Dynamic evolution of ceftazidime-avibactam resistance from a single patient through the IncX3_NDM-5 plasmid transfer and blaKPC mutation.

The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and blaKPC mutation from blaKPC-2 to blaKPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10-3-10-5, while the mutation frequency of K. pneumoniae 5298 was 10-6-10-8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of blaNDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: ISKox3-IS26-blaNDM-5-IS5-ISAba125-Tn3000-Tn3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including blaNDM-5 tranfer and blaKPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment.

Unveiling the genetic architecture and transmission dynamics of a novel multidrug-resistant plasmid harboring blaNDM-5 in E. Coli ST167: implications for antibiotic resistance management.

BACKGROUND: The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi metallo-ß-lactamase (NDM) gene, which confer resistance to carbapenems, a critical class of last-resort antibiotics. This study investigates the genetic characteristics and implications of a novel blaNDM-5-carrying plasmid pNDM-5-0083 isolated from an E. coli strain GZ04-0083 from clinical specimen in Zhongshan, China. RESULTS: Phenotypic and genotypic evaluations confirmed that the E. coli ST167 strain GZ04-0083 is a multidrug-resistant organism, showing resistance to diverse classes of antibiotics including ß-lactams, carbapenems, fluoroquinolones, aminoglycosides, and sulfonamides, while maintaining susceptibility to monobactams. Investigations involving S1 pulsed-field gel electrophoresis, Southern blot analysis, and conjugation experiments, alongside genomic sequencing, confirmed the presence of the blaNDM-5 gene within a 146-kb IncFIB plasmid pNDM-5-0083. This evidence underscores a significant risk for the horizontal transfer of resistance genes among bacterial populations. Detailed annotations of genetic elements-such as resistance genes, transposons, and insertion sequences-and comparative BLAST analyses with other blaNDM-5-carrying plasmids, revealed a unique architectural configuration in the pNDM-5-0083. The MDR region of this plasmid shares a conserved gene arrangement (repA-IS15DIV-blaNDM-5-bleMBL-IS91-suI2-aadA2-dfrA12) with three previously reported plasmids, indicating a potential for dynamic genetic recombination and evolution within the MDR region. Additionally, the integration of virulence factors, including the iro and sit gene clusters and enolase, into its genetic architecture poses further therapeutic challenges by enhancing the strain's pathogenicity through improved host tissue colonization, immune evasion, and increased infection severity. CONCLUSIONS: The detailed identification and characterization of pNDM-5-0083 enhance our understanding of the mechanisms facilitating the spread of carbapenem resistance. This study illuminates the intricate interplay among various genetic elements within the novel blaNDM-5-carrying plasmid, which are crucial for the stability and mobility of resistance genes across bacterial populations. These insights highlight the urgent need for ongoing surveillance and the development of effective strategies to curb the proliferation of antibiotic resistance.

Genome sequence of a sequence type 1 NDM-5-producing carbapenem -resistant Klebsiella pneumoniae in China.

OBJECTIVE: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents significant health challenges. Here, we present the structural genome sequence of an NDM-5-producing K. pneumoniae (HZKP2) in China. METHODS: Antimicrobial susceptibility tests were conducted via broth microdilution. Whole-genome sequencing (WGS) was performed for genomic analysis. Wzi and capsular polysaccharide (KL) were analysed using Kaptive. Resistance genes, virulence factors, and comparative genomics analyses were also conducted. Multilocus sequence typing (MLST), replicons type, and core genome multilocus sequence typing (cgMLST) analysis were further conducted using BacWGSTdb server. RESULTS: HZKP2 was resistant to cefepime, ceftazidime, ciprofloxacin, ciprofloxacin, meropenem, and ertapenem. It harbored fosA, blaSHV-187, oqxA, oqxB, sul1, dfrA1, tet(A), floR, aph(6)-Id, aph(3'')-Ib, sul2, blaCTX-M-55, and blaNDM-5. Based on the RAST results, 5563 genes that belonged to 398 subsystems were annotated. The complete genome sequence of HZKP2 was characterized as ST1, wzi 19, and KL19, with five contigs totaling 5,654,446 bp, including one chromosome and four plasmids. Further analysis found that blaNDM-5 was located in a 46,161 bp IncX3 plasmid (pHZKP2-3). The genetic structure of blaNDM-5 gene was ISKox3-IS26-bleMBL-blaNDM-5-IS5-ISAb125-IS3000. Further analysis revealed that insertion sequences mediated the dissemination of blaNDM-5 from other species of Enterobacterales. Phylogenetic analysis showed that the closest relative was from a human stool specimen in China, which differed by 53 cgMLST alleles. CONCLUSION: Our study provides the first structural perspective of the ST1 K. pneumoniae isolate producing NDM-5 in China. These results could provide valuable insights into the genetic characteristics, antimicrobial resistance mechanisms, and transmission dynamics of CRKP in clinical settings.

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.

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).

Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5.

PURPOSE: The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. METHODS: The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. RESULTS: Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. CONCLUSION: Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most ß-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

The effects of NDM-5 on Escherichia coli and the screening of interacting proteins.

Carbapenem-resistant Escherichia coli (E. coli) strains are widely distributed and spreading rapidly, creating significant challenges for clinical therapeutics. NDM-5, a novel mutant of New Delhi Metallo-ß-Lactamase-1 (NDM-1), exhibits high hydrolase activity toward carbapenems. Since the genetic backgrounds of clinically isolated carbapenem-resistant E. coli are heterogeneous, it is difficult to accurately evaluate the impact of blaNDM-5 on antibiotic resistance. Herein, E. coli BL21 was transformed with a plasmid harboring blaNDM-5, and the resultant strain was named BL21 (pET-28a-blaNDM-5). Consistent with the findings of previous studies, the introduction of exogenous blaNDM-5 resulted in markedly greater resistance of E. coli to multiple ß-lactam antibiotics. Compared with BL21 (pET-28a), BL21 (pET-28a-blaNDM-5) exhibited reduced motility but a significant increase in biofilm formation capacity. Furthermore, transcriptome sequencing was conducted to compare the transcriptional differences between BL21 (pET-28a) and BL21 (pET-28a-blaNDM-5). A total of 461 differentially expressed genes were identified, including those related to antibiotic resistance, such as genes associated with the active efflux system (yddA, mcbR and emrY), pili (csgC, csgF and fimD), biofilm formation (csgD, csgB and ecpR) and antioxidant processes (nuoG). Finally, the pGS21a plasmid harboring blaNDM-5 was transformed into E. coli Rosetta2, after which the expression of the NDM-5 protein was induced using isopropyl-ß-D-thiogalactoside (IPTG). Using glutathione-S-transferase (GST) pull-down assays, total proteins from E. coli were scanned to screen out 82 proteins that potentially interacted with NDM-5. Our findings provide new insight into the identified proteins to identify potential antibiotic targets and design novel inhibitors of carbapenem-resistant bacteria.

First report of a blaNDM-5-carrying Escherichia coli sequence type 12 isolated from a dog with pyometra in Japan.

Carbapenemase-producing Enterobacterales (CPE) are a serious concern in human clinical settings. Companion animal-origin CPE have been only rarely identified in several countries, but they have not yet been identified in Japan. In this study, we present the first case of a canine infected with CPE in Japan. The patient was hospitalized due to pyometra. The pus discharged from the patient's uterus was subjected to bacteriological analysis. As a result, E. coli was identified in the pus and exhibited resistance to piperacillin, amoxicillin-clavulanic acid, cefazolin, ceftazidime, cefepime, meropenem, amikacin, and sulfamethoxazole-trimethoprim and susceptibility to aztreonam, minocycline, and levofloxacin. Results of the sodium mercaptoacetic acid double-disk synergy test showed that the E. coli isolate was positive for metallo-ß-lactamases. Next-generation sequencing identified the blaNDM-5 gene, which was located in the IncFII-type plasmid together with blaTEM-1b, rmtB, aadA2, bleMBL, sul1, qacE, and dfrA12. The case was treated successfully with doxycycline and orbifloxacin. Our finding emphasizes that close attention should be paid to the significance of CPE harboring multidrug-resistance plasmid in companion animals, based on the perspective of One Health approach in Japan as well as in other countries.

Genomic insights into a blaNDM-5-carrying Escherichia coli ST167 isolate recovered from faecal sample of a healthy individual in China.

OBJECTIVES: Since its discovery, blaNDM-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 blaNDM-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 blaNDM gene with blaNDM-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 blaNDM-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.

Emergence of KPC-2 and NDM-5-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae with high-risk sequence types ST11 and ST15.

The emergence of Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-ß-lactamase (NDM)-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae (KPC-2-NDM-hv-CRKP) poses a certain threat to public health. Currently, only a few sporadic reports of such double-positive hv-CRKPs were available. In this study, we isolated two KPC-2-NDM-5-hv-CRKPs from elderly patients with serious underlying diseases and poor prognoses. We found both FK3122 and FK3127 were typical multidrug-resistant (MDR) isolates, exhibiting high-level resistance to both carbapenems and novel ß-lactamase inhibitors ceftazidime/avibactam. Notably, FK3122 is even resistant to cefiderocol due to multiple blaNDM-5 elements. Besides the MDR phenotype, A549 human lung epithelial cells and Galleria mellonella infection model all indicated that FK3122 and FK3127 were highly pathogenic. According to the whole-genome sequencing analysis, we observed over 10 resistant elements, and the uncommon co-existence of blaKPC-2, blaNDM-5, and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: rmpA and iuc operon. We also found that the resistance and virulence plasmids in FK3127 could not only be transferred to Escherichia coli EC600 independently but also together as a co-transfer, which was additionally confirmed by the S1-pulsed-field gel electrophoresis plasmid profile. Moreover, polymorphic mobile genetic elements were found surrounding resistance genes, which may stimulate the mobilization of resistance genes and result in the duplication of these elements. Considering the combination of high pathogenicity, limited therapy options, and easy transmission of KPC-2-NDM-5-hv-CRKP, our study emphasizes the need for underscores the imperative for ongoing surveillance of these pathogens.IMPORTANCEHypervirulent Klebsiella pneumoniae drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP). However, little information is available on the virulence characteristics of the New Delhi metallo-ß-lactamase (NDM) and Klebsiella pneumoniae carbapenemase-2 (KPC-2) co-producing K. pneumoniae strains. In this study, we obtained two KPC-2-NDM-hv-CRKPs from elderly patients, each with distinct capsule types and sequence types: ST11-KL64 and ST15-KL24; these ST-type lineages are recognized as classical multidrug-resistant (MDR) K. pneumoniae. We found these KPC-2-NDM-hv-CRKPs were not only typical MDR isolates, including resistance to ceftazidime/avibactam and cefiderocol, but also displayed exceptionally high levels of pathogenicity. In addition, these high-risk factors can also be transferred to other isolates. Consequently, our study underscores the need for ongoing surveillance of these isolates due to their heightened pathogenicity, limited therapeutic options, and potential for easy transmission.

Genomic characterization of New Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022.

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1-producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum ß-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1-producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 ß-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Genomic characterization of a carbapenem-resistant Citrobacter freundii clinical isolate from China carrying blaNDM-5 on a novel IncC-IncFIB-IncX3 plasmid.

OBJECTIVES: Citrobacter freundii is one of the important pathogens that can cause nosocomial infections. The advent of carbapenem-resistant C. freundii complicates clinical treatment. Here, we reported the genome sequence of a carbapenem-resistant C. freundii strain carrying a novel IncC-IncFIB-IncX3 plasmid in China. METHODS: The genome sequence of C. freundii CRNMS1 was obtained using the Illumina NovaSeq 6000 platform and the long-read Nanopore sequencer. Multilocus sequence typing was identified using MLST (v.2.23.0). The identification of antimicrobial resistance genes (ARGs) and plasmid replicons was performed using the resfinder and plasmidfinder of ABRicate (v.1.0.1). Circular comparisons of plasmids were performed using the BLAST Ring Image Generator (BRIG). RESULTS: CRNMS1 belongs to ST116 in the C. freundii MLST scheme. Thirteen ARGs were predicted in all, including blaNDM-5, which was located in a plasmid. The plasmid pblaNDM5-S1, which carried the blaNDM-5 gene, was discovered to be a novel plasmid including three plasmid replicons (IncC, IncFIB, and IncX3) as well as seven ARGs (sul1, sul2, floR, dfrA17, aadA5, qnrA1, and blaNDM-5). A total of 38 blaNDM-5-bearing C. freundii strains can be retrieved from the NCBI database. Phylogenetic analysis revealed a worldwide distribution of C. freundii strains carrying the blaNDM-5 gene, with China having the highest prevalence (39%, 15/38). However, they were distantly related to CRNMS1 with SNP differences >2545. CONCLUSION: In summary, we reported a novel IncC-IncFIB-IncX3 plasmid carrying blaNDM-5 in a carbapenem-resistant C. freundii strain in China. The development of such hybrid plasmids facilitates the transmission of ARGs.

Analysis of the hypovirulent Klebsiella pneumoniae with the NDM-5 gene on IncN plasmids.

IMPORTANCE: It is crucial to strengthen the ongoing clinical surveillance of non-highly virulent, multi-resistant Klebsiella pneumoniae.

Draft genome sequence of NDM-5, CMY-42 carrying carbapenem-resistant Escherichia coli.

We report the draft genome sequence of carbapenem-resistant Escherichia coli (CREC-1) of sequence type (ST2083) isolated from a urine sample of a 2-year-old female toddler carrying antimicrobial resistance genes blaNDM-5 and blaCMY-42 which displays resistance against multiple classes of antibiotics notably ß-lactam antibiotics, cephalosporins, and carbapenem and codes for several virulence factors.

Multidrug-resistant pathogens contaminate river water used in irrigation in disenfranchised communities.

OBJECTIVES: The contamination of fresh surface waters poses a significant burden on human health and prosperity, especially in marginalized communities with limited resources and inadequate infrastructure. Here, we performed in-depth genomic analyses of multidrug-resistant bacteria (MDR-B) isolated from Al-Oueik river water that is used for irrigation of agricultural fields in a disenfranchised area that also hosts a makeshift Syrian refugee camp. METHODS: A composite freshwater sample was filtered. Faecal coliforms were counted and extended spectrum cephalosporins and/or ertapenem resistant bacteria were screened. Isolates were identified using MALDI-TOF-MS and analysed using whole-genome sequencing (WGS) to identify the resistome, sequence types, plasmid types, and virulence genes. RESULTS: Approximately 106 CFU/100 mL of faecal coliforms were detected in the water. Four drug-resistant Gram-negative bacteria were identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, and Pseudomonas otitidis. Notably, the E. coli isolate harboured blaNDM-5 and a YRIN-inserted PBP3, representing an emerging public health challenge. The K. pneumoniae isolate carried blaSHV-187 as well as mutations in the gene encoding the OmpK37 porin. Enterobacter hormaechei and P. otitidis harboured blaACT-16 and blaPOM-1, respectively. CONCLUSION: This report provides comprehensive genomic analyses of MDR-B in irrigation water in Lebanon. Our results further support that irrigation water contaminated with faecal material can be a reservoir of important MDR-B, which can spread to adjacent agricultural fields and other water bodies, posing both public health and food safety issues. Therefore, there is an urgent need to implement effective water quality monitoring and management programs to control the proliferation of antibiotic-resistant pathogens in irrigation water in Lebanon.

Whole genome structure and resistance genes in carbapenemase-producing multidrug resistant ST378 Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation in the nosocomial environment. CPKP carried by companion dogs, which are close to human beings, should be considered a common threat to public health. However, CPKP dissemination through companion animals is still under consideration of major diagnosis and surveillance systems. METHODS: Two CPKP isolates which were genotyped to harbor bla NDM-5-encoding IncX3 plasmids, were subjected to the whole-genome study. Whole bacterial DNA was isolated, sequenced, and assembled with Oxford Nanopore long reads and corrected with short reads from the Illumina NovaSeq 6000 platform. The whole-genome structure and positions of antimicrobial resistance (AMR) genes were identified and visualized using CGView. Worldwide datasets were downloaded from the NCBI GenBank database for whole-genome comparative analysis. The whole-genome phylogenetic analysis was constructed using the identified whole-chromosome SNP sites from K. pneumoniae HS11286. RESULTS: As a result of the whole-genome identification, 4 heterogenous plasmids and a single chromosome were identified, each carrying various AMR genes. Multiple novel structures were identified from the AMR genes, coupled with mobile gene elements (MGE). The comparative whole-genome epidemiology revealed that ST378 K. pneumoniae is a novel type of CPKP, carrying a higher prevalence of AMR genes. CONCLUSIONS: The characterized whole-genome analysis of this study shows the emergence of a novel type of CPKP strain carrying various AMR genes with variated genomic structures. The presented data in this study show the necessity to develop additional surveillance programs and control measures for a novel type of CPKP strain.

Dissemination of blaNDM-5-carrying IncX3-type plasmid among non-clonal Escherichia coli strains colonising a dog with a skin infection caused by a carbapenem-resistant Klebsiella pneumoniae, United Kingdom.

A successful outcome of a post-surgical wound infection management by a carbapenem-resistant Klebsiella pneumoniae is described in a dog. Four multidrug-resistant and carbapenem-resistant Escherichia coli strains belonging to ST410 (n = 1) and ST648 (n = 3) were isolated from faecal samples and nasal swabs of this dog at admission to a veterinary hospital in the United Kingdom, and one month after discharge. Whole-genome sequencing analysis suggests dissemination of a 46,161-bp IncX3 blaNDM-5-carrying plasmid among E. coli strains from the different lineages. In this study, the E. coli ST648 strains were virtually identical to each other (5 SNPs difference) indicating dissemination and persistence of this clone over time and across different anatomical sites in the same dog maybe due to the prolonged antimicrobial therapy. The carbapenemase carrying plasmid also showed homology with other publicly available plasmid sequences from Asian countries. These results suggests that plasmids may be a major vehicle in mediating the dissemination of carbapenem-resistance. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst companion animals are needed as it may further contaminate other environments posing a threat to public health.

Genomic Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Isolated from Neonatal Patients in Southwest China During 2017-2021.

Objective: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is spreading worldwide, becoming a serious threat to public health. The present study aimed to analyze the molecular epidemiology and drug resistance mechanism of CRKP isolated from neonatal patients in Sichuan, Southwest China. Methods: CRKP isolates were collected from neonatal patients of West China Second University Hospital from June 2017 to June 2021. Antimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing of all isolates were performed to determine the antimicrobial resistance genes, sequence typing, phylogenetic relationships. Results: In total, 41 nonduplicate CRKP isolates were collected. All isolates were highly resistant to the cephalosporins and carbapenems, however, they were all susceptible to amikacin, tigecycline, ciprofloxacin, and colistin. Various resistance genes were detected, blaNDM-5 (n = 35, 85.4%) was the predominant carbapenemase genes. The most common replicon type was IncX3, which was harbored by 36 (87.8%) isolates, followed by IncFIB (n = 34, 82.9%), and IncFII (n = 32, 78.0%). The 41 CRKP isolates belonged to 8 sequence types (STs) and ST789 (n = 29, all had blaNDM-5) was the dominant sequence type. Conclusion: The study revealed that blaNDM was the most dominant carbapenemase resistance gene. ST789 CRKP strains carrying blaNDM-5 were a tremendous menace to neonates in this hospital. Therefore, effectively implement prevention and control measures need to be taken for the prevention and treatment of CRKP infection in the neonatal ward.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2 K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2 K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2 K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2 K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, bla CTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.