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Objectives: While Escherichia coli phylogroup-A is typically associated with commensal strains, some isolates can harbour virulence and exhibit multidrug-resistant (MDR) phenotypes. We report the draft genome of a rare instance of carbapenem, fosfomycin and colistin resistant E. coli phylogroup-A, isolated as part of routine screening of a human patient in a clinical setting in Ireland. Methods: E. coli E230738 was identified using MALDI-ToF/MS. Antibiotic susceptibility testing was performed using the Sensititre-EUMDRXXF plate. Whole-genome-sequencing was conducted with NextSeq1000, and genomic analysis identified antibiotic-resistance-genes (ARGs) and virulence-factors (VFs). Phylogenetic analysis was performed using whole-genome-multilocus-sequence-typing (wgMLST). Results: E. coli E230738 genome was identified to belong to phylogroup-A/ST10 complex and to harbour 63 ARGs, 17 of which acquired. Resistance to beta-lactams, including carbapenems and cephalosporins was likely due to chromosomally identified blaNDM-5. Colistin resistance appeared associated with acquired mcr-1.1. Despite lacking fosfomycin-inactivating-enzymes, fosfomycin resistance was observed, possibly due to efflux pumps. Forty-seven chromosomal VFs were identified, involved in adhesion and iron acquisition amongst other properties. Plasmid replicons associated with the spread of MDR genes such as IncHI2/HI2A were detected. wgMLST analysis showed the closest relative being a strain from the UK, exhibiting differences in the sequences of 851 genes. Conclusion: This is a first detected instance of a blaNDM-5 and mcr-1.1 co-occurring in E. coli in Ireland. The MDR profile of E. coli E230738 highlights the growing public health concern posed by the dissemination of MDR E. coli lineages with limited treatment options and underscores the need for clinical screening coupled with genomic surveillance to better understand evolving MDR patterns in E. coli.

Objective: Highly carbapenem-resistant Salmonella has emerged worldwide in recent years and is largely associated with the multiform transmission of resistance genes, which poses a huge challenge in clinical practice. Our study delves into the resistance mechanisms and epidemiology of blaNDM-carrying plasmids. Methods: Whole-genome sequencing was utilised to analyse the molecular characteristics and antimicrobial resistance mechanisms of Salmonella isolates recovered from the faeces of a paediatric patient at the Children's Hospital of Nanjing Medical University. Moreover, we conducted an epidemiologic analysis and focused on studying the mechanisms of plasmid-mediated blaNDM transmission, incorporating genomes deposited in the NCBI Pathogen Detection database. Results: The clinical isolate 23S9 belonged to serovar Typhimurium, antigenic profile 4:i:-, ST34, and carried pNDM_23S9 harbouring several antimicrobial resistance genes, including aac(6′)-Ib-cr6, OXA-1, catB3, arr-3, qacEdeltal and blaNDM. Comparative analysis revealed that blaNDM-5 can exist in different plasmids of different isolates, proving its transmission through plasmids. Furthermore, blaNDM-carrying isolates are mostly resistant to beta-lactams, aminoglycosides, sulphonamide, macrolides, and trimethoprim. Conclusions: These findings provided thorough and intuitive insights into the intercontinental spread of blaNDM-carrying Salmonella. Ongoing surveillance is essential for effectively monitoring the worldwide dissemination of this high-risk carbapenem-resistant Salmonella.

ABSTRACT: Objectives: The emergence of carbapenem-resistant Klebsiella pneumoniae 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 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 MLST analysis were further conducted using BacWGSTdb server. Results: HZKP2 was resistant to cefepime, ceftazidime, ciprofloxacin, ciprofloxacin, meropenem, and ertapenem. It harboured 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, 5 five contigs totalling 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 core genome MLST alleles. Conclusions: 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 carbapenem-resistant K. pneumoniae in clinical settings.

Yuanzhong Fang,1 Juan Jin,1 Minfei Peng,2 Lidong Xu,1 Linyuan Gu,1 Danni Bao,3 Qiuying Zhang,4 Kainan Jin5 1Department of Clinical Laboratory, Hangzhou Linping District Women & Children Hospital, Hangzhou, Zhejiang, People’s Republic of China; 2Department of Clinical Laboratory, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Taizhou, 317000, People’s Republic of China; 3Department of Clinical Laboratory, Sanmen People’s Hospital, Sanmen Bay Branch of The First Affiliated Hospital, Zhejiang University School of Medicine, Taizhou, Zhejiang, 317100, People’s Republic of China; 4Department of Clinical Laboratory, Suizhou Hospital, Hubei University of Medicine, Suizhou, Hubei, People’s Republic of China; 5Department of Gastroenterology, Linhai First People’s Hospital, Taizhou, Zhejiang, 317000, People’s Republic of ChinaCorrespondence: Qiuying Zhang, Department of Clinical Laboratory, Suizhou Hospital, Hubei University of Medicine, Suizhou, Hubei, People’s Republic of China, Email 26067889@qq.com Kainan Jin, Department of Gastroenterology, Linhai first People’s hospital, Taizhou, Zhejiang, 317000, People’s Republic of China, Email 353803262@qq.comBackground: Hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) has been spreading rapidly worldwide. Here, we investigated the genomic characteristics of ST11 K. pneumoniae isolate SM117 with capsular serotype KL25, co-carrying blaNDM-5, two copies of blaKPC-2 and multiple plasmid-borne virulence genes from a county level hospital in China.Methods: Antimicrobial susceptibility of K. pneumoniae SM117 was evaluated. The Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms were applied to sequence the genome and then de novo assembled. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline and further subjected to identify the sequence type (ST), capsular type, antibiotic resistance genes, plasmid replicon types and virulence genes. The phylogenetic analysis was performed based on the core genome single nucleotide polymorphisms (cgSNPs) using CSI Phylogeny 1.4, and further visualized by Interactive Tree of Life (iTOL) V5 web server.Results: The whole-genome sequence of K. pneumoniae SM117 is made up of eight contigs totaling 6,104,486 bp, contain a 5,612,620 bp single chromosome and seven plasmids. The isolate was assigned to ST11 with capsular serotype KL25, co-carrying including blaNDM-5, blaKPC-2 and multiple plasmid-borne virulence genes including rmpA2 and aerobactin genes iucABCD-iutA. The coexistence of blaKPC and blaNDM in K. pneumoniae strains exhibit a high degree of resistance to β-lactam antibiotics. The strain SM117 also carries multiple antibiotic resistance genes, making it resistant to all antibiotics except polymyxin. The closest relative of K. pneumoniae C793 was identified in 2023 from a hospital surface sample in Zhejiang, China, with just 52 SNPs difference.Conclusion: This study reported the genomic characteristics of a multidrug-resistant ST11 K. pneumoniae with capsular serotype KL25, co-carrying blaNDM-5, two copies of blaKPC-2 genes and multiple plasmid-borne virulence genes in China. These findings will provide important knowledge of the antibiotic resistance mechanisms, genomic epidemiological characteristics and transmission dynamics of multidrug-resistant K. pneumoniae.Keywords: whole-genome sequencing, K. pneumoniae, multidrug-resistance, blaNDM-5, blaKPC-2, rmpA2, iucABCD-iutA, hvCRKP

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-β-lactamases. Next-generation sequencing identified the bla NDM-5 gene, which was located in the IncFII-type plasmid together with bla TEM-1b , rmtB , aadA2 , ble MBL, 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. [ABSTRACT FROM AUTHOR]

Importance: Carbapenem-resistant Enterobacteriaceae are emerging as a global public health risk. Therefore, assessing the prevalence of carbapenem-resistant Escherichia coli (CRE) in both humans and animals is important. Objective: We aimed to ascertain the occurrence and characteristics of CRE isolated from companion animals, dogs and cats. Methods: E. coli strains were tested for antimicrobial susceptibility using the broth microdilution technique. Antimicrobial resistance genes were detected by polymerase chain reaction and sequencing analysis. The molecular characteristics of CRE were determined using multi-locus sequence typing, replicon typing, and pulsed-field gel electrophoresis (PFGE). Results: In total, 13 CRE isolates (0.13%) were identified from dogs possessing blaNDM-5 along with ß-lactamase genes, mostly blaCMY-2 (92.2%) and blaTEM-1 (53.8%). The commonly observed mutations were S83L and D87N in gyrA, S80I in parC, and S458A in parE. CRE carried nonbeta-lactam resistance genes, with the majority being tet(B) (100%), sul (84.6%), and aac(3)-II (53.8%). Nine different PFGE patterns (P1-P9), IncX3-type plasmids (69.2%), and ST410 (84.6%) were predominantly detected. Conclusions and Relevance: This investigation provides significant insight into the prevalence and molecular characteristics of blaNDM-5-carrying E. coli in dogs. The co-existence of blaNDM-5 and other antimicrobial resistance genes in E. coli potentially poses severe health hazards to humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In this study, Escherichia coli, Klebsiella oxytoca, and Citrobacter amalonaticus carrying blaNDM-5 were isolated from a single patient. Methods: The antibiotic susceptibility of the isolates was evaluated by using E-test and agar dilution methods, and blaNDM-5 was identified in genomic and plasmid DNA by using polymerase chain reaction and sequencing. Whole genome sequencing and de novo assembly were used for species characterization, resistance gene identification, and plasmid analysis. Results: All three species had identical plasmids, which were similar to pEC463-NDM5, a plasmid harboring blaNDM-5. Transconjugation experiments confirmed the horizontal gene transfer of blaNDM-5, highlighting the need for a close monitoring of Enterobacteriaceae species harboring this gene. Conclusions: This study conclusively demonstrates the propensity for horizontal gene transfer of blaNDM-5 among Enterobacteriaceae species, underlining the importance of vigilant monitoring to combat antibiotic resistance. [ABSTRACT FROM AUTHOR]

Escherichia coli (E. coli) serves as a critical indicator microorganism for assessing the prevalence and dissemination of antibiotic resistance, notably harboring various antibiotic-resistant genes (ARGs). Among these, the emergence of the blaNDM gene represents a significant threat to public health, especially since carbapenem antibiotics are vital for treating severe infections caused by Gram-negative bacteria. This study aimed to characterize the antibiotic resistance features of blaNDM-5-positive E. coli strains isolated from waterfowl in several regions of China and elucidate the dissemination patterns of the blaNDM-5 gene. We successfully isolated 103 blaNDM-5-positive E. coli strains from 431 intestinal fecal samples obtained from waterfowl across five provincial-level units in China, with all strains exhibiting multidrug resistance (MDR). Notably, the blaNDM-5 gene was identified on plasmids, which facilitate efficient and stable horizontal gene transfer (HGT). Our adaptability assays indicated that while the blaNDM-5-positive plasmid imposed a fitness cost on the host bacteria, the NDM-5 protein was successfully induced and purified, exhibiting significant enzymatic activity. One strain, designated DY51, exhibited a minimum inhibitory concentration (MIC) for imipenem of 4 mg/L, which escalated to 512 mg/L following exposure to increasing imipenem doses. This altered strain demonstrated stable resistance to imipenem alongside improved adaptability, correlating with elevated relative expression levels of the blaNDM-5 and overexpression of efflux pumps. Collectively, this study highlights the horizontal dissemination of the blaNDM-5 plasmid among E. coli strains, confirms the associated fitness costs, and provides insights into the mechanisms underlying the stable increase in antibiotic resistance to imipenem. These findings offer a theoretical framework for understanding the dissemination dynamics of blaNDM-5 in E. coli, which is essential for developing effective strategies to combat carbapenem antibiotic resistance.

Shaohua Tan,1,* Xiangchen Li,2,* Bing Lu,1,3 Yibin Lin,3 Yunxiang Cai,1 Jie He,4 Sisi Chen,2 Junli Gao,2 Junshun Gao,2 Xinhua Qiang1 1Department of Clinical Laboratory, The First People’s Hospital of Huzhou, Huzhou, Zhejiang, 313000, People’s Republic of China; 2Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, Zhejiang, 310020, People’s Republic of China; 3School of Medicine, Huzhou University, Huzhou, Zhejiang, 313000, People’s Republic of China; 4Department of Infectious Diseases, The First People’s Hospital of Huzhou, Huzhou, Zhejiang, 313000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xinhua Qiang, Department of Clinical Laboratory, The First People’s Hospital of Huzhou, No. 158 Back Square Road, Wuxing District, Huzhou, Zhejiang, 313000, People’s Republic of China, Email qiangxinhua@zjhu.edu.cnAbstract: 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 IS 26 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-IS 26-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.Keywords: blaNDM-5, Salmonella London, ST155, carbapenem resistance, IncFIB, integron

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 β-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: -. [ABSTRACT FROM AUTHOR]

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 β-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: -. [ABSTRACT FROM AUTHOR]

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 IS 26 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-IS 26-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. [ABSTRACT FROM AUTHOR]

ABSTRACT: 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.

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. [ABSTRACT FROM AUTHOR]

IntroductionIn 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. MethodsThis 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). ResultsOur 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. DiscussionThe 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.

Lingyan Sun,1– 3,* Yan Chen,4– 6,* Tingting Qu,7,8 Keren Shi,4– 6 Xinhong Han,4– 6 Wenhao Wu,7,8 Yan Jiang,4– 6 Yunsong Yu4– 6 1Department of Laboratory Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 2Key Laboratory of Clinical in Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, People’s Republic of China; 3Institute of Laboratory Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 4Department of Infectious Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 5Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, People’s Republic of China; 6Regional Medical Center for National Institute of Respiratory Diseases, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 7State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 8Infection Control Department, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yunsong Yu, Tel +86 138 0579 0432, Email yvys119@zju.edu.cnPurpose: 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.Keywords: blaNDM-5, blaKPC-2, Klebsiella pneumoniae, hybrid plasmid, IS 26

Background: The increase in antimicrobial resistance includes emerging mechanisms such as 16S ribosomal RNA methylases, which confer high-level resistance to aminoglycosides. In this regard, the most predominant genes observed worldwide are rmtB and armA, but their presence in Uruguay is unknown. Objectives: We describe the genomic characterization of isolates carrying rmtB and rmtC, together with blaNDM-5 and blaNDM-1, respectively, and rmtD in our country. Methology: Five isolates from patients admitted to three hospitals were studied. Identification and antibiotic susceptibility testing were performed using the Vitek2 System. Whole Genome Sequencing was conducted, and hybrid assembly was performed with Unicycler. In silico analysis using the Center for Genomic Epidemiology’s tools was undertaken to predict antibiotic resistance determinants, plasmid incompatibility groups, and sequence types. Results: We report three K. pneumoniae ST307 isolates with an IncR plasmid carrying blaNDM-5/blaCTX-M-15/blaTEM-1B/rmtB/dfrA14/dfrA12/sul1/qacEΔ1/ermB/mphA, one K. pneumoniae ST258 harboring an IncC plasmid containing rmtC/blaNDM-1/blaCMY-6/aac(6′)-Ib/sul1, and one E. cloacae ST88 isolate with an IncFIB/II plasmid hosting rmtD, within a novel Tn21-like transposon named Tn7825, alongside blaOXA-101/sul1/tet(G)/floR, and a new variant of blaTEM assigned as blaTEM-258. One of the strains, named UH_B2, also carried an IncM1 plasmid encoding qnrE1/blaTEM-1/blaCTX-M-8 associated with ISEcp1. Conclusions: This is the first description of plasmids harboring 16S rRNA methyltransferases in Uruguay. The association and dissemination of diverse antibiotic-resistant genes underpin the health threat they represent, highlighting the lack of available antibiotics effective against multidrug-resistant microorganisms.

Eman R Abdelbary,1 Ashraf M Elsaghier,2 Rehab M Abd El-Baky,3,4 Nancy GFM Waly,3 Mohammed Ramadan,1 Fatma S Abd- Elsamea,5 Mohamed E Ali,1 Hayat A Alzahrani,6 Mohammed Salah7 1Microbiology and Immunology Department, Faculty of Pharmacy, Al-Azhar University-Assiut Branch, Assiut, 11651, Egypt; 2Gastroenterology and Hepatology Unit, University Children Hospital, Faculty of Medicine, Assiut University, Assiut, 11651, Egypt; 3Microbiology and Immunology Department, Faculty of Pharmacy, Minia University, Minia, 61519, Egypt; 4Microbiology and Immunology Department, Faculty of Pharmacy, Deraya University, Minia, 11566, Egypt; 5Medical Microbiology and Immunology Department, Faculty of Medicine, Assiut University, Assiut, 11651, Egypt; 6Department of Medical Laboratory Technology, Faculty of Applied Medical Science, Northern Border University, Arar, 91431, Saudi Arabia; 7Microbiology and Immunology Department, Faculty of Pharmacy, Port Said University, Port Said City, 42526, EgyptCorrespondence: Rehab M Abd El-Baky, Email rehab.mahmoud@mu.edu.egIntroduction: New Delhi metallo-β-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.Keywords: blaNDM-5, OqxAB, carbapenem resistant K. pneumoniae, efflux pumps

Sijia Pan,1,* Shuangqing Liu,2,* Shuhong Tai,1 Jing Yu,1 Enwu Yuan,1 Yitao Duan1 1Department of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 2Department of Clinical Laboratory, The Second Hospital of Tianjin Medical University, Tianjin, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yitao Duan; Enwu Yuan, Department of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou University, No. 7 Front Kangfu Street, Zhengzhou, Henan, 450052, People’s Republic of China, Email duanyitao@zzu.edu.cn; yuanenwu@126.comPurpose: 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.Keywords: Escherichia coli, ST167, blaNDM-5, phylogenetic analysis, MDR conjugative plasmids

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. [ABSTRACT FROM AUTHOR]

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-5Escherichia coli and 40 (9.1%) blaKPC-2Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5E. 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-5E. 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. [ABSTRACT FROM AUTHOR]

ABSTRACTThe detection rate of carbapenem-resistant nontyphoidal Salmonella (NTS) is relatively low. However, carbapenem-sensitive and carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium) were isolated from a clinical outpatient within a span of 10 days, named 1104–65 and 1104–75. We aimed to reveal the mechanism of carbapenem resistance in S. enterica serovar Typhimurium isolates in this case. The resistance phenotype of S. enterica serovar Typhimurium was analyzed by the VITEK-2 Compact equipment and Kirby-Bauer disc diffusion method. Whole-genome sequencing was used to analyze the serotype, ST type, genetic relationship, resistance genes, plasmid replicon, the genetic environment of blaNDM-5, and the characteristics of IncFII plasmid carrying blaNDM-5 in S. enterica serovar Typhimurium. The transferability of the IncFII plasmid carrying blaNDM-5 was verified by the conjugation assay and PCR. The serotypes of both NTS are S. enterica serovar Typhimurium, belonging to ST34. Single nucleotide polymorphisms analysis showed that they were the same clone. A conjugative IncFII plasmid pIncFII-NDM5 with transferability was detected in isolate 1104–75, which harboring the blaNDM-5 gene was the primary mechanism responsible for mediating carbapenem resistance in S. enterica serovar Typhimurium. The genetic environment of blaNDM-5 on pIncFII-NDM5 is “IS26-ΔIS3000-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-dsbC -cutA-IS26”, which was confirmed as a novel structure not previously reported in the NCBI database. Although the mechanism is rarely reported in NTS, the prevalence of IncFII plasmid pIncFII-NDM5 will pose a great threat to the clinical treatment of S. enterica serovar Typhimurium. Meanwhile, the coexistence of blaCTX-M-55, qnrS1, blaNDM-5, and mph(A) in S. enterica serovar Typhimurium ST34 warrants additional attention.IMPORTANCEIn 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.

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 blaNDM-5 by more than twofold 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. [ABSTRACT FROM AUTHOR]

Mengzhu Hou,1,* Nan Chen,2,* Lili Dong,3,* Yulian Fang,1 Rui Pan,2 Wei Wang,1 Lu Wang,1 Jing Ning,3 Hanquan Dong3 1Tianjin Pediatric Research Institute, Tianjin Children’s Hospital (Children’s Hospital of Tianjin University), Tianjin, People’s Republic of China; 2Department of Clinical Lab, Tianjin Children’s Hospital (Children’s Hospital of Tianjin University), Tianjin, People’s Republic of China; 3Department of Respiratory, Tianjin Children’s Hospital (Children’s Hospital of Tianjin University), Tianjin, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing Ning; Hanquan Dong, Department of Respiratory, Tianjin Children’s Hospital (Children’s Hospital of Tianjin University), No. 238 Longyan Road, Beichen District, Tianjin, 300134, People’s Republic of China, Tel +86 13752230008 ; +86 13820476506, Email jingning2022@126.com; mifits@sina.comPurpose: 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 blaNDM-5 and blaKPC-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 blaNDM-5 and blaKPC-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.Keywords: carbapenem-resistantKlebsiella pneumonia, blaKPC-2, blaNDM-5, ST11, children, bloodstream infection

Background: Co-harbouring of carbapenem and colistin resistance genes in multidrug-resistant Enterobacterales strains poses a serious public health problem. In this study, an MCR-1.1 and NDM-5 coproducing Escherichia coli strain named EC6563 was isolated and characterized. Objectives: This study aimed to characterize a clinical carbapenem-resistant E. coli isolate which co-harbours mcr-1.1 and blaNDM-5 on separate plasmids, and explored the phenotypic and genotypic characteristics of the mcr-1.1- and blaNDM-5-harbouring plasmids. Methods: E. coli isolate EC6563 was subjected to antimicrobial susceptibility testing, conjugation assay, stability of the plasmid and growth rate determination. In addition, the whole genome sequence of this strain was obtained and the genetic characteristics of the mcr-1.1- and blaNDM-5-harbouring plasmids were analyzed. Results: Carbapenem-resistant E. coli isolate EC6563 was resistant to all the tested antibiotics except tigecycline. Bioinformatic analysis confirmed that the IncHI2 plasmid carrying mcr-1.1 was highly similar to the previously reported mcr-1.1-harbouring plasmid pGDP37–4, and carried multiple drug resistance genes and the IncI1-I plasmid carrying blaNDM-5 had low similarity to the published blaNDM-5-carrying IncI1-I plasmid pEC-16–10-NDM-5. The pEC6563-NDM5 plasmid was capable of conjugation with an efficiency of 1.34 × 10−2 in a filter mating experiment. The transconjugant J53/pEC6563-NDM5 was able to be stably inherited after 12 days of passage. Conclusions: To the best of our knowledge, this is the first time that an IncHI2 plasmid carrying mcr-1.1 and an IncI1-I plasmid carrying blaNDM-5 is found to coexist in an E. coli isolate. Our research expands the known diversity of plasmids in NDM-5-producing Enterobacterales strains. Meanwhile, effective measures should be taken to prevent the spread of these plasmids.

Min Yang,1,* Gufeng Xu,1,* Zhi Ruan,2 Yue Wang1 1Department of Ambulatory Surgery, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; 2Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yue Wang, Women’s hospital, Zhejiang University School of Medicine, 1 Xueshi Road, Hangzhou, Zhejiang, 310000, People’s Republic of China, Email misswangyue@zju.edu.cn Zhi Ruan, Sir Run Run Shaw hospital, Zhejiang University School of Medicine, 3 East Qingchun Road, Hangzhou, Zhejiang, 310016, People’s Republic of China, Email r_z@zju.edu.cnBackground: 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 blaNDM-5 and blaCTX-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 blaNDM-5, which is located on a 46,161 bp IncX3 plasmid and the blaCTX-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 blaNDM-5 and blaCTX-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.Keywords: whole-genome sequencing, Escherichia coli, multidrug-resistant, blaNDM-5, blaCTX-M-14, pediatrics

Although carbapenem use is prohibited in animals in China, carbapenem-resistant Escherichia coli (CREC), especially New Delhi metallo-β-lactamase (NDM)-producing strains, are widely prevalent in food-producing animals. At present, the impact of livestock-associated CREC strains on human populations at the national level is unknown. Here, we conduct a retrospective cross-sectional study to investigate the prevalence of CREC from clinical settings across 22 Chinese provinces or municipalities and analyze anthropogenic factors associated with their presence. We also ascertain the blaNDM and blaKPC abundance among pig and chicken farms and present a detailed genomic framework for CREC of animal and human origin. Overall, 631/29 799 (2.1%) clinical Escherichia coli (E. coli) isolates were identified as CREC. Multivariable analysis revealed that being male, an age below 1, an age between 13 and 18, provinces with greater chicken production, and provinces with higher pig production were associated with higher odds of CREC infection. In general, 73.8% (n = 45/61) of pig farms and 62.2% (n = 28/45) of chicken farms had a blaNDM abundance of 1 × 10−5 to 1 × 10−3 and 1 × 10−3 to 1 × 10−2, respectively. Among all the Chinese NDM-positive E. coli (n = 463) available at the National Center for Biotechnology Information (NCBI), the genomic analysis revealed that blaNDM-5 and IncX3 were the predominant carbapenemase gene-plasmid combination, while a highly homogeneous relationship between NDM-positive isolates from humans and animals was demonstrated at the plasmid and core genome levels. All the findings suggest frequent CREC transmission between humans and animals, indicating that further discussions on the use of antibiotics in animals and humans are needed, both in China and across the globe.

In this study, we described the carbapenem blaNDM-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 (blaNDM-5, blaCTX-M, blaSHV, and blaTEM), 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 bleMBL at upstream and downstream, respectively, could play a significant role in the horizontal transmission of the blaNDM-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 blaNDM-5 K. pneumoniae ST437 and demands extensive molecular surveillance of river and nearby hospitals for better community health. [ABSTRACT FROM AUTHOR]

Aquatic environment can act as reservoir and disseminator of antimicrobial resistance and resistant pathogens. Novel high-risk carbapenem resistant E. coli (CREC) are continuously emerging worldwide; however, the occurrence of CREC in freshwater aquaculture environment is largely unexplored. To fill this gap, large scale sampling of freshwater pond sites and retail fish markets was done between Oct 2020 and Oct 2021 to investigate the CREC contamination in fish. The frequency of CREC contamination in the freshwater fish was 6.99% (95% CI: 3.78–10.20%). All the isolates were MDR and harbored carbapenemase encoding gene, blaNDM-5 along with other antimicrobial resistance genes (ARGs), blaTEM (64.7%), blaCTX-M-15 (35.3%), blaOXA-1 (5.9%), tet(A) (100%), sul1 (94.1%), qnrS (82.3%), cat1 (35.3%), and cat2 (23.5%). The isolates belonged to phylogroup C and showed low virulence gene profile. ERIC-PCR grouped the isolates into five clusters (I-V). The isolates of clusters I, II, and III were identified as ST167 (76.4%) and of cluster IV as ST361 (17.6%). This is the first report documenting the contamination of NDM-5 producing E. coli ST167 and ST361 of clinical/livestock lineage in freshwater fish from India. The blaNDM-5 was significantly associated with ARGs, tet(A), and sul1; and plasmid replicons, IncF, IncI1, and IncP, signifying the presence of blaNDM-5 and associated ARGs on these transferable plasmids. These findings were validated by the successful conjugal transfer of blaNDM-5 and associated ARGs into non-CREC strain (J53). Our study highlights the ability of CREC to disseminate antimicrobial resistance which has health implications and environmental concerns. [ABSTRACT FROM AUTHOR]

The coexistence of mcr-1 and blaNDM-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 blaNDM-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 blaNDM-5 in E. coli isolated from lesion-bearing animal organs. Nine mcr-1-and blaNDM-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 blaNDM-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 blaNDM-5 gene on the IncX3-type plasmid. The genetic structure of mcr-1 located on the chromosome was an intact Tn6330, and blaNDM-5-carrying IncX3-type plasmid was ISAb125-IS5-blaNDM-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. [ABSTRACT FROM AUTHOR]

Goutam Chowdhury,1,2 Thandavarayan Ramamurthy,1 Bhabatosh Das,3 Debjani Ghosh,1 Keinosuke Okamoto,2 Shin-ichi Miyoshi,2,4 Shanta Dutta,1 Asish K Mukhopadhyay1 1Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, India; 2Collaborative Research Centre of Okayama University for Infectious Diseases, ICMR-National Institute of Cholera and Enteric Diseases, Kolkata, India; 3Department of Infection and Immunology, Translational Health Science and Technology Institute, Faridabad, India; 4Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, JapanCorrespondence: Goutam Chowdhury; Asish K Mukhopadhyay, Division of Bacteriology, ICMR-National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010, India, Email goutambiotech@gmail.com; asish_mukhopadhyay@yahoo.comPurpose: The multidrug resistance Enterobacteriaceae cause many serious infections resulting in prolonged hospitalization, increased treatment charges and mortality rate. In this study, we characterized blaNDM-5-positive multidrug resistance commensal Escherichia coli (CE) isolated from diarrheal patients in Kolkata, India.Methods: Three CE strains were isolated from diarrheal stools, which were negative for different pathogroups of diarrheagenic E. coli (DEC). The presence of carbapenemases encoding genes and other antimicrobial resistance genes (ARGs) was detected using PCR. The genetic arrangement adjoining blaNDM-5 was investigated by plasmid genome sequencing. The genetic relatedness of the strains was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods.Results: In addition to colistin, the blaNDM-5-positive CE strains showed resistance to most of the antibiotics. Higher MICs were detected for ciprofloxacin (> 32 mg/L) and imipenem (8 mg/L). Molecular typing revealed that three CE strains belonged to two different STs (ST 101 and ST 648) but they were 95% similar in the PFGE analysis. Screening for ARGs revealed that CE strains harbored Int-1, blaTEM, blaCTX-M3, blaOXA-1, blaOXA-7, blaOXA-9, tetA, strA, aadA1, aadB, sul2, floR, mph(A), and aac(6´)-Ib-cr. In conjugation experiment, transfer frequencies ranged from 2.5× 10− 3 to 8.4x10− 5. The blaNDM-5 gene was located on a 94-kb pNDM-TC-CE-89 type plasmid, which is highly similar to the IncFII plasmid harboring an IS26-IS30-blaNDM-5-bleMBL-trpF-dsbd-IS 91-dhps structure.Conclusion: To the best of our knowledge, this is the first report on carbapenem resistance involving the blaNDM-5 gene in CE from diarrheal patients. The circulation of blaNDM-5 gene in CE is worrisome, since it has the potential to transfer blaNDM-5 gene to other enteric pathogens.Keywords: antimicrobial resistance, carbapenem-resistance, commensal E. coli, plasmid; blaNDM-5

This study conducted a four-month monitoring of carbapenem resistance in a broiler breeding farm in China. A total of 185 carbapenem-resistant bacterial isolates were obtained from 2298 cloacal swabs from broiler breeders and their offspring within a production cycle. The detection rate of carbapenem-resistant isolates was higher during the brooding period. Whole-genome sequencing (WGS) was performed on 133 isolates based on sampling stages, including 113 carbapenem-resistant Enterobacterales (CRE) isolates and 20 Stenotrophomonas pavanii isolates, which have intrinsic resistance to carbapenems. A total of 69 antibiotic resistance genes (ARGs), including bla NDM-1 , mcr-1 , and bla NDM-5 , were identified among the sequenced CRE isolates. Notably, bla NDM-5 (92.0 %, 104/113) was the primary contributor to carbapenem resistance. CRE isolates from the same breeding stage exhibited close genomic relationships, and the bla NDM-5 genes were observed in similar genetic backgrounds, indicating the transmission of CRE strains and bla NDM-5 during the broiler breeding process. No CRE was isolated from 0 d broiler offspring, suggesting that broiler breeders were not the direct source of CRE in their offspring. Tracing the feeding process revealed that brooder and rearing houses were likely key factors in the cross-transmission of CRE between broiler breeders and their offspring. CRE pose a significant threat to public health and food safety. China is one of the world's leading poultry producing and consuming countries. This study provided insights into the epidemiological trends and key transmission nodes of carbapenem resistance and CRE within the broiler breeding process, which could help the control of antibiotic resistance and bacterial infections in the broiler industry. • This study investigated the carbapenem resistance during the broiler breeding process. • CRE and bla NDM-5 could transfer in the broiler breeding process. • Brooder and rearing houses were the potential reservoirs of CRE. [ABSTRACT FROM AUTHOR]

Danni Bao,1,2 Linyao Huang,3 Jianxin Yan,3 Yexuzi Li,4 Zhi Ruan,2 Tian Jiang2,3 1Department of Clinical Laboratory, Sanmen People’s Hospital, Taizhou, Zhejiang, 317100, People’s Republic of China; 2Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, People’s Republic of China; 3Department of Clinical Laboratory, The Affiliated Wenling Hospital, Wenzhou Medical University, Wenling, 317500, People’s Republic of China; 4Department of Critical Care Medicine, The Affiliated Wenling Hospital, Wenzhou Medical University, Wenling, 317500, People’s Republic of ChinaCorrespondence: Tian Jiang; Zhi Ruan, Email jiangtianhw@zju.edu.cn; r_z@zju.edu.cnAbstract: The emergence of multidrug-resistant Pseudomonas spp. in the clinical settings has heightened public awareness. Here, we described the genomic characteristics of a P. putida isolate co-carrying five β-lactam resistance genes recovered from a urinary tract infection in China. Whole-genome sequencing was performed using Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms. The genome sequence was annotated and further subjected to identify the sequence type (ST), antibiotic resistance and virulence genes. Phylogenetic analysis of 193 P. putida strains stored in NCBI public database based on core genome single nucleotide polymorphism (cgSNP) strategy were also performed and visualized. Our study indicated that P. putida PP_2463 was resistant to a wide range of antimicrobial agents tested, including aminoglycosides, carbapenems and fluoroquinolones. The complete genome sequence of P. putida PP_2463 is made up of one chromosome and two plasmids, which could be assigned to a new sequence type (ST) 148. The co-occurrence of β-lactam resistance genes blaIPM-15, blaPME-1, blaCARB-2, and blaNDM-1 were first identified in P. putida, and a novel β-lactamase gene located in the chromosome were among the antimicrobial resistance genes discovered. The closest relative of P. putida PP_2463 was identified in 2012 from a urine sample in China, with a difference of 143 SNPs. Along with the presence of multiple β-lactamase genes and mobile genetic elements, the multidrug-resistant phenotype suggests a significant potential as an antibiotic resistance reservoir for Pseudomonas spp.Keywords: Pseudomonas putida, whole genome sequencing, multidrug-resistance, blaPME-1, blaNDM-5, blaIPM-15, blaCARB-2, urinary tract infection

Danni Bao, Xiaohong Xu, Yizhang Wang, Fengjiao Zhu Department of Clinical Laboratory, Sanmen People’s Hospital, Taizhou, Zhejiang, People’s Republic of ChinaCorrespondence: Fengjiao Zhu, Department of Clinical Laboratory, Sanmen People’s Hospital, Taizhou, Zhejiang, People’s Republic of China, Email sm.zfj@foxmail.comBackground: Carbapenem-resistant Enterobacterales (CRE) are a significant threat to worldwide public health, resulting in increased morbidity, death, hospitalization time and healthcare expenses. Here, the genomic and phylogenetic characteristics of a multidrug-resistant Escherichia coli isolate carrying both the new mcr-1.33 variant and blaNDM-5 gene obtained from a urinary tract infection in China are investigated.Methods: Antimicrobial susceptibility of E. coli 779 was evaluated by using the broth microdilution method. Short-read Illumina NovaSeq 6000 and long-read Oxford Nanopore MinION platforms were applied to sequence the bacterial whole genomic DNA and then de novo assembled. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline and further subjected to identify the sequence type (ST), capsular type, and antibiotic resistance genes. BacWGSTdb 2.0 was used to perform the core genome multilocus sequence typing (cgMLST) analysis with other closely related E. coli isolates deposited in the public database.Results: E. coli 779 was resistant to aztreonam, levofloxacin, fosfomycin, cefoxitin, cefepime, cefotaxime, imipenem, meropenem, polymyxin, and tigecycline. The complete genome sequence of E. coli 779 is made up of nine contigs totaling 5,667,876 bp, including one chromosome and eight plasmids. The isolate was assigned to ST101, serotype O-:H31, and phylogroup B1. The colistin resistance gene mcr-1.33 (located in a 242,460 bp IncHI2/IncHI2A plasmid) and the β-lactam resistance gene blaNDM-5 (located in a 46,161 bp IncX3 plasmid) were among the 27 antimicrobial resistance genes discovered. The closest relative of E. coli 779, another ST101 strain (E. coli 443) obtained from a sewage sample in Shandong, China in 2015, differs by only 24 cgMLST alleles.Conclusion: We discovered the first multidrug-resistant ST101 E. coli strain with plasmid-mediated mcr-1.33 variant and blaNDM-5 gene in China. These findings would help us to better understanding the genomic traits, antimicrobial resistance mechanisms and epidemiological aspects of this bacterial pathogen.Keywords: whole genome sequencing, Escherichia coli, multidrug-resistance, mcr-1.33, blaNDM-5, urinary tract infection

The coexistence of mcr-1 and blaNDM-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 blaNDM-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 blaNDM-5 in E. coli isolated from lesion-bearing animal organs. Nine mcr-1- and blaNDM-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 blaNDM-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 blaNDM-5 gene on the IncX3-type plasmid. The genetic structure of mcr-1 located on the chromosome was an intact Tn6330, and blaNDM-5-carrying IncX3-type plasmid was ISAb125-IS5-blaNDM-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.

Background: Urinary tract infection (UTI) is one of the most common outpatient bacterial infections. In this study, we isolated and characterized an extensively-drug resistant (XDR) NDM-5-producing Escherichia coli EC1390 from a UTI patient by using whole-genome sequencing (WGS) in combination with phenotypic assays. Methods: Antimicrobial susceptibility to 23 drugs was determined by disk diffusion method. The genome sequence of EC1390 was determined by Nanopore MinION MK1C platform. Conjugation assays were performed to test the transferability of EC1390 plasmids to E. coli recipient C600. Phenotypic assays, including growth curve, biofilm formation, iron acquisition ability, and cell adhesion, were performed to characterize the function of EC1390 plasmids. Results: Our results showed that EC1390 was only susceptible to tigecycline and colistin, and thus was classified as XDR E. coli. A de novo genome assembly was generated using Nanopore 73,050 reads with an N50 value of 20,936 bp and an N90 value of 7,624 bp. WGS analysis showed that EC1390 belonged to the O101-H10 serotype and phylogenetic group A E. coli. Moreover, EC1390 contained 2 conjugative plasmids with a replicon IncFIA (pEC1390-1 with 156,286 bp) and IncFII (pEC1390-2 with 71,840 bp), respectively. No significant difference was observed in the bacterial growth rate in LB broth and iron acquisition ability between C600, C600 containing pEC1390-1, C600 containing pEC1390-2, and C600 containing pEC1390-1 and pEC1390-2. However, the bacterial growth rate in nutrition-limited M9 broth was increased in C600 containing pEC1390-2, and the cell adhesion ability was increased in C600 containing both pEC1390-1 and pEC1390-2. Moreover, these plasmids modulated the biofilm formation under different conditions. Conclusions: In summary, we characterized the genome of XDR-E. coli EC1390 and identified two plasmids contributing to the antimicrobial resistance, growth of bacteria in a nutrition-limited medium, biofilm formation, and cell adhesion. [ABSTRACT FROM AUTHOR]

Co-harbouring of carbapenem and colistin resistance genes in multidrug-resistant Enterobacterales strains poses a serious public health problem. In this study, an MCR-1.1 and NDM-5 coproducing Escherichia coli strain named EC6563 was isolated and characterized. This study aimed to characterize a clinical carbapenem-resistant E. coli isolate which co-harbours mcr-1.1 and bla NDM-5 on separate plasmids, and explored the phenotypic and genotypic characteristics of the mcr-1.1- and bla NDM-5 -harbouring plasmids. E. coli isolate EC6563 was subjected to antimicrobial susceptibility testing, conjugation assay, stability of the plasmid and growth rate determination. In addition, the whole genome sequence of this strain was obtained and the genetic characteristics of the mcr-1.1 - and bla NDM-5 -harbouring plasmids were analyzed. Carbapenem-resistant E. coli isolate EC6563 was resistant to all the tested antibiotics except tigecycline. Bioinformatic analysis confirmed that the IncHI2 plasmid carrying mcr-1.1 was highly similar to the previously reported mcr-1.1 -harbouring plasmid pGDP37–4, and carried multiple drug resistance genes and the IncI1-I plasmid carrying bla NDM-5 had low similarity to the published bla NDM-5 -carrying IncI1-I plasmid pEC-16–10-NDM-5. The pEC6563-NDM5 plasmid was capable of conjugation with an efficiency of 1.34 × 10−2 in a filter mating experiment. The transconjugant J53/pEC6563-NDM5 was able to be stably inherited after 12 days of passage. To the best of our knowledge, this is the first time that an IncHI2 plasmid carrying mcr-1.1 and an IncI1-I plasmid carrying bla NDM-5 is found to coexist in an E. coli isolate. Our research expands the known diversity of plasmids in NDM-5-producing Enterobacterales strains. Meanwhile, effective measures should be taken to prevent the spread of these plasmids. [ABSTRACT FROM AUTHOR]

Escherichia coli ( E. coli ) serves as a critical indicator microorganism for assessing the prevalence and dissemination of antibiotic resistance, notably harboring various antibiotic-resistant genes (ARGs). Among these, the emergence of the bla NDM gene represents a significant threat to public health, especially since carbapenem antibiotics are vital for treating severe infections caused by Gram-negative bacteria. This study aimed to characterize the antibiotic resistance features of bla NDM-5 -positive E. coli strains isolated from waterfowl in several regions of China and elucidate the dissemination patterns of the bla NDM-5 gene. We successfully isolated 103 bla NDM-5 -positive E. coli strains from 431 intestinal fecal samples obtained from waterfowl across five provincial-level units in China, with all strains exhibiting multidrug resistance (MDR). Notably, the bla NDM-5 gene was identified on plasmids, which facilitate efficient and stable horizontal gene transfer (HGT). Our adaptability assays indicated that while the bla NDM-5 -positive plasmid imposed a fitness cost on the host bacteria, the NDM-5 protein was successfully induced and purified, exhibiting significant enzymatic activity. One strain, designated DY51, exhibited a minimum inhibitory concentration (MIC) for imipenem of 4 mg/L, which escalated to 512 mg/L following exposure to increasing imipenem doses. This altered strain demonstrated stable resistance to imipenem alongside improved adaptability, correlating with elevated relative expression levels of the bla NDM-5 and overexpression of efflux pumps. Collectively, this study highlights the horizontal dissemination of the bla NDM-5 plasmid among E. coli strains, confirms the associated fitness costs, and provides insights into the mechanisms underlying the stable increase in antibiotic resistance to imipenem. These findings offer a theoretical framework for understanding the dissemination dynamics of bla NDM-5 in E. coli , which is essential for developing effective strategies to combat carbapenem antibiotic resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Shu, Yang, Zhong, Wang, Jia, Chen, Liu, Zhu, Zhao, Wu, Yang, Huang, Ou, Sun, Tian, Wu, He and Cheng.)

ABSTRACT: Objectives: The aim of this study was to characterise the co-occurrence of blaKPC and blaNDM in a K64-ST11 carbapenem-resistant Klebsiella pneumoniae strain. Methods: Antimicrobial susceptibility was determined by the disk diffusion method. Whole-genome sequencing was performed using Illumina MiSeq and PacBio II sequencers. High-quality reads were de novo assembled using the SOAPdenovo package. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and genome characteristics were analysed using bioinformatics methods. Results: Klebsiella pneumoniae strain KPWX136 was resistant to most of the tested antibiotics, being susceptible only to polymyxin B and tigecycline. The genome of strain KPWX136 is composed of a single chromosome (5 473 976 bp) and six plasmids including pA (191 359 bp), pB (134 972 bp), pC (117 844 bp), pD (87 095 bp), pE (11 970 bp) and pF (5596 bp). Complete sequence analysis revealed the resistome of isolate KPWX136, which included blaKPC-2 and blaNDM-5 together with 23 other resistance genes, of which 6 resistance genes were located on the chromosome and 19 on plasmids. Virulome analysis showed that KPWX136 carried a large number of virulence-associated genes. Meanwhile, 26 genomic islands and 6 prophages were predicted within the genome. Conclusion: Genetic characterisation of K. pneumoniae KPWX136 co-harbouring blaNDM-5 and blaKPC-2 showed that it carried not only 25 resistance genes and a large number virulence factors but also various mobile genetic elements (MGEs) such as plasmids and genomic islands. Therefore, we must be alert to the transmission of resistance genes and virulence determinants via MGEs.

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 blaNDM–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., blaTEM–1B, blaNDM–5, blaCMY–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 blaNDM–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.

Animal farms have become one of the most important reservoirs of carbapenemresistant 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 blaNDM-5, which was flanked by the same core genetic structure (IS5-blaNDM-5-bleMBL-trpF-dsbDIS26-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-baeSdgkA-orf-IS903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying-blaNDM-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. [ABSTRACT FROM AUTHOR]

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 blaNDM 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), blaNDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a blaNDM-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 nontigecycline 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. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: The emergence of carbapenem-resistant Enterobacteriaceae has become a serious public-health threat. Here we report the complete genome sequence of a multidrug-resistant (MDR) Escherichia coli carrying blaNDM-5 and two copies of blaCTX-M-14 recovered from a bloodstream infection in China. Methods: Whole-genome sequencing of E. coli strain 2D was performed both using Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. De novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes and plasmid replicons were identified from the genome sequence. Core genome MLST (cgMLST) analysis between E. coli 2D and all ST354 E. coli strains retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. Results: The complete genome sequence of E. coli 2D consists of six contigs comprising 5 363 300 bp, including one chromosome and five plasmids, and was assigned to ST354. Six antimicrobial resistance genes were identified, including blaNDM-5 located in a 46 161-bp IncX3 plasmid and two copies of the blaCTX-M-14 gene located both in the chromosome and in a 96 499-bp IncB plasmid. The closest relative of E. coli 2D was another isolate recovered from Lebanon, which differed by 162 cgMLST loci. Conclusion: This study reports the first genome sequence of a MDR E. coli carrying blaNDM-5 and two copies of blaCTX-M-14 in China. These data may help to understand the antimicrobial resistance mechanisms and transmission dynamics of carbapenem-resistant Enterobacteriaceae in clinical settings.

Xingwei Cao,1,* Qiaoshi Zhong,1,* Yinjuan Guo,2,3 Yaping Hang,1 Yanhui Chen,1 Xueyao Fang,1 Yanping Xiao,1 Hongying Zhu,1 Hong Luo,1 Fangyou Yu,2,3 Longhua Hu1 1Jiangxi Provincial Key Laboratory of Medicine, Clinical Laboratory of the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s Republic of China; 3Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s Republic of China*These authors contributed equally to this workCorrespondence: Longhua Hu; Fangyou Yu Email longhuahu@163.com; wzjxyfy@163.comIntroduction: Polymyxin resistance caused by the plasmid-mediated mcr-1 gene in gram-negative bacilli poses a huge threat to our health. In recent years, many regions have reported that mcr-1 and β-lactamase genes can coexist in a single strain.Methods: In this study, 107 nonduplicate Klebsiella pneumoniae (K. pneumoniae) isolates were collected from a tertiary hospital in Jiangxi, China. Antimicrobial susceptibility testing of isolates was performed using gram-negative susceptibility cards on the VITEK system. The minimum inhibitory concentrations (MICs) of polymyxin B was detected using the microdilution broth method. The presence of resistance genes was assessed using polymerase chain reaction (PCR). We subjected isolates to genotyping using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and analyzed the transferability of plasmids with filter mating and electroporation. Subsequently, whole-genome sequencing was performed for plasmids.Results: Of the 107 K. pneumoniae isolates, 15 (14.0%) were resistant to polymyxin B. All polymyxin B-resistant isolates harbored at least one of the extended-spectrum β-lactamase genes tested. Only one isolate simultaneously harbored mcr-1, blaNDM− 5, blaCTX-M-55, and blaSHV-27 genes. MLST results showed that 15 carbapenem-resistant K. pneumoniae isolates belonged to five sequence types (STs). PFGE results displayed nine different PFGE clusters. Conjugation and transformation experiments and sequencing analysis showed that the strain had three plasmids, and mcr-1, blaNDM− 5, and blaCTX-M-55 were located on different plasmids.Conclusion: The present study demonstrated for the first time the coexistence of mcr-1, blaNDM− 5, and blaCTX-M-55 in a K. pneumoniae ST485 isolate. The three plasmids carrying the mcr-1, blaNDM− 5, and blaCTX-M-55 genes can be transmitted in Enterobacteriaceae strains, which may lead to more severe bacterial resistance.Keywords: Klebsiella pneumoniae, polymyxin B, mcr-1, blaNDM-5, blaCTX-M-55, plasmid