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Jiamin Long,1,2,* Jiali Chen,3,* Yue Yuan,4,* Shaozhen Yang,5,* Xinya Xie,1,2 Xuelian Wu,1,2 Yuan Liu,1,2 Jinpeng Guo,2 Yong Chen,1,2 Changjun Wang,1,2 Xiong Liu2 1School of Public Health, China Medical University, Shenyang, 110122, People’s Republic of China; 2Chinese PLA Center for Disease Control and Prevention, Beijing, 100071, People’s Republic of China; 3School of Medicine, NanKai University, Tianjin, 300071, People’s Republic of China; 4Fifth Medical Center of Chinese PLA General Hospital, Beijing, 100039, People’s Republic of China; 5School of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing, 400038, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiong Liu; Changjun Wang, Chinese PLA Center for Disease Control and Prevention, Beijing, 100071, People’s Republic of China, Email liuxiong714@163.com; science2008@hotmail.comBackground: Klebsiella michiganensis is an emerging human pathogen that causes nosocomial infections. Its prevalence and spread in the environment should not be ignored. This study identified and characterized Klebsiella michiganensis co-harboring blaKPC-2 and TmexCD2-ToprJ2 in hospital wastewater samples.Methods: Twelve K. michiganensis strains were isolated from wastewater samples collected at a tertiary hospital in Beijing, China. The genomic characteristics of K. michiganensis strains were analyzed using whole-genome sequences, providing information on the comparison between the genome of K. michiganensis strains and the reference genome, antibiotic resistance genes (ARGs), virulence genes, secretion systems, and mobile genetic elements (plasmids, insertion sequences [ISs], and prophages).Results: Genome analysis showed that the twelve multi-drug resistant (MDR) strains carried a variety of ARGs and virulence genes, as well as four macromolecular secretion systems (T1SS, T2SS, T5aSS, T5bSS, and T4aP). The genetic environments of both the TmexCD2-ToprJ2 gene cluster and blaKPC-2 gene contained ISs. The plasmids carrying TmexCD2-ToprJ2 gene cluster of nine strains in clade 1 and two strains in clade 2 were annotated as IncR plasmid and rep_cluster_1254 type, respectively. The plasmids carrying blaKPC-2 in 10 strains in clade 1 were identified as IncU, and the plasmids carrying blaKPC-2 in the k11 and k12 strains in clade 2 were IncU and IncX6. The phylogenetic tree and heatmap revealed that the secretion system of type VI (T6SSi) existed in 10 strains in clade 1, and Type IV (T4SS) only existed in the k11 strain in clade 2. In addition, K. michiganensis strains carried 13 plasmids, 14 ISs, and 138 prophages.Conclusion: In this study, the whole genome sequencing demonstrated the diversity of K. michiganensis genome despite 12 K. michiganensis strains from a hospital wastewater, which lays the foundation for further genetic research and drug resistance gene transmission.Keywords: Klebsiella michiganensis, hospital wastewater, whole-genome sequencing, blaKPC-2, TmexCD2-ToprJ2

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

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The blaKPC-2-bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these blaKPC-2-bearing plasmids. The blaKPC-2 was highly consistent in seven blaKPC-2-bearing plasmids (ISKpn6-blaKPC-2-ISKpn27-ISYps3-IS26). In addition, we found a region composed of ISIR, Tn5393, and IS26. It was located upstream of the blaCTX-M-15 gene and presented in six blaKPC-2-bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains.IMPORTANCEWe reported seven CR-hvKP strains all carried a highly homologous blaKPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

ABSTRACT Colistin is used as a last-line therapy against carbapenem-resistant Klebsiella pneumoniae (CRKP). However, colistin resistance in Klebsiella pneumoniae is increasingly reported worldwide. This study aims to investigate the instrumental role of insertion sequence (IS) elements in colistin resistance through mgrB disruption in K. pneumoniae during treatment. Five clinical isolates of CRKP, designated KPN1~KPN5 were collected from the lower respiratory tract of a patient with chest infection before and after treatment with colistin. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole genome sequencing and bioinformatics were used to analyze the sequence types (STs), resistance genes, and genetic characteristics of the five isolates of K. pneumoniae. Antimicrobial susceptibility testing indicated that all five K. pneumoniae isolates were resistant to cephalosporins (ceftriaxone, ceftazidime, and cefepime), several carbapenems (imipenem, meropenem), cefoperazone–sulbactam, piperacillin–tazobactam, ciprofloxacin, and fosfomycin, whereas they were sensitive to amikacin and tigecycline. In addition, three of these isolates were resistant to colistin, with minimum inhibitory concentration values of >8 mg/L. Whole genome sequencing revealed that all five K. pneumoniae isolates belonged to sequence type 1 (ST1), which shared an identical blaKPC-2. Notably, disruption of mgrB by the ISKpn26 insertion sequence was shown to be the primary colistin resistance mechanism during the treatment. To our knowledge, this is the first report of ISKpn26 element mediating mgrB disruption in the ST1 colistin and CRKP obtained from a patient with chest infection in mainland China. This study provides new research ideas to explore the clinical drug resistance mechanism of CRKP and the critical need to monitor and understand resistance mechanisms to preserve the efficacy of last-line antibiotics such as colistin.IMPORTANCEOf note, this chapter gives an update on colistin resistance in sequence type 1 Klebsiella pneumoniae, by focusing on the mgrB disrupted by ISKpn26 element.

Hang Jin,1,2,* Zhongqiang Yan,3,* Xin Ge,4,* Qi Wang,2 Hui Wang,2 Xinying Du,2 Hongbo Liu,2 Chaojie Yang,2 Ying Xiang,2 Sai Tian,2 Shaofu Qiu,1,2 Yu Zhou5 1School of Public Health, Zhengzhou University, Zhengzhou, People’s Republic of China; 2Department of Infectious Disease Control and Prevention, Center for Disease Control and Prevention of Chinese PLA, Beijing, People’s Republic of China; 3Department of Disease Prevention and Control, The Second Medical Center of PLA General Hospital, Beijing, People’s Republic of China; 4School of Public Health, Anhui Medical University, Hefei, People’s Republic of China; 5Department of Clinical Laboratory, National Clinical Research Center for Geriatric Diseases, The Second Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shaofu Qiu, Center for Disease Control and Prevention of Chinese PLA, Beijing, People’s Republic of China, Email qiushf0613@hotmail.com Yu Zhou, Department of Clinical Laboratory, National Clinical Research Center for Geriatric Diseases, The Second Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China, Email zhouy427@163.comPurpose: This study conducted an phenotypic and whole-genome sequencing analysis with Klebsiella aerogenes to elucidate its clinical epidemiological characteristics, antimicrobial resistance (AMR) phenotype, biofilm formation ability and hemolytic activity testing, AMR genes and phylogenetic relationships, so as to provide a further understanding of the intra-hospital strain transmission.Methods: Samples were collected from a hospital in Beijing between 2020 and 2022. All strains underwent bacterial identification, antimicrobial susceptibility testing (AST) using the VITEK-2 compact system. Biofilm formation ability and hemolytic activity were tested. Second-generation sequencing was applied to all strains, with those carrying the blaKPC gene were selected for third-generation sequencing. Whole-genome analysis identified resistance genes, plasmid types, MLST typing, and phylogenetic relationships. Plasmids were assembled to detect plasmid structures and AMR gene location.Results: Among the 42 K. aerogenes isolates, 21 were carbapenem-resistant K. aerogenes (CRKA). All strains exhibited strong biofilm formation and no hemolytic activity. Most were sourced from sputum (83.3%). CRKA demonstrated extensive resistance to antibiotics, particularly β-lactamase inhibitors and Cefotetan. This resistance pattern was closely associated with the presence of an IncFII(pHN7A8) plasmid, which carried multiple resistance genes, including blaKPC-2, blaCTX-M-65, blaTEM-1, rmtB and a large number of mobile elements. The majority of CRKA strains clustered within the same branch of the phylogenetic tree, exhibiting minimal single nucleotide polymorphism (0– 13 SNPs) differences, and they shared the same sequence type (ST292), resistance genes, and plasmids, originating from different departments, suggesting clonal transmission among the hospital.Conclusion: Our research reveals that the clonal transmission of CRKA occurs across various departments within the hospital. The widespread resistance observed in CRKA, attributed to the presence of blaKPC and ESBLs genes, underscores the need for heightened vigilance to prevent the further dissemination of CRKA within the hospital and, potentially, throughout the wider community.Keywords: Klebsiella aerogenes, carbapenem resistance, blaKPC-2, clonal transmission

Huiqian Guo,1,2,* Jing Luo,1,3,* Suming Chen,1 Ting Yu,4 Xiaofei Mu,4 Fangzhou Chen,4 Xiuhui Lu,4 Jiaqi He,4 Yali Zheng,4 Chunmei Bao,2 Peng Wang,4 Zhe Yin,4 Boan Li1– 3 1Department of Clinical Laboratory, the Fifth Medical Center of PLA General Hospital, Beijing, 100039, People’s Republic of China; 2School of Medical Laboratory, Weifang Medical University, Weifang, 261053, People’s Republic of China; 3Medical School of Chinese PLA, Beijing, 100853, People’s Republic of China; 4State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, People’s Republic of China*These authors contributed equally to this workCorrespondence: Boan Li, Department of Clinical Laboratory, the Fifth Medical Center of PLA General Hospital, Beijing, 100039, People’s Republic of China, Email lba@263.net Zhe Yin, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, People’s Republic of China, Email jerry9yin@163.comPurpose: To investigate the genetic diversity of IncG plasmids, we have proposed a typing scheme based on replicon repA and performed comparative genomic analysis of five IncG plasmids from China.Methods: p30860-KPC, p116965-KPC, pA1705-KPC, pA1706-KPC and pNY5520-KPC total in five IncG plasmids from clinical isolates of Pseudomonas and Enterobacteriaceae, respectively, were fully sequenced and were compared with the previously collected reference plasmid p10265-KPC.Results: Based on phylogeny, IncG-type plasmids are divided into IncG-I to IncG-VIII, the five plasmids belong to IncG-VIII. A detailed sequence comparison was then presented that the IncG plasmid involved accessory region I (Tn 5563a/b/c/d/e), accessory region II (ISpa19), and accessory region III (blaKPC-2-region). Expect for the pNY5520-KPC, the rest of the plasmids had the same backbone structure as the reference one. Within the plasmids, insertion sequences Tn 5563d and Tn 5563e were identified, a novel unknown insertion region was found in Tn 5563b/c/d/e. In addition, Tn 6376b and Tn 6376c were newly designated in the study.Conclusion: The data presented here including a typing scheme and detailed genetic comparison which provide an insight into the diversification and evolution history of IncG plasmids.Keywords: IncG plasmids, blaKPC-2, phylogenetic tree, multidrug resistance, mobile elements

ABSTRACT: Objectives: Herein, we detected one multidrug-resistant Aeromonas hydrophila strain K522 co-carrying two blaKPC-2 genes together with a novel chromosomal integrative and mobilizable element (IME) Tn7548 from China. To reveal the genetic characteristics of the novel reservoir of blaKPC-2 and IME in Aeromonas, a detailed genomic characterization of K522 was performed, and a phylogenetic analysis of Tn7412-related IMEs was carried out. Methods: Carbapenemases were detected by using the immunocolloidal gold technique and antimicrobial susceptibility was tested by using VITEK 2. The whole-genome sequences of K522 were analysed using phylogenetics, detailed dissection, and comparison. Results: Strain K522 carried a Tn7412-related chromosomal IME Tn7548 and three resistance plasmids pK522-A-KPC, pK522-B-KPC, and pK522-MOX. A phylogenetic tree of 82 Tn7412-related IMEs was constructed, and five families of IMEs were divided. These IMEs shared four key backbone genes: int, repC, and hipAB, and carried various profiles of antimicrobial resistance genes (ARGs). pK522-A-KPC and pK522-B-KPC carried blaKPC-2 and belonged to IncG and unclassified type plasmid, respectively. The blaKPC-2 regions of these two plasmids were the truncated version derived from Tn6296, resulting in the carbapenem resistance of K522. Conclusion: We first reported A. hydrophila harbouring a novel Tn7412-related IME Tn7548 together with two blaKPC-2 carrying plasmids and a MDR plasmid. Three of these four mobile genetic elements (MGEs) discovered in A. hydrophila K522 were novel. The emergence of novel MGEs carrying ARGs indicated the rapid evolution of the resistance gene vectors in A. hydrophila under selection pressure and would contribute to the further dissemination of various ARGs in Aeromonas.

Background: The escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably ß-lactams, constitutes a formidable challenge for healthcare and global public health management. Methods: This research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features. Results: The study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (=5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments. Conclusion: In summary, our study highlights the extensive spread of antibioticresistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmidmediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to investigate the clinical and molecular characteristics of Klebsiella pneumoniae infection from a tertiary general hospital in Wuhan, China. Methods: From December 2019 to August 2022, 311 non-duplicate isolates of K. pneumoniae were collected from a tertiary hospital in Wuhan. These comprised 140 carbapenem-resistant K. pneumoniae (CRKP) isolates and 171 carbapenem-susceptible K. pneumoniae (CSKP) isolates. The clinical characteristics of patients with K. pneumoniae infection were retrospectively collected. Polymerase chain reaction (PCR) assays were used to identify the main carbapenem resistance genes, virulence genes and multi-locus sequence typing (MLST) profiles of the isolates, and the Galleria mellonella infection model was used to determine their virulence phenotypes. Results: Independent risk factors for CRKP infection were hypertension, neurological disorders, being admitted to the intensive care unit (ICU) and prior use of antibiotics. Patient with CRKP infection had higher mortality than those with CSKP infection (23.6% vs 14.0%, P < 0.05). One hundred and two sequence types (STs) were identified among the K. pneumoniae isolates, and the most prevalent ST type was ST11 (112/311, 36.0%). All of the ST11 isolates were CRKP. Among the 112 ST11 isolates, 105 (93.8%) harboured the carbapenem resistance gene blaKPC-2 (ST11-KPC-2), and of these isolates, 78 (74.3%, 78/105) contained all of the four virulence genes, namely rmpA, rmpA2, iroN and iucA, suggesting that these genes were widespread among the isolates responsible for K. pneumoniae infections. Conclusion: In this study, ST11-KPC-2 was responsible for most of the K. pneumoniae infection cases. Carbapenem resistance rather than the co-occurrence of the virulence genes rmpA, rmpA2, iroN and iucA was associated with K. pneumoniae infection-related mortality during hospitalisation. Furthermore, a high proportion of ST11-KPC-2 isolates carried all of the four virulence genes. [ABSTRACT FROM AUTHOR]

ABSTRACTHospital sewage serves as a crucial reservoir for antibiotic resistance genes. As colistin and carbapenems are the last-resort antibiotics, the emergence of their resistance genes has become a significant concern in clinical settings. In this study, we found that two novel mcr alleles (mcr-3.43 and mcr-7.2) with two carbapenemase genes (blaNDM-1 and blaKPC-2) were encoded in a single Aeromonas caviae strain isolated from hospital sewage. Our phylogenetic analysis revealed that the mcr-3.43 gene clustered with mcr-3.17 (with 95.55% amino acid identity), while the mcr-7.2 gene clustered with mcr-7.1 (with 68.68% amino acid identity). BLAST search against GenBank showed that mcr-7.2 was exclusively detected in Aeromonas spp. Mobile genetic elements were not found in the genetic context of mcr-7.2, suggesting that the dissemination of mcr-7.2 in Aeromonas spp. may be dependent on vertical transfer or recombination. The blaNDM-1 was adjacent to a recombinase gene and flanked by two IS91 elements, indicating a potential mobilization mechanism mediated by recombination and/or ISs. The blaKPC-2 gene was located on an IncU plasmid and adjacent to an ISKpn6. In summary, our study provides evidence for Aeromonas spp. as one of the potential reservoirs of colistin and carbapenem resistance genes.IMPORTANCEThe study discovered two novel mcr genes (mcr-3.43 and mcr-7.2) and two carbapenemase genes (blaNDM-1 and blaKPC-2) in a single Aeromonas caviae strain retrieved from hospital sewage. Using phylogenetic analysis and comparative data evaluation, the study revealed the genetic relatedness and dissemination potential of the detected resistance genes. With the exclusive discovery that mcr-7.2 is only present in Aeromonas spp. and the lack of mobile genetic elements in its genetic context, there is a strong indication of limited dissemination. The identification of these four resistance genes in a single strain of Aeromonas provided valuable insights into their potential presence in this genus. This study revealed that hospital sewage functions as a significant reservoir for antibiotic resistance genes, including colistin and carbapenem resistance genes.

Lingyan Sun,1– 3,&ast; Yan Chen,4– 6,&ast; 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&ast;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

Xu M, Fu Y, Fang Y, et al. Infect Drug Resist. 2019;12:641–653. Page 647, Molecular characteristics of the K. pneumoniae isolates, first paragraph, right column, second sentence from the bottom, the text “The PFGE results showed that the homology of 44 isolates was diverse (Figure 3)” should read “The PFGE results showed that the homology of 44 isolates was diverse (Figure 4)”. Page 648, left column, last sentence, the text “In general, ST11 KPC-2-producing hvKP strains harbored more antibioticresistant genes and less virulence factors when compared with the non-ST11 KPC-2-producing hvKP (Figure 5)” should read “In general, ST11 KPC-2-producing hvKP strains harbored more antibioticresistant genes and less virulence factors when compared with the non-ST11 KPC-2-producing hvKP (Figure 6)”. Page 648, right column, first sentence, the text “Among the 15 KPC-2-producing hvKP strains, 4 strains (KP35, KP37, KP43, and KP44) shared >75% similarity and classified into one cluster (Figure 3), while the remaining 11 strains exhibited distinct genetic relationship” should read “Among the 15 KPC-2-producing hvKP strains, 4 strains (KP35, KP37, KP43, and KP44) shared >75% similarity and classified into one cluster (Figure 4), while the remaining 11 strains exhibited distinct genetic relationship”. Page 649, left column, second paragraph, first sentence, the text “Consistent with the epidemiology trend of K. pneumoniae meningitis in some Asian countries especially in Taiwan,3,4” should read “Consistent with the epidemiology trend of K. pneumoniae meningitis in Taiwan,3,4”. The authors apologize for these errors.

We report the clonal spread and evolution of high-risk Pseudomonas aeruginosa sequence type 463 co-producing KPC-2 and AFM-1 carbapenemases isolated from hospital patients in China during 2020–2022. Those strains pose a substantial public health threat and surveillance and stricter infection-control measures are essential to prevent further infections.

ABSTRACT: Objectives: The emergence and spread of colistin resistance in carbapenem-resistant Enterobacteriaceae pose a serious threat to human and animal health. This work aimed to characterise the genetic features of antimicrobial resistance of the carbapenem- and colistin-resistant Enterobacter kobei strain SCLZS19, isolated from hospital sewage, by using whole genome sequencing. Methods: Antimicrobial susceptibility tests were performed using the disk diffusion method. Whole genome sequencing of SCLZS19 was carried out on the HiSeq 2000 combined with PacBio RSII platforms. Sequence type, plasmid incompatibility types, resistance genes, and insertion elements were identified using multilocus sequence typing, PlasmidFinder, ResFinder, and ISfinder, respectively. Conjugation assays were performed using both broth- and filter-based methods with the azide-resistant Escherichia coli J53 as the recipient. The function of the mcr-9-like variant was determined by gene cloning. Results: E. kobei SCLZS19 had a 4 862 177-bp circular chromosome and nine circular plasmids ranging in size from 4120 bp to 282 472 bp. It carried 11 antibiotic resistance genes, and 10 of them were located on plasmids. The colistin resistance gene mcr-10 was located on a 118 766-bp non-transferable IncF (Y3:A-:B-) plasmid. The carbapenemase gene blaKPC-2 was carried by a self-transmissible IncP6 plasmid, which is epidemic in China. In addition, SCLZS19 also carried an mcr-9-like variant on a IncHI2 (ST1) plasmid. The cloning assay showed that the mcr-9-like variant did not mediate colistin resistance in E. coli DH5α. Conclusion: The findings highlight that carbapenem- and colistin-resistant Enterobacterales from water environments may serve as a reservoir for clinically significant antibiotic resistance genes, and continuous surveillance is required.

ABSTRACT: Objectives: We isolated a strain of Enterobacter hormaechei, ECC2783, co-harbouring blaNDM-1, blaKPC-2 and mcr-9 plasmids from a bloodstream infection and investigated its biological features. Methods: The presence of carbapenemase genes and mcr-9 was confirmed by polymerase chain reaction amplification. Whole genome sequencing and genomic analysis were performed on ECC2783. Experiments assessing the conjugation and stability of plasmids carrying the carbapenemase gene were performed. We also performed a colistin resistance induction experiment and studied the fitness cost of transconjugants. Results: ECC2783 has an extensive drug resistance phenotype. Multilocus sequence typing analysis results showed that ECC2783 belongs to sequence type 93. Bioinformatics analysis confirmed that ECC2783 has four plasmids, of which pECC2783_a, carrying mcr-9, is the IncHI2 type, and pECC2783_c, carrying blaNDM-1, is the IncX3 type. pECC2783_d, carrying blaKPC-2, is an unclassified type. We successfully obtained two transconjugants (J53/ECC2783_1, carrying blaNDM-1, and J53/ECC2783_2, carrying blaKPC-2 and blaNDM-1). There was no statistically significant difference in the relative growth rate between J53 and J53/ECC2783_2. Conclusion: For the first time, we isolated carbapenem-resistant E. hormaechei plasmids co-harbouring blaNDM-1, blaKPC-2, and mcr-9 from a patient with a blood stream infection. This isolate has a survival advantage in a hospital environment, and its clinical monitoring should be strengthened.

BackgroundThe escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably β-lactams, constitutes a formidable challenge for healthcare and global public health management.MethodsThis research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features.ResultsThe study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments.ConclusionIn summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.

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]

Aim of this study was to explore molecular characteristics and resistance mechanisms of carbapenem-resistant Raoultella ornithinolytica (CR-ROR) isolated from patients in a hospital in China. Three CR-ROR strains were collected and bacterial identification was done by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) Vitek-MS and by digital DDH analysis. VITEK 2 compact system and Kirby-Bauer (K–B) disk diffusion were used for antimicrobial susceptibility testing. Whole genome sequencing was carried out using the Illumina platform NovaSeq sequencer. Abricate software was used for the prediction of antibiotic resistance genes of three CR-ROR strains. The phylogenetic tree was constructed through genome SNPs to investigate the genetic relationship of three CR-ROR strains. Three CR-ROR (WF1357, WF2441, and WF3367) strains were collected in this study. Two strains were isolated from neurosurgery (WF1357 and WF2441), and one was isolated from pulmonology department (WF3367). All strains harboured multiple antibiotic resistance genes. Two strains (WF1357, WF2441) carried the bla NDM-1 gene, one of the strains (WF3367) carried the bla KPC-2 gene. Three CR-RORs were resistant to different antimicrobial agents including carbapenems. The three CR-ROR strains collected in this study and 51 CR-ROR strain genomes downloaded from NCBI, were divided into six evolutionary groups (A-F). In this study, three CR-ROR strains were found to have a higher level of resistance to antibacterial agents and carried multiple antibiotic resistance genes. The CR-ROR strains carrying multiple antibacterial resistant genes require the stringent monitoring to avoid the spread of multidrug-resistant bacterial strains. [ABSTRACT FROM AUTHOR]

Background: Carbapenemase is the predominant enzyme in the mechanism leading to Enterobacterales resistance to carbapenems, and the rapid spread of the blaKPC gene is a major public health concern. Here, we describe a carbapenem-resistant Proteus mirabilis strain XH983, which harbored a blaKPC-2-producing IncN plasmid, isolated from a bloodstream infection. Methods: Whole-genome sequencing and bioinformatics analysis were performed to assess the genetic environment of P. mirabilis XH983. Conjugation and transfer experiments were performed and the corresponding strains were confirmed by antimicrobial susceptibility testing. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant P. mirabilis isolates worldwide. Results: P. mirabilis XH983 was isolated from the blood of a patient in Hangzhou, China. The genome of XH983 contained one 4128,916 bp circular chromosome and one 24,225 bp IncN plasmid harboring blaKPC-2. P. mirabilis XH983 had multiple resistance genes, conferring resistance to aminoglycosides [aph(3')-Ia, aph(3'')-Ib, aph(6)-Id, aac(3)-IId, aadA5, aadA1], β-lactams (blaKPC-2, blaTEM-1B), phenicol (cat, catA1), sulphonamide/trimethoprim (drfA1, drfA17, sul1, sul2) and tetracycline [tet(J)]. The phylogenetic tree showed that XH983 was present in a cluster of 30 isolates, all of which carried blaKPC-2 and most of them came from the same hospital as XH983, indicating the clonal spread of the cluster. Conclusion: We characterized carbapenem-resistant P. mirabilis clinical isolate XH983. The genome sequence of P. mirabilis XH983 provides information about resistance mechanisms of P. mirabilis carrying the blaKPC-2 plasmid and the potential spread of blaKPC-2.

Xianhong He, Liwei Xu, Hangdong Dai, Minxia Ge, Jufang Zhu, Hangyu Fu, Shuilong Zhu, Jiayu Shao Department of Clinical Laboratory, The Third People’s Hospital of Xiaoshan, Hangzhou, People’s Republic of ChinaCorrespondence: Jiayu Shao; Shuilong Zhu, Department of Clinical Laboratory, The Third People’s Hospital of Xiaoshan, 76 Zhishan North Road, Hangzhou, Zhejiang, People’s Republic of China, Tel +86 18505812918 ; +86 18057155360, Email 583744598@qq.com; zslong1976@163.comBackground: The transmission of carbapenem-resistant Enterobacterales pose a significant threat to global public health, which weakens the effectiveness of most antimicrobial agents. The aim of this study is to present the genomic characteristics of a multidrug-resistant Escherichia coli, which contains both blaKPC-2 and blaCTX-M-15 genes, discovered from a respiratory infection in China.Methods: The antimicrobial susceptibility of E. coli isolate 488 was measured by using the broth microdilution method. The Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms were applied to determine the whole-genome sequence of this isolate. De novo assembly of short Illumina reads and long MinION reads were performed by Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes and plasmid replicon types were determined using the genome sequencing data. Additionally, a pairwise core genome single nucleotide polymorphism (cgSNP) comparison between E. coli 488 and all ST648 E. coli strains retrieved from NCBI GenBank database were conducted using the BacWGSTdb 2.0 server.Results: E. coli 488 was resistant to aztreonam, levofloxacin, cefepime, fosfomycin, amikacin, imipenem, cefotaxime, and meropenem. The complete genome sequence of E. coli 488 (belong to ST648) is made up of eleven contigs totaling 5,573,915 bp, including one chromosome and ten plasmids. Eight antimicrobial resistance genes were identified, including blaKPC-2 located in a 46,161 bp IncI1-type plasmid and the blaCTX-M-15 gene situated in the chromosome. Other two E. coli S617-2 and R616-1 isolates, recovered from China in 2018, are the closest relatives of E. coli 488, with only 52 SNPs difference. The genome also contains at least 57 genomic islands and several IS elements.Conclusion: Our study reveals the first ST648 E. coli isolate containing both blaKPC-2 and blaCTX-M-15 in China. These results could provide valuable insights into the genetic characteristics, antimicrobial resistance mechanisms, and transmission dynamics of carbapenem-resistant Enterobacterales in clinical settings.Keywords: blaKPC-2, blaCTX-M-15, whole genome sequencing, multidrug-resistant, Escherichia coli

Fang Rong,1,2,&ast; Ziyi Liu,3,4,&ast; Pengbin Yang,3,4 Feng Wu,5 Yu Sun,5 Xuewei Sun,5 Jun Zhou5 1Department of General Practice, The Affiliated Hospital of Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China; 2Graduate School Department of Dalian Medical University, Dalian, Liaoning, People’s Republic of China; 3Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China; 4Institute of Comparative Medicine, Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China; 5Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou, Jiangsu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jun Zhou, Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Yangzhou University, Yangzhou University, 368 Hanjiang Middle Road, Yangzhou, Jiangsu, 225009, People’s Republic of China, Email zj662121@163.comObjective: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged and spread worldwide. It can usually cause a serious threat complicating treatment options in clinical settings. However, treatment options are limited. The present study investigates the prevalence and genetic characteristics of blaNDM-1 and blaKPC-2 co-harboring clinical isolates of Klebsiella pneumoniae.Methods: In this study, Multiplex polymerase chain reaction (PCR) was performed to detect the carbapenem-resistant genes, and the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of antibacterial drugs. The transferability of carbapenem-resistant phenotypes was examined using filter mating assays. Overall, we used Illumina sequencing to evaluate the epidemiological and molecular characteristics of blaNDM-1 and blaKPC-2 (genes encoding carbapenemase) co-occurrence in CRKP strains.Results: All strains exhibited resistance to carbapenems and other antibiotics. However, they were still susceptible to polymyxin E. Among them, 18 isolates were positive for blaKPC-2, blaNDM-1, and multiple virulence determinants, such as genes encoding the virulence factor aerobactin, yersiniabactin, and the regulator of the mucoid phenotype (rmpA and rmpA2). Whole genome sequencing revealed that the 18 CRKP strains belonged to ST11 and capsular serotype KL64, and could be grouped into two evolutionary branches. Furthermore, these strains displayed hypervirulence potential since all of them carried pLVPK-like plasmid.Conclusion: These findings suggested that ST11-KL64 CRKP strains are major threats in terms of nosocomial infections in this hospital. Hence, new strategies should be urgently developed to monitor, diagnose, and treat this high-risk CRKP clone.Keywords: Klebsiella pneumoniae, carbapenem resistance, illumina sequencing, blaKPC-2, blaNDM-1

ABSTRACT: Objectives: The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. Methods: Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. Results: Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. Conclusion: Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to β-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.

Objectives: This study aimed to elucidate resistance to carbapenems and fluoroquinolones, the transmission mechanism of blaKPC-2, and the virulence characteristics of a Pseudomonas aeruginosa strain (TL3773) isolated in East China. Methods: The virulence and resistance mechanisms of TL3773 were investigated by whole genome sequencing (WGS), comparative genomic analysis, conjugation experiments, and virulence assays. Results: This study isolated carbapenem-resistant P. aeruginosa from blood resistant to carbapenems. The patient's clinical data showed poor prognosis compounded by multiple sites of infection. WGS showed that TL3773 carried aph (3′)-IIb, blaPAO, blaOXA-486, fosA, catB7, and two crpP resistance genes on chromosome, and the carbapenem resistance gene blaKPC-2 on plasmid. We identified a novel crpP gene named TL3773-crpP2. Cloning experiments proved that TL3773-crpP2 was not the primary cause of fluoroquinolone resistance in TL3773. GyrA and ParC mutations may confer fluoroquinolone resistance. The blaKPC-2 genetic environment was IS26-TnpR-ISKpn27-blaKPC-2-ISKpn6-IS26-Tn3-IS26, potentially mediating the transmission of blaKPC-2 in P. aeruginosa. The overall virulence of TL3773 was lower than that of PAO1. However, the pyocyanin and biofilm formation of TL3773 was higher than that of PAO1. WGS further indicated that TL3773 was less virulent than PAO1. Phylogenetic analysis showed that TL3773 was most similar to the P. aeruginosa isolate ZYPA29 from Hangzhou, China. These observations further indicate that ST463 P. aeruginosa is rapidly spreading. Conclusions: The threat of ST463 P. aeruginosa harbouring blaKPC-2 is emergent and may pose a threat to human health. More extensive surveillance and effective actions are urgently needed to control its further spread.

ABSTRACT: Objectives: Pseudomonas aeruginosa is a widely distributed opportunistic pathogen that can cause a variety of infections. The emergence of multidrug-resistant P. aeruginosa has complicated clinical treatment. Here, we report the genome sequence of a P. aeruginosa strain co-carrying blaKPC-2, blaOXA-246, and blaOXA-486. Genetic and phylogenetic characteristics of this strain were investigated. Methods: The Illumina NovaSeq 6000 platform was used to sequence the strain's genome. MLST v.2.22.1 was used to analyse the multilocus sequence typing (MLST). Antimicrobial resistance genes (ARGs) and virulence genes were identified using ABRicate v.1.0.1. Phylogenetic relationship analysis was performed using snippy. Results: The genomic sequence of P. aeruginosa PA23 consists of 229 contigs with a combined length of 7 193 462 bp. It was determined that PA23 belonged to ST463 and serotype O4, which is associated with a high-risk clone. The strain was resistant to almost all antibiotics tested, except colistin. The genome contained 23 antimicrobial resistance genes, including blaKPC-2, blaOXA-246, and blaOXA-486. A total of five P. aeruginosa strains both carrying blaKPC-2, blaOXA-246, and blaOXA-486 can be retrieved from the NCBI database. All of these strains are ST463 and serotype O4. With the exception of one strain, the other strains were spread across two neighbouring Chinese provinces and were clonal related. Conclusions: In conclusion, we reported the genome sequence of a multidrug-resistant P. aeruginosa ST463 strain containing 23 ARGs in China. This clone has the potential to become a dominant endemic clone in eastern China. To prevent clonal dissemination, continuous surveillance is necessary in the future.

An extensively drug-resistant (XDR) Escherichia coli strain 258E was isolated from an anal swab sample of a chicken farm of Anhui Province in China. Genomic analyses indicated that the strain 258E harbors an incompatibility group N (IncN) plasmid pEC258-3, which co-produces blaCTX-M-3, blaKPC-2, blaTEM-1B, qnrS1, aac(6´)-Ib-cr, dfrA14, arr-3, and aac(6´)-Ib3. Multiple genome arrangement analyses indicated that pEC258-3 is highly homologous with pCRKP-1-KPC discovered in Klebsiella pneumoniae from a patient. Furthermore, conjugation experiments proved that plasmid pEC258-3 can be transferred horizontally and may pose a significant potential threat in animals, community and hospital settings.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales.

Mengzhu Hou,1,&ast; Nan Chen,2,&ast; Lili Dong,3,&ast; 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&ast;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

ABSTRACT: Objectives: Klebsiella pneumoniae carbapenemase (KPC)–producing sequence type (ST) 463 Pseudomonas aeruginosa are increasingly prevalent in China. This study aims to investigate how blaKPC-2 is acquired in ST463 P. aeruginosa during antimicrobial therapy. Methods: Two extensively drug-resistant P. aeruginosa strains, B1122 and U1121, were respectively isolated from blood and urine of a patient during carbapenem therapy. Whole-genome sequences were obtained, and minimum inhibitory concentrations (MICs) were determined. Plasmid transferability and stability were examined. Bacterial growth kinetics, biofilm formation, and virulence level was assessed. Results: U1121 and B1122 were only susceptible to amikacin and intermediately susceptible to colistin. They were isogenic ST463 P. aeruginosa strains and shared the same chromosome-encoded resistance genes, including blaAFM-1. This is the first report of chromosomal integration of blaAFM-1 in P. aeruginosa mediated by ISCR29. pU1121 and pB1122, which shared almost identical backbone, were the sole plasmids in U1121 and B1122, respectively, differing by an insertion region containing two copies of blaKPC-2 genes observed on pU1121. Sequence alignment revealed that pU1121 might evolve in vivo from pB1122 via IS26-mediated continuous genetic rearrangement in response to selective challenge from carbapenem. pU1121 was not self-transmissible and could be stably maintained in the host in the absence of antibiotic. Both U1121 and B1122 were hypervirulent, and no differences on virulence were recorded between them. However, U1121 exhibited significant impaired growth in comparison with B1122. Conclusion: ST463 P. aeruginosa can capture blaKPC-2 through horizontal transfer of insertion sequence under antibiotic selection pressure, which does decrease the fitness but does not impair the virulence of the ancestor.

Qin Ni,1,2,&ast; Xingwei Yao,3,&ast; Jinhui Li,2,&ast; Jinghan Ma,3,&ast; Kaiying Wang,2 Xiong Liu,2 Peihan Li,2 Lang Yang,2 Peng Li,2 Shenlong Li1,2 1Epidemiology, Zhengzhou University, Zhengzhou, People’s Republic of China; 2Biosafety, Chinese PLA Center for Disease Control and Prevention, Beijing, People’s Republic of China; 3Medical Clinical Laboratory, Dong Zhimen Hospital Beijing University of Chinese Medicine, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Peng Li; Shenlong Li, Email jiekenlee@126.com; lishenlong@sohu.comObjective: Klebsiella pneumoniae is a common multidrug-resistant pathogen that jeopardizes the health of hospitalized patients. We aimed to study the phenotypic and genotypic characteristics of carbapenem-resistant K. pneumoniae (CRKP) isolates from a hospital in Beijing.Methods: Twenty-four CRKP clinical isolates were collected within a half-year to investigate antimicrobial resistance and genomic characteristics. Illumina and Nanopore sequencing were performed to assemble and annotate genomes.Results: All strains were multi-drug resistant. Twenty-two strains carried the blaKPC-2 gene and two harbored blaNDM-5. Multilocus sequence type(MLST) analysis identified five sequence types; most isolates belonged to ST11. Three strains were isolated from the same patient; each carried a different plasmid replicon, either IncFII (pHN7A8), IncX, or IncFIB (K).Conclusion: This study furthers the understanding of CRKP antimicrobial resistance genotypes, and may facilitate the control of nosocomial infections caused by antimicrobial-resistant pathogens.Keywords: Klebsiella pneumoniae, drug resistance, carbapenemase, ST11, blaKPC-2

Chunlei Chen,1,&ast; Qingmiao Shi,1,&ast; Xinjun Hu,2 Xiaojing Liu,1,3 Yi Liu,1 Ruishan Liu1 1Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 2Department of Infectious Diseases, The First Affiliated Hospital, College of Clinical Medicine, Henan University of Science and Technology, Luoyang, People’s Republic of China; 3Department of Structure and Morphology, Jinan Microecological Biomedicine Shandong Laboratory, Jinan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ruishan Liu, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang, 310000, People’s Republic of China, Tel +86 571 872 364 23, Fax +86 571 872 364 21, Email 924172261@qq.comPurpose: Beta-lactamase-producing Klebsiella pneumoniae is common in the clinic, but research associated with the co-existence of KPC-2, LAP-2, and CTX-M-65 in K. pneumoniae is still rare. In this study, the phenotypic and genetic characteristics of a multidrug-resistant K. pneumoniae strain SJ25 co-harboring blaKPC-2, blaLAP-2, and blaCTX-M-65 with rare ST1469 were investigated.Methods and Results: Antimicrobial susceptibility testing revealed that strain SJ25 was resistant to various common antibiotics, except ciprofloxacin, fosfomycin, colistin, and tigecycline. Whole-genome analysis revealed that strain SJ25 carries a variety of antimicrobial resistance genes and virulence determinants. Plasmid analysis confirmed that the blaKPC-2 and blaCTX-M-65 genes were located on an ~136 kb transferrable IncFII/IncR plasmid and that blaLAP-2 was located on an untypeable plasmid.Conclusion: Our findings emphasized the need for continuous surveillance of β-lactamase-bearing K. pneumoniae in the clinic to control potential dissemination and outbreak.Keywords: Klebsiella pneumoniae, multidrug-resistant, blaKPC-2, blaLAP-2, blaCTX-M-65

Carbapenemase is the predominant enzyme in the mechanism leading to Enterobacterales resistance to carbapenems, and the rapid spread of the bla KPC gene is a major public health concern. Here, we describe a carbapenem-resistant Proteus mirabilis strain XH983, which harbored a bla KPC-2 -producing IncN plasmid, isolated from a bloodstream infection. Whole-genome sequencing and bioinformatics analysis were performed to assess the genetic environment of P. mirabilis XH983. Conjugation and transfer experiments were performed and the corresponding strains were confirmed by antimicrobial susceptibility testing. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant P. mirabilis isolates worldwide. P. mirabilis XH983 was isolated from the blood of a patient in Hangzhou, China. The genome of XH983 contained one 4128,916 bp circular chromosome and one 24,225 bp IncN plasmid harboring bla KPC-2. P. mirabilis XH983 had multiple resistance genes, conferring resistance to aminoglycosides [ aph(3')-Ia , aph(3'')-Ib , aph(6)-Id , aac(3)-IId , aadA5 , aadA1 ], β-lactams (bla KPC-2 , bla TEM-1B), phenicol (cat , catA1), sulphonamide/trimethoprim (drfA1 , drfA17 , sul1 , sul2) and tetracycline [ tet(J) ]. The phylogenetic tree showed that XH983 was present in a cluster of 30 isolates, all of which carried bla KPC-2 and most of them came from the same hospital as XH983, indicating the clonal spread of the cluster. We characterized carbapenem-resistant P. mirabilis clinical isolate XH983. The genome sequence of P. mirabilis XH983 provides information about resistance mechanisms of P. mirabilis carrying the bla KPC-2 plasmid and the potential spread of bla KPC-2. [ABSTRACT FROM AUTHOR]

Purpose: To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9. Methods: MALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL. Results: Citrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3-, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacE-1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcAkorC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34\u00B0C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang. Conclusion: We found C. freundii carrying blaIMP-4, blaNDM-1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs). Methods: WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism. Results: High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66 × 101. Conclusions: Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC–2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.

IntroductionThe raise of multi-drug resistant bacteria involving carbapenem, colistin, or tigecycline resistance constitutes a threat to public health, which partly results from the transmission of corresponding mobile resistance genes, such as blaKPC and blaNDM for carbapenem, mcr for colistin, and tmexCD-toprJ gene cluster for tigecycline. Herein, we described the emergence of an Aeromonas veronii strain HD6454 co-harboring blaKPC–2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster from hospital sewage.MethodsWhole genome sequencing (WGS) was used to determine the genome sequence of HD6454, and the detailed genomic analysis of genetic elements or regions carrying key antimicrobial resistance genes (ARGs) from HD6454 were performed. Cloning experiment was conducted to confirm the function of key ARGs in mediating antimicrobial resistance. Conjugation experiment was conducted to determine the mobility of the plasmid.ResultsThe results showed that this strain belonged to a novel sequence type (ST) variant ST1016, and carried 18 important ARGs. Among them, the blaKPC–2 was carried by non-self-transmissible IncP-6 plasmid, while tmexC3.2-tmexD3.3-toprJ1b gene cluster and mcr-3.17 were carried by integrative and mobilizable element (IME) or IME-related region in chromosome. The mcr-3.17, mcr-3.6, and mcr-3-like3 genes were further inferred to originate from IMEs of Aeromonas species. Additionally, for the first time, the mcr-3.17 was confirmed to confer low-level resistance to colistin under inducible expression, while tmexC3.2-tmexD3.3-toprJ1b gene cluster was confirmed to confer low-level resistance to tigecycline.DiscussionThis is the first report of a strain co-harboring blaKPC–2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster. Although the resistance and/or mobility of these ARGs are limited in this strain, the emergence of this multiple important ARGs-carrying strain deserves further attention.

Background: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) attracted extensive attention. Information on CRKP from hospital wastewater (HWW) is limited. The aims of this study were to investigate the genomic characteristics and to evaluate the survivability characteristics of 11 CRKP from HWW in a Chinese teaching hospital in Fujian province. Results: A total of 11 CRKP from HWW were recovered in this study. All CRKP from HWW were resistant to most antibiotics. Comparative genetic analysis demonstrated that all CRKP isolates were clustered into the three distinct phylogenetic clades and clade 2 and clade 3 were mixtures of samples collected from both HWW and clinical settings. Varieties of resistance genes, virulence genes and plasmid replicon types were detected in CRKP from HWW. In vitro transfer of blaKPC-2 was successful for 3 blaKPC-2-positive CRKP from HWW with high conjugation frequency. Our study demonstrated that the genetic environments of blaKPC−2 shared core structure with ISKpn27-blaKPC−2-ISKpn6. Group analysis showed that CRKP from HWW had a lower survivability in serum compared to clinical CRKP (p < 005); and CRKP from HWW had no significant difference in survivability in HWW compared to clinical CRKP (p > 005). Conclusions: We analyzed the genomic and survivability characteristics of CRKP from HWW in a Chinese teaching hospital. These genomes represent a significant addition of genomic data from the genus and could serve as a valuable resource for future genomic studies about CRKP from HWW. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: Enterobacter cloacae complex (ECC) has awakened interest recently because of its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence types (STs) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis, and phylogenetic studies of a Klebsiella pneumoniae carbapenemase (KPC) -producing multidrug-resistant (MDR) ECC isolate from Argentina. Methods: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I (Illumina, San Diego, CA). The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST. Results: The strain was identified as Enterobacter hormaechei, an important emerging human pathogen. No ST could be assigned; six of seven alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes, including blaKPC-2 in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria. Conclusion: Because of its MLST profile being so close to that of E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.

ABSTRACT: Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasingly reported worldwide and has posed a serious challenge for public health. Here we report the complete genome sequence of a multidrug-resistant (MDR) K. pneumoniae carrying one blaNDM-1 and two copies of blaKPC-2 genes isolated from a cerebrospinal fluid specimen in China. Methods: The minimal inhibitory concentrations (MICs) of 26 antimicrobial agents against K. pneumoniae strain KP46 were measured. The complete genome sequence of KP46 was determined using Illumina and Nanopore platforms. The derived short and long reads were assembled using Unicycler. Multilocus sequence typing (MLST), antimicrobial resistance genes, virulence genes, and plasmid replicons were predicted in silico using the BacWGSTdb server. The phylogenetic relationship between KP46 and 454 ST15 K. pneumoniae strains obtained from NCBI GenBank database was analysed based on a core genome MLST (cgMLST) strategy. Results: K. pneumoniae strain KP46 was resistant to all antimicrobial agents tested, except for tigecycline, colistin, cefiderocol, and fosfomycin. The genome sequence of KP46 belonged to sequence type 15 (ST15), which contained seven circularized contigs comprising 5 674 521 bp, including one chromosome and six plasmids. Serval antimicrobial resistance genes were identified, including a blaNDM-1 gene located in a 53 096 bp IncX3 plasmid, and two copies of blaKPC-2 gene located both in a 103 807 bp IncX6 and an 88 164 bp IncFII plasmid, respectively. The most closely related strain was another ST15 strain also isolated from China with five cgMLST loci differences. Conclusion: We reported the first complete genome sequence of a K. pneumoniae ST15 clinical isolate coharbouring blaNDM-1 and two copies of blaKPC-2 in China. This study will provide insight into the antimicrobial resistance mechanisms and phylogeny of carbapenem-resistant ST15 K. pneumoniae.

ABSTRACT: Objectives: This study investigated the underlying mechanism of the evolution of tigecycline resistance during treatment in a patient infected with Klebsiella pneumoniae harbouring blaKPC-2. Methods: A total of seven clonal K. pneumoniae strains were continuously isolated from a patient during hospitalisation. Antimicrobial resistance in the strains was determined by antimicrobial susceptibility testing. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to explore the homology of the isolates. Whole-genome shotgun (WGS) analysis and cloning experiments were used to investigate the underlying mechanism of the evolution of tigecycline resistance. Results: All of the isolates had a minimum inhibitory concentration (MIC) for tigecycline of 4 µg/mL, except strain FK6768 that had a MIC of 32 µg/mL. Carbapenem-resistant K. pneumoniae strains (FK6614, FK6768 and FK6809) were consecutively isolated from faeces at different times. Antimicrobial susceptibility testing indicated that tigecycline resistance increased in FK6768 and subsequently decreased in FK6809, which attracted our attention. WGS and further bioinformatics analysis showed a homology for the three faecal isolates of >99%. The blaKPC-2 carbapenemase gene and a tet(A) mutation were found in tigecycline-resistant isolate FK6768. Subsequent cloning experiments confirmed the contribution of a tet(A) variant to reduced tigecycline susceptibility. Conclusion: Here we report a K. pneumoniae isolate carrying both tet(A) mutation and the blaKPC-2 gene, which led to increased tigecycline resistance during tigecycline treatment. This is the first report describing tigecycline resistance of K. pneumoniae first increasing and subsequently decreasing in vivo.

ABSTRACT Generation of hybrid MDR plasmids accelerated the evolution and transmission of resistance genes. In this study, we characterized a blaKPC-2- and blaIMP-4-coharboring conjugative hybrid plasmid constituted of an IncHI5 plasmid-like region, an IncFII(Yp)/IncFIA plasmid-like region, and a KPN1344 chromosome-like region from a clinical ST852-KL18 Klebsiella quasipneumoniae strain. The blaIMP-4 gene was captured by a novel integron In1965, and the blaKPC-2 gene was located on a new non-Tn4401 group I NTEKPC element. Both blaKPC-2- and blaIMP-4-containing genetic architectures were distinguished from classical structures, highlighting the constant evolution of these genetic elements. IMPORTANCE The emergence of carbapenem-resistant Enterobacterales (CRE) that coexpress serine- and metallo-carbapenemases is a severe threat to the efficacy of ceftazidime-avibactam (CZA), which has been proven to be extremely effective against KPC-producing Enterobacterales strains. Our study described the cooccurrence of KPC-2, a serine β-lactamase, and IMP-4, a metallo-β-lactamase (MBL), on a conjugative hybrid plasmid from a clinical carbapenem-resistant K. quasipneumoniae strain, and it revealed an alternative route for IncHI5 plasmid to evolve by recombining with other plasmids to form a hybrid plasmid. Moreover, this hybrid plasmid can be transferred into other Klebsiella species and stably persist during passage. The propagation of two important carbapenemase genes with a new genetic background using well-evolved plasmids in the clinical setting promotes the emergence of superbugs that require careful monitoring.

Gram-negative bacteria containing three different carbapenemases are extremely rare. Klebsiella pneumoniae (N22-925) with KPC-2, NDM-1, and OXA-48 was obtained from a Canadian patient with recent hospitalization in Romania. Short and long read whole genome sequencing showed that the blaKPC-2 was situated on a 214 kb IncFIB(K)/IncFII(K) plasmid, the blaNDM-1 on a 104 kb IncFIB (pQil)/IncFII(K) plasmid, and the blaOXA-48 on a 64 kb IncL plasmid. These plasmids were conjugated to Escherichia coli J53. N22-925 belonged to a unique ST147 cluster that is likely endemic in Romania. This case emphasizes the need for rapid carbapenemase screening in patients from endemic regions. We described the first complete genome sequence of a K. pneumoniae isolate with three different carbapenemases, providing a reference for future studies on this rarely reported occurrence. [ABSTRACT FROM AUTHOR]

Background: Klebsiella michiganensis is an emerging human pathogen that causes nosocomial infections. Its prevalence and spread in the environment should not be ignored. This study identified and characterized Klebsiella michiganensis co-harboring bla KPC-2 and TmexCD2-ToprJ2 in hospital wastewater samples., Methods: Twelve K. michiganensis strains were isolated from wastewater samples collected at a tertiary hospital in Beijing, China. The genomic characteristics of K. michiganensis strains were analyzed using whole-genome sequences, providing information on the comparison between the genome of K. michiganensis strains and the reference genome, antibiotic resistance genes (ARGs), virulence genes, secretion systems, and mobile genetic elements (plasmids, insertion sequences [ISs], and prophages)., Results: Genome analysis showed that the twelve multi-drug resistant (MDR) strains carried a variety of ARGs and virulence genes, as well as four macromolecular secretion systems (T1SS, T2SS, T5aSS, T5bSS, and T4aP). The genetic environments of both the TmexCD2-ToprJ2 gene cluster and bla KPC-2 gene contained ISs. The plasmids carrying TmexCD2-ToprJ2 gene cluster of nine strains in clade 1 and two strains in clade 2 were annotated as IncR plasmid and rep&#95;cluster&#95;1254 type, respectively. The plasmids carrying bla KPC-2 in 10 strains in clade 1 were identified as IncU, and the plasmids carrying bla KPC-2 in the k11 and k12 strains in clade 2 were IncU and IncX6. The phylogenetic tree and heatmap revealed that the secretion system of type VI (T6SSi) existed in 10 strains in clade 1, and Type IV (T4SS) only existed in the k11 strain in clade 2. In addition, K. michiganensis strains carried 13 plasmids, 14 ISs, and 138 prophages., Conclusion: In this study, the whole genome sequencing demonstrated the diversity of K. michiganensis genome despite 12 K. michiganensis strains from a hospital wastewater, which lays the foundation for further genetic research and drug resistance gene transmission., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Long et al.)

Yingying Du,1,&ast; Shikui Mu,1,&ast; Yan Liu,2 Yinghua Yuan,2 Yunlou Zhu,2 Lijie Ma,2 Qixing Wang,2 Zhengfang Zhu,2 Yuhao Liu,1 Sheng Wang1 1Department of Critical Care Medicine, Shanghai Tenth People’s Hospital, Tongji University, School of Medicine, Shanghai, 200072, People’s Republic of China; 2Department of Clinical Microbiology, Shanghai Tenth People’s Hospital, Tongji University, School of Medicine, Shanghai, 200072, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Sheng Wang; Yuhao LiuDepartment of Critical Care Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 200072, People’s Republic of ChinaTel +86-21-6630 7153; +86-21-6630 7162Fax +86-21-6630 3983Email wangsheng@tongji.edu.cn; lyh-7906@163.comPurpose: This study retrospectively analyzed the genome characteristics of blaKPC-2 in multidrug-resistant Klebsiella pneumoniae collected from the ICU of a teaching hospital in Shanghai, China.Methods: From February 2018 to December 2019, 36 strains of multidrug-resistant Klebsiella pneumoniae were collected from the bronchoalveolar lavage fluid of critically ill patients. The genome of all isolates was obtained through the Illumina sequence, and single nucleotide polymorphisms of the blaKPC-2 gene were analyzed to explore blaKPC-2’s evolutionary characteristics. Different strains’ genetic relationships and homology were studied by constructing an evolutionary tree on a single copy orthologue. Pacbio combined Illumina sequence was conducted to evaluate the structure and potential mobility of drug-resistant plasmids of the strain KP-s26.Results: The distribution of resistance and virulence genes had little difference, but most strains had significant differences in the plasmid-encoded region. Most strains (31/36) carried the carbapenemase gene blaKPC-2, with no single nucleotide polymorphism in different strains. Extended-spectrum β-lactamase resistance genes, such as blaCTX-M and blaSHV, were found in the isolates, but no metallo-β-lactamases were detected. All strains with blaKPC-2 coexisted with chromosomal-associated fosfomycin resistance genes fosA6, and the coexistence of blaKPC-2 and blaCTX variants (blaCTX-M-15, blaCTX-M-65, and blaCTX-M-27) was also detected in 29/31 strains. The isolate KP-s26 carried five circular plasmids. pA and pB were conjugate plasmids, as they carried drug resistance genes and contained a complete IV secretion system.Conclusion: The blaKPC-2 carbapenemase gene is relatively conservative in the process of evolution; drug-resistant plasmids containing conjugated transfer elements contribute to the spreading of drug resistance. The coexistence of blaKPC-2 with fosA6 or blaCTX-M variants was associated with increased fosfomycin resistance and broad-spectrum β-lactam resistance, respectively.Clinical Trials Registration: Clinical Trials.gov Identifier: NCT03950544Keywords: Klebsiella pneumoniae, blaKPC-2, whole-genome sequencing, single nucleotide polymorphism, drug-resistant plasmids

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.

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has received considerable attention. Typically, the genetic elements that confer virulence are harbored by nonconjugative plasmids. In this study, we report a CR-hvKP strain, CY814036, of high-risk sequence type 25 (ST25) and the K2 serotype, which is uncommon among K. pneumoniae isolates but caused serious lung infection in a tertiary teaching hospital in China. Whole-genome sequencing (WGS) revealed a rare conjugative plasmid, pCY814036-iucA, carrying a virulence-associated iuc operon (iucABCD-iutA) coding for aerobactin and determinants of multidrug resistance (MDR), coexisting with a conjugative blaKPC-2-bearing plasmid, pCY814036-KPC2, in the same strain. A conjugation assay showed that pCY814036-iucA and pCY814036-KPC2 could be efficiently cotransmitted from CY814036 to Escherichia coli EC600. Further phenotypic investigation, including antimicrobial susceptibility tests, serum resistance assays, and mouse infection models, confirmed that pCY814036-iucA was capable of cotransferring multidrug resistance and hypervirulence features to the recipient. pCY814036-KPC2 also conferred resistance to antibiotics, including β-lactams and aminoglycosides. Overall, the rare coexistence of a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in a K. pneumoniae isolate offers a possible mechanism for the formation of CR-hvKP strains and the potential to significantly accelerate the propagation of high-risk phenotypes. IMPORTANCE The increased reporting of carbapenem-resistant hypervirulent K. pneumoniae is considered a worrisome concern to human health care and has restricted the choice of effective antibiotics for clinical treatment. Moreover, virulence plasmids with complete conjugation modules have been identified, which evolved via homologous recombination. Here, we characterize an ST25 CR-hvKP strain, CY814036, harboring both a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in China. This study highlights that the cotransmission of drug resistance and virulence plasmids increases therapeutic difficulties and worsens clinical prognoses. Also, active surveillance of the conjugative MDR-virulence plasmid is necessary.

ABSTRACT: Objective: The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. Methods: Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. Results: PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. Conclusion: This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.