was firstly isolated from pediatric patients. The association of efflux pump genes such as OqxAB is involved in resistance to ciprofloxacin. This highlighted the severity risk of blaNDM-5-positive K. pneumonia as it could transfer blaNDM-5 to other bacteria and has more resistance against carbapenems. This underlines the importance of continuous monitoring of infection control guidelines, and the urgent need for a national antimicrobial stewardship plan in Egyptian hospitals.

Genomic Analysis of an Escherichia coli Sequence Type 167 Isolate Harboring a Multidrug-Resistant Conjugative Plasmid, Suggesting the Potential Transmission of the Type Strains from Animals to Humans.

Purpose: The E. coli ST167 clone is the globally dominant ST among extraintestinal pathogenic E. coli (ExPEC) and is frequently associated with carbapenem resistance. This study reports genomic characterization of a pandrug-resistant E. coli ST167 isolate (ECO3183) and the possibility of the type strains' transmission. Materials and Methods: Antibiotic susceptibility testing was performed using disk diffusion and the VITEK 2 automated system. The E. coli ECO3183 genome was sequenced. We used the genome to analyze the phylogenetic relationship, phylogenetic group, sequence type (ST), acquired antibiotic resistance genes (ARGs), IS elements, genomics islands, the replicon type and transferability of the plasmids. The conjugative transfer of plasmids was assessed using filter mating experiments. Results: ECO3183 contained a 4.87-Mb chromosome and two plasmids [pECO3183-1 (167.63 Kb) and pECO3183-2 (46.16 Kb)]. It belonged to phylogenetic group A, clonal complex 10 (CC10), and ST167. ECO3183 is a pandrug-resistant strain nonsusceptible to 24 tested antimicrobials representing 8 different antimicrobial classes. Among 55 E. coli isolates phylogenetically related to ECO3183, 47% (26/55) were from humans, while 35% (19/55) were from animals. Further analysis revealed that among 1140 ST167 isolates (in the EnteroBase database), 4% (47/1140) originated from environments, 17% (192/1140) were isolated from humans, and 78% (890/1140) were obtained from animals. The pECO3183-1 contained two identical repeats of a 9633 bp region (IS6100-sul1-ΔaadA16-dfrA27-arr-3-aac(6')-Ib-cr-IS26) and a 17.88-kb resistance island (sul2-aph(3″)-Ib-aph(6)-Id-IS26-Δaph(3')-Ia-IS26-tet(A)-ΔfloR-ΔISVsa3-IS26-Δaac(3)-IId-IS26-mph(A)), and these three regions contained most of ECO3183 carrying ARGs. It was identified as a conjugative plasmid, which confers MDR resistance and has the potential to spread. Conclusion: ECO3183 exhibited pandrug-resistance phenotype that was mediated by pECO3183-1 carrying MDR ARGs and pECO3183-2 carrying blaNDM-5. Source analysis of strains indicated that ST167 E. coli might be transmitted between species from animals to humans, which needs continued monitoring.

Dihydroartemisinin inhibits plasmid transfer in drug-resistant Escherichia coli via limiting energy supply.

Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( mcr-1) by more than 160-fold in vitro in Escherichia coli, and more than two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla NDM-5 by more than two-fold in vitro. Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.

NDM-5-carrying Klebsiella pneumoniae ST437 belonging to high-risk clonal complex (CC11) from an urban river in eastern India.

In this study, we described the carbapenem bla NDM-5-carrying extensive drug-resistant (XDR) K. pneumoniae ST437 from an urban river water Kathajodi in Odisha, India. The presence of carbapenem and co-occurrence of other resistance determinants (bla NDM-5, bla CTX-M, bla SHV, and bla TEM), virulence factors (fimH, mrkD, entB, irp-1, and ybtS), and capsular serotype (K54) represent its pathogenic potential. The insertion sequence ISAba125 and the bleomycin resistance gene ble MBL at upstream and downstream, respectively, could play a significant role in the horizontal transmission of the bla NDM-5. Its biofilm formation ability contributes toward environmental protection and its survivability. MLST analysis assigned the isolate to ST437 and clonal lineage to ST11 (CC11) with a single locus variant. The ST437 K. pneumoniae, a global epidemic clone, has been reported in North America, Europe, and Asia. This work contributes in understanding of the mechanisms behind the spread of bla NDM-5 K. pneumoniae ST437 and demands extensive molecular surveillance of river and nearby hospitals for better community health. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03556-5.

Genetic characteristic of coexisting of mcr-1 and bla NDM-5 in Escherichia coli isolates from lesion-bearing animal organs.

The coexistence of mcr-1 and bla NDM-5 in the plasmid of Escherichia coli has been widely reported and such strains have been mainly isolated from animal and human feces. However, few reports have focused on the genetic diversity of mcr-1-carrying chromosomes and bla NDM-5-carrying plasmids in E. coli isolates from lesion-bearing animal organs. This study investigated the genetic characteristics of chromosome-mediated mcr-1 and plasmid-mediated bla NDM-5 in E. coli isolated from lesion-bearing animal organs. Nine mcr-1- and bla NDM-5-positive E. coli strains (MNPECs) showed extensive drug resistance (XDR). The predominant clonal complexes (CC) mainly belonged to CC156, CC10, and CC165 from the 56 MNEPCs (including nine strains in this study) retrieved from the literature. These strains were widely distributed in China, and originated from pig fecal samples, human stool/urine samples as well as intestinal contents of chicken. Two transconjugants harboring bla NDM-5 gene were also successfully obtained from two donors (J-8 and N-14) and this transfer increased the MIC for meropenem by 256 times. However, conjugative transfer of mcr-1 gene failed. Both J-8 and N-14 strains contained point mutations associated with quinolone resistance and more than three types of AMR genes, including the mcr-1 gene on the chromosome and the bla NDM-5 gene on the IncX3-type plasmid. The genetic structure of mcr-1 located on the chromosome was an intact Tn6330, and bla NDM-5-carrying IncX3-type plasmid was ISAb125-IS5-bla NDM-5-bleO-trpF-tat-cutA-IS26 gene cassette. Moreover, differences between chromosomes included additional partial sequence of phage integrated into host genome and the different genes associated with O-antigen synthesis.

Molecular Epidemiology, Clinical Characteristics and Risk Factors for Bloodstream Infection of Multidrug-Resistant Klebsiella pneumoniae Infections in Pediatric Patients from Tianjin, China.

Purpose: The data on pediatrics with Multidrug-Resistant (MDR) Klebsiella pneumoniae infections are scarce. This study aims to investigate the molecular epidemiology of MDR Klebsiella pneumoniae, detect the mechanism of drug resistance, and determine the clinical risk factors for carbapenem-resistant Klebsiella pneumonia (CRKP) bloodstream infections (BSIs) in a children's hospital. Methods: A total of 62 strains were collected from Tianjin Children's Hospital. Carba NP and polymerase chain reactions (PCR) were performed to detect MDR mechanisms. Multilocus sequence typing (MLST) was used for analyzing strain homology. Clinical data were collected and logistic regression was used for BSI risk factors. Results: ST11 was the principal ST among the CRKP isolates clinically, accounting for 56.45% (35/62); there were also 57.14% (20/35) ST11 CRKP strains co-carrying bla NDM-5 and bla KPC-2, which were resistant to most of the tested antibiotics, being susceptible only to cotrimoxazole and tigecycline. The clinical data showed that 72.73% (40/55) of children with CRKP infection had serious underlying diseases; 20.00% (11/55) patients developed BSIs with the potential to cause multiple organ failure, shock and death. The logistic regression showed that the risk of BSIs caused by CRKP strain infections in children with hematological malignancies after chemotherapy was 7 times that of other children (95%Cl: 1.298-45.415, P=0.025). Conclusion: ST11 was the prevalent clone in our hospital. The emergence of ST11 CRKP co-carrying bla NDM-5 and bla KPC-2 should be a cause for alarm as they were resistant to most of the tested antibiotics. CRKP strain infections are mainly occurring in young immunocompromised patients and the chemotherapy for hematological malignancies is an independent risk factor for BSIs.

Dissemination of bla NDM-5 and mcr-8.1 in carbapenem-resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae in an animal breeding area in Eastern China.

Animal farms have become one of the most important reservoirs of carbapenem-resistant Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. "One Health"-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of "One-Health," 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene bla NDM-5, which was flanked by the same core genetic structure (IS5-bla NDM-5-ble MBL-trpF-dsbD-IS26-ISKox3) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS903-orf-mcr-8.1-copR-baeS-dgkA-orf-IS903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying-bla NDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.

Persistence of plasmid and tet(X4) in an Escherichia coli isolate coharboring bla NDM-5 and mcr-1 after acquiring an IncFII tet(X4)-positive plasmid.

The prevalence of plasmid-mediated tigecycline resistance gene tet(X4) is presenting an increasing trend. Once tet(X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as bla NDM and mcr-coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet(X4), bla NDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a bla NDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet(X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet(X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet(X4)-positive plasmid. However, the stability of tet(X4) gene showed different scenarios in the two types of evolved strains. The tet(X4) gene in non-tigecycline treated strains was stable whereas the tet(X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet(X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet(X4)-bearing plasmid and tet(X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet(X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria.

Genomic Characterization of a Multidrug-Resistant Escherichia coli Isolate Co-Carrying bla NDM-5 and bla CTX-M-14 Genes Recovered from a Pediatric Patient in China.

Background: Public health is seriously threatened by the rise of carbapenem-resistant Enterobacterales (CRE). However, the genomic characteristics of CRE detected in pediatric patients are largely unknown. Here, we reported the genomic characteristics of a multidrug-resistant Escherichia coli strain containing the plasmid-borne bla NDM-5 and bla CTX-M-14 genes recovered from a Chinese pediatric patient. Methods: The genome sequence of E. coli strain B379 was determined using Illumina NovaSeq 6000 and Oxford Nanopore MinION. Multiple bioinformatics tools were used to annotate the genome sequence, identify antimicrobial resistance genes and plasmid replicons and perform the in silico multilocus sequence typing (MLST) analysis. Using BacWGSTdb 2.0 server, a core genome MLST (cgMLST) comparison was made between E. coli B379 and all ST746 E. coli strains downloaded from the public database. Results: The E. coli B379 genome sequence is comprised of six contigs totaling 5,152,502 bp, including one chromosome and five plasmids. Nineteen antimicrobial resistance genes were predicted. The bla NDM-5, which is located on a 46,161 bp IncX3 plasmid and the bla CTX-M-14 gene which is located on a 147,204 bp IncFII/IncFIA/IncFIB plasmid are two examples of these 19 genes. E. coli B379 was resistant to ampicillin, ampicillin/sulbactam, ceftriaxone, ceftazidime, imipenem, cefepime, ciprofloxacin, ertapenem, trimethoprim-sulfamethoxazole. This isolate belonged to ST746 and the closest relative was another one originating from a human material specimen in Denmark, which differed by 273 cgMLST alleles. Conclusion: Our study reports the emergence of a multidrug-resistant E. coli strain co-carrying bla NDM-5 and bla CTX-M-14 recovered from a pediatric patient in China. These data would help us better understand the prevalence, genetic characteristics, and mechanisms of antimicrobial resistance of this recently identified multidrug-resistant bacteria in children.

Coexistence of bla NDM-5 and tet(X4) in international high-risk Escherichia coli clone ST648 of human origin in China.

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new "superbugs." This study reports the first incident of a bla NDM-5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., bla TEM-1B , bla NDM-5, bla CMY-2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that bla NDM-5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting.

Convergence of MCR-8.2 and Chromosome-Mediated Resistance to Colistin and Tigecycline in an NDM-5-Producing ST656 Klebsiella pneumoniae Isolate From a Lung Transplant Patient in China.

We characterized the first NDM-5 and MCR-8.2 co-harboring ST656 Klebsiella pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant patient. Complete genome sequences were obtained through Illumina HiSeq sequencing and nanopore sequencing. The acquired resistance genes and mutations in chromosome-encoded genes associated with colistin and tigecycline resistance were analyzed. Comparative genomic analysis was conducted between mcr-8.2-carrying plasmids. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonging to ST656, and harbored plasmid-encoded blaNDM-5 and mcr-8.2 genes. The blaNDM-5 gene was located on an IncX3 type plasmid. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8, which had a common ancestor with another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. The MIC of KP32558 for colistin was 256 mg/L. The mcr-8.2 gene and mutations in the two-component system, pmrA and crrB, and the regulator mgrB, had a synergistic effect on the high-level colistin resistance. The truncation in the acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.