Search

Yingying Du,1,* Shikui Mu,1,* 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*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

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.

Objectives The aim of this study was to investigate the potential for transmission and heritability of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains. Methods Potential for transmission of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains was tested in three conjugation models, namely filter-mating conjugation in laboratory conditions, a meat product model and the gastrointestinal (GI) tract of rats. Plasmid stability in Enterobacteriaceae strains was also determined. Results We demonstrated that plasmids carrying a carbapenemase gene (blaKPC-2 or blaNDM-1) could be efficiently conjugated to strains carrying the mcr-1 gene and vice versa, and that these plasmids could stably co-exist in clinical Enterobacteriaceae strains. These findings suggest that Enterobacteriaceae can readily acquire phenotypic resistance to both carbapenems and colistin in natural environments such as food products and the GI tract of human and animals. Conclusion Gene transfer events are common among members of the Enterobacteriaceae and serve as a key mechanism facilitating adaptation to new environments. Development of innovative strategies and surveillance measures to curtail the dissemination of multidrug resistance plasmids is necessary. Transmission and stable inheritance of these two types of plasmids would lead to the emergence of multidrug-resistant pathogens that are resistant to all currently available last-line antibiotics for Gram-negative bacterial infections.

Jun Li,1 Ziyan Huang,1 Mengli Tang,1 Changhang Min,1 Fengjun Xia,1 Yongmei Hu,1 Haichen Wang,1 Haijian Zhou,2 Mingxiang Zou1 1Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, People’s Republic of China; 2State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, People’s Republic of ChinaCorrespondence: Mingxiang ZouDepartment of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, People’s Republic of ChinaTel +86 13907496278Email zoumingxiang@csu.edu.cnBackground: Carbapenem-resistant Enterobacterales (CRE) are rapidly increasing worldwide in last two decades and lead few antibiotics for treatment. The molecular epidemiology of CRE in China was investigated to provide basis for clinical rational use of antibiotics and prevent its spread.Methods: All CRE isolates in this study were collected from 11 hospitals from October 2015 to July 2018. The isolates were subjected to antimicrobial susceptibility tests, PCR molecular identification, pulsed-field gel electrophoresis, and multilocus sequence typing.Results: Among the 399 CRE isolates, 51.6% (206/399) harbored carbapenemase genes. Three carbapenemase genes were detected, namely blaKPC-2, blaNDM-1, and blaIMP at rates of 29.8% (119/399), 17.5% (70/399), and 4.0% (16/399), respectively. In Klebsiella pneumoniae (350) and Escherichia coli (26), blaKPC-2 (33.4%, 117/350) and blaNDM-1 (61.5%, 16/26) were the predominant genes. The most common genes in the CRE isolates were blaKPC (85.5%) and blaNDM-1 (76.5%) from adults and children, respectively. Particularly, ST11 K. pneumoniae with blaKPC-2 harbored by IncFII plasmids were distributed in both general and primary hospitals, suggesting a clonal transmission pattern at these sites. In addition, the clonal distribution of ST2407 K. pneumoniae with blaNDM-1 located on IncX3 plasmids and blaIMP-38-positive ST307 K. pneumoniae were detected in a children’s hospital.Conclusion: The distribution of carbapenemase genes differed among strains and age groups. Multiple carbapenemase genes in the CRE strains were clonally disseminated in the tested regions mediated by multiple plasmids. Therefore, CRE monitoring should be increased and measures should be adopted to prevent its transmission.Keywords: carbapenem-resistant Enterobacterales, blaKPC-2, blaNDM-1, blaIMP, IncFII, ST11

Hazrat Bilal,1 Tayyab Ur Rehman,2 Muhammad Asif Khan,2 Fareeha Hameed,2 Zhang Gao Jian,1 Jianxiong Han,1 Xingyuan Yang1 1Faculty of Health Sciences, Institute of Physical Sciences and Information Technology, Anhui University, Hefei, People’s Republic of China; 2Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, PakistanCorrespondence: Xingyuan YangFaculty of Health Sciences, Institute of Physical Science and Information Technology, Anhui University, No, 111 Jiulong Road, Hefei, Anhui, 230601, People’s Republic of ChinaEmail xingyuan@ahu.edu.cnPurpose: The multiple-drug resistant Escherichia coli are among the deadliest pathogens causing life-threatening infections. This study was planned to determine the molecular epidemiology of mcr-1, blaKPC-2, and blaNDM-1 harboring clinically isolated E. coli from Pakistan.Methods: In total, 545 strains of E. coli from clinical samples were collected from June 2018 to September 2019. All the isolates were screened for colistin-resistance, extended-spectrum-β-lactamases (ESBL), and carbapenemases through the micro-dilution method, Double-Disk-Synergy-Test (DDST), and Modified-Hodge-Test (MHT). The detection, sequence-typing, conjugal transfer, S1-PFGE, plasmid-replicon-typing, and southern-blotting for mcr, ESBL, and carbapenemase-encoding genes were performed.Findings: A total of four (0.73%) colistin-resistant strains carrying alongside mcr-1 and blaCTX-M-15 genes, three of these strains also had the blaTEM-1 gene. The presence of ESBL genes was detected in 139 (25.5%) isolates harboring blaCTXM-15 (74.82%), blaTEM (34.53%), blaSHV (28.06%) and blaOXA-1 (28.78%). In 129 carbapenemase-producers, 35.83% possessed blaNDM-1, 26.67% blaKPC-2, 8.3% blaOXA-48, 25% blaVIM-1, and 20.83% blaIMP-1 genes. The sequence typing revealed that mcr-1 harboring isolates belonged to ST405, ST117, and ST156. Fifty percent of blaKPC-2 and 48.83% of blaNDM-1 were found on ST131 and ST1196, respectively. Two rare types of STs, ST7584, and ST8671 were also identified in this study. The mcr-1 gene was located on Incl2 (60-kb) plasmid. The blaKPC-2 was present on (140-kb) IncH12, (100-kb) IncN, (90-kb) Incl1, while blaNDM-1 was located on (70-kb) IncFIIK, (140-kb) IncH12, (100-kb) IncN, (60-kb) IncA/C, and (45-kb) IncFII plasmids, which were successfully trans-conjugated. Among the plasmid types, the Incl1 carrying blaKPC-2, IncH12 harboring blaKPC-2 and blaNDM-1, and IncFIIK carrying blaNDM-1 were for the first time detected in Pakistan.Conclusion: The mcr-1, blaKPC-2, and blaNDM-1 genes finding in various clonal and plasmids types indicate that a substantial selection of the resistance genes had occurred in our clinical strains.Keywords: colistin-resistance, extended-spectrum-β-lactamases, carbapenemases, mcr1, blaKPC-2, blaNDM-1, plasmids, E. coli

Hongmao Liu,1– 3,* Hailong Lin,1– 3,* Zhewei Sun,2,3 Xinyi Zhu,1– 3 Xueya Zhang,1– 3 Qiaoling Li,1– 3 Junwan Lu,2,3 Xi Lin,2,3 Li Lin,1,3 Kewei Li,2,3 Mei Zhu,4 Qiyu Bao,2,3 Teng Xu,5 Yunliang Hu,1– 3 Hailin Zhang1,3 1The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, People’s Republic of China; 2Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, People’s Republic of China; 3Institute of Biomedical Informatics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, People’s Republic of China; 4Department of Clinical Laboratory, Zhejiang Hospital, Hangzhou, Zhejiang 310013, People’s Republic of China; 5Institute of Translational Medicine, Baotou Central Hospital, Baotou 014040, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yunliang Hu; Hailin ZhangThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, People’s Republic of ChinaTel/Fax +86-577-86699398; Tel +86-577-88002134Email huyunliang66@163.com; zhlwz97@hotmail.comBackground: This study was designed to characterize the dissemination mechanism and genetic context of Klebsiella pneumoniae carbapenemase (KPC) genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates.Methods: A retrospective analysis was performed on CRKP strains isolated from a teaching hospital of Wenzhou Medical University during 2015– 2017. Polymerase chain reaction (PCR)-based amplification and whole-genome sequencing (WGS) were used to analyze the genetic context of the blaKPC-2 gene. Conjugation experiments were performed to evaluate the transferability of blaKPC-2-bearing plasmids. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to investigate the clonal relatedness of blaKPC-2-producing strains.Results: The blaKPC-2 gene was identified from 13.61% (40/294) of clinical K. pneumoniae isolates. Three different sequence types (ST11, ST15 and ST656) and 5 PFGE subtypes (A to E) were classified among them. ST11 was the dominant sequence type (92.50%, 37/40). Plasmid-oriented antibiotic resistance genes, such as extended spectrum-β-lactamases (ESBLs) and other antimicrobial resistance genes, were also found in KPC-positive K. pneumoniae (KPC-Kp) isolates. Mapping PCR and genomic sequencing revealed that the blaKPC-2-bearing sequence regions, which are related to different mobile elements, including Tn1721- and IS26-based transposons, were mainly located in but not restricted to IncFII-like plasmids and were structurally divergent.Conclusion: The blaKPC-2 genes related to divergent mobile genetic elements encoded on transferable plasmids may transfer widely, facilitating the spread of carbapenem resistance among bacteria with different genetic backgrounds. The dissemination of blaKPC-bearing plasmids that collectively carry additional multidrug resistance genes has caused widespread public concern, further limiting the antibiotics available to treat infections caused by KPC-producing pathogens.Keywords: carbapenemase, CRKP, blaKPC-2, KPC-Kp, ST11, Tn 1721

Objectives The aim of this study was to explore the genomic content of a blaKPC-2- and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance. Methods Antimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing. Results Escherichia coli strain QE11−421 was an mcr-1-positive colistin-resistant isolate that co-harboured the blaKPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11−421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The blaKPC-2 gene was located on a Tn3–Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon. Conclusions Horizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat.

Lingtong Tang,1,2,* Jianglian Huang,3,* Junping She,1 Kelei Zhao,4 Yingshun Zhou1 1Department of Pathogenic Biology, School of Basic Medicine, Southwest Medical University, Luzhou 646000, Sichuan, People’s Republic of China; 2Department of Clinical Laboratory, People’s Hospital of Gao County, Yibing 644000, Sichuan, People’s Republic of China; 3Department of Clinical Laboratory, The Second Affiliated Hospital of Xiamen Medical College, Xiaman 361600, People’s Republic of China; 4Antibiotics Research and Re-Evaluation Key Laboratory of Sichuan Province, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610052, Sichuan, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yingshun ZhouDepartment of Pathogenic Biology, School of Basic Medicine, Southwest Medical University, Luzhou, Sichuan 646000, People’s Republic of China, No. 319, Zhongshan Road, Tel +86-0830-3160073Email yingshunzhou@swmu.edu.cnKelei ZhaoAntibiotics Research and Re-Evaluation Key Laboratory of Sichuan Province, Sichuan Industrial Institute of Antibiotics, No. 168, Huaguan Road, Chengdu 610052, Sichuan, People’s Republic of ChinaTel +86– 028– 84216035Email zhaokelei@cdu.edu.cnPurpose: To characterize the genetic feature of a multi-drug-resistant Aeromonas caviae strain isolated from the diarrhea sample of a 45-year-old male patient with acute diarrhea.Materials and Methods: Whole-genome of the A. caviae strain SCAc2001 was sequenced via the Illumina system, followed by a series of bioinformatic analyses to describe the genetic feature.Results: The genome sequence of A. caviae SCAc2001 was assembled into 340 scaffolds (305 of them were > 1000 bp in length and 4,487,370 bp in total) with an average G+C content of 61.09%. Phylogenetic analysis showed that the A. caviae SCAc2001 strain was highly similar to the A. caviae strain R25-2 and T25-39. Resistome analysis identified that A. caviae SCAc2001 carried 13 antimicrobial resistance genes, including β-lactams (blaKPC, blaCTX-M-14, blaTEM-1, blaOXA-10, blaOXA-427, blaVEB-3 and blaMOX-6), aminoglycosides (aadA1), fluoroquinolones (aac(6ʹ)-Ib-cr), phenicol resistance (catB3), sulfonamide (sul1), trimethoprim (dfrA5) and colistin resistance (mcr-3.3).And also, A. caviae ScAc2001 carried 54 putative virulence genes including the type IV pilus, fimbria, flagellarthe, and hemolysin A encoding genes, and 12 pathogen–host interactions (PHI) genes. There were also four genomic islands and eight prophages in the genome of A. caviae ScAc2001. In addition, A. caviae SCAc2001 also carried three secondary metabolism products coding clusters including nonribosomal peptide synthetases (nrps), hserlactone and bacteriocin.Conclusion: A. caviae ScAc2001 carries many resistance genes, a variety of virulence factors, PHI genes and four genomic islands and eight prophages, which poses a severe threat to infectious diseases control strategies, diagnosis methods and clinical treatment.Keywords: Aeromonas caviae, blaKPC-2, mcr-3.3, virulence factors, secondary metabolism products &nbsp

Shujin Xie,1,2,* Shiming Fu,3,* Mengwei Li,4 Zhusheng Guo,2 Xuehai Zhu,2 Jianmin Ren,5 Fupin Hu1,6 1Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Tungwah Hospital of Sun Yat-Sen University, Dongguan, People’s Republic of China; 3Department of Clinical Laboratory, The Traditional Chinese Medical Hospital of Suichang, Zhejiang, People’s Republic of China; 4Clinical Microbiology Laboratory, Department of Liuzhou Worker Hospital, Guangxi, People’s Republic of China; 5Institute of Antibiotics, The Central Hospital of Lishui City, Zhejiang, People’s Republic of China; 6Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fupin HuInstitute of Antibiotics, Huashan Hospital, Fudan University, 12 M. Wulumuqi Road, Shanghai 200040, People’s Republic of ChinaTel +86-21-52888186Fax +86-21-62488290Email hufupin@fudan.edu.cnObjective: To investigate the molecular characteristics of carbapenem-resistant Enterobacteriaceae (CRE) from county hospitals in China.MaterialsandMethods: Forty-three sequential non-duplicate CRE strains (including 33 Klebsiella pneumoniae isolates, 4 Enterobacter cloacae isolates, 3 Escherichia coli isolates, 1 Serratia marcescens, 1 Morganella morganii and 1 Citrobacter freundii) were collected from 4 county hospitals and 2 municipal hospitals. Antimicrobial susceptibility testing was conducted by broth microdilution method, using 3-aminophenylboronic acid and EDTA and the modified carbapenem inactivation method (mCIM) to screen phenotype of carbapenemase. β-Lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of blaNDM-5 was investigated by transformation experiment. Clonal relatedness was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing .Results: The results of antimicrobial susceptibility testing indicatedthat 43 CRE strains were resistant to most of the antimicrobial agents, except tigecycline and colistin. Overall, 93%, 93%, and 97.7% of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. PCR and DNA sequencing indicated that 67.4% (29/43) were blaKPC-2 positive isolates, in which 3.4% (1/29) was coproduced with blaNDM-1. In addition, 7.0% (3/43), 4.7% (2/43), 4.7% (2/43), 2.3% (1/43), 2.3% (1/43) were blaNDM-1, blaNDM-16, blaNDM-4, blaNDM-5, blaIMP-4 positive isolates, respectively. The 29 blaKPC-2-positive isolates belonged to 12 different PFGE type and designated as ST11 (n=20) and ST15, ST39, ST116, ST667, ST2245, ST2338. The plasmid bearing blaNDM-5 could be transferred into recipient E. coli J53 through transformation.Conclusion: Our study indicated the dissemination of CRE between the tertiary hospitals and secondary hospitals.Keywords: Enterobacteriaceae, blaKPC-2, blaNDM-1

Juan Xu, 1,* Zhao Zhao, 2,* Yumei Ge, 2 Fang He 2 1Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou Medical College, Hangzhou, Zhejiang 310013, People’s Republic of China; 2Department of Clinical Laboratory, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fang He Tel/Fax +86-571-8821-5596Email hetrue@163.comPurpose: Pandrug-resistant K. pneumoniae is still rare in China, and the lack of studies limits our understanding of the emergence mechanism of pandrug-resistant K. pneumoniae. Here, we report the rapid emergence of a pandrug-resistant K. pneumoniae ST11 strain in an inpatient after treatment with multiple broad-spectrum antibiotics in China.Patients and Methods: K. pneumoniae strain KP65 was isolated from a 55-year-old male patient hospitalized in the Department of Intensive Care Unit (ICU) of a teaching hospital in China. Antimicrobial susceptibility testing was conducted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The complete genome sequence of the strain was determined using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Genomic features and resistance mechanisms of the strain were comprehensively analysed using various bioinformatics approaches.Results: K. pneumoniae strain KP65 was found to be resistant to all antibiotics tested, including beta-lactams, aminoglycosides, quinolones, tigecycline and colistin. Seven resistance genes were identified in the genome. The carbapenem-resistant gene blaKPC-2 and extended-spectrum β-lactamase (ESBL)-producing gene blaCTX-M-65 are located on the IncFII-type plasmid pKPC-2-KP65. No mcr genes were detected in the genome, but an IS 5 insertion element was found at position 117 of the mgrB gene. Regarding the rpsJ gene, single-base substitution, G169C, leading to the amino acid substitution V57L was also identified. According to in silico MLST analysis, K. pneumoniae KP65 belongs to sequence type ST11. The closest relative of K. pneumoniae KP65 is another ST11 K. pneumoniae strain, which was isolated from a bloodstream infection in Hangzhou, differing by only 53 cgMLST loci.Conclusion: Under the selective pressure of antibiotics, the KPC-2-producing K. pneumoniae ST11 strain can easily evolve pandrug resistance through chromosomal mutations. More attention is required to monitor the prevalence of the KPC-2-producing K. pneumoniae ST11 strain in China.Keywords: Klebsiella pneumoniae, mgrB, rpsJ, ST11, blaKPC-2

De-Yu Ma, 1,* Han-Yu Huang, 2,* Hua Zou, 1 Meng-Lu Wu, 1 Qiu-Xia Lin, 1 Bo Liu, 3 Shi-Feng Huang 1 1Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China; 2Department of Radiology, Chongqing Health Center for Women and Children, Chongqing, People’s Republic of China; 3Department of Burn and Plastic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Bo LiuDepartment of Burn and Plastic Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China, No. 1 Friendship Road, Yuzhong District, Chongqing 400016, People’s Republic of ChinaTel +86-18623007069Fax +86-023-89012093Email fcz69@sina.com Shi-Feng HuangDepartment of Clinical Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China, No. 1 Friendship Road, Yuzhong District, Chongqing 400016, People’s Republic of ChinaTel +86-18623027077Fax +86-023-89012513Email sfhuang@hospital.cqmu.edu.cnPurpose: Little is known about the epidemiology and carbapenem-resistance determinants of carbapenem-resistant K. aerogenes (CRKA) isolated from a single medical center. The present study was initiated to characterize the molecular epidemiology and the carbapenem-resistance mechanisms of CRKA isolated during 2012– 2018 from a teaching hospital in southwest China, and to investigate the risk factors and clinical outcomes of CRKA infections as well.Methods: Pulsed-field gel electrophoresis (PFGE) was employed for epidemiological analysis. PCR amplification and DNA sequencing were used to examine the antibiotic-resistance determinants. Plasmids were extracted and characterized by PCR-based replicon typing and conjugation assays. In order to further investigate the risk factors and clinical outcomes of CRKA infections, a retrospective case–control study was also performed.Results: PFGE analysis showed 32 different PFGE patterns among the 36 non-duplicated CRKA strains collected. Most of the isolates harbored multi-drug resistance (MDR) genes, including 2 (5.6%) carrying blaNDM-1, 1 (2.8%) harboring blaKPC-2, 13 (36.1%) carrying ESBL genes, 23 (63.9%) carrying ampC genes, 34 (94.4%) carrying quinolone resistance determinants (QRD) genes and 9 (25%) carrying aminoglycoside resistance determinants (ARD) genes. The outer membrane porins, OmpE35 and OmpE36, were, respectively, lost in 4 and 2 isolates. The efflux pump inhibition experiments were positive in 25 (69.4%) of the CRKA strains. Multivariate analysis indicated that hypo-albuminaemia, invasive procedures, and carbapenem exposure were independent risk factors for acquiring CRKA infections.Conclusion: No clonality relationship was identified among most of the 36 CRKA isolates. The over-expression of ESBLs and AmpC coupled with the efflux pumps contributed to carbapenem resistance in K. aerogenes. Additionally, this is the first report of CRKA isolate co-harboring blaNDM-1, blaCTX-M-15, blaEBC, blaACC, acc (6ʹ)-Ib, armA, qnrD and loss of OmpE36 in China. Hypo-albuminaemia, invasive procedures and carbapenem exposure were associated with acquisition of CRKA infections.Keywords: case-control study, blaNDM-1, blaKPC-2, porins, efflux pumps

Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa (KPC-PA) has been reported sporadically. However, epidemiological and antimicrobial susceptibility data specific for KPC-PA are lacking. We collected 374 carbapenem-resistant P. aeruginosa (CRPA) isolates from seven hospitals in China from June 2016 to February 2019 and identified the blaKPC-2 gene in 40.4% (n = 151/374) of the isolates. Approximately one-half of all KPC-PA isolates (n = 76/151; 50.3%) were resistant to ceftazidime-avibactam (CAZ-AVI). Combining Kraken2 taxonomy identification and Nanopore sequencing, we identified eight plasmid types, five of which carried blaKPC-2, and 13 combination patterns of these plasmid types. In addition, we identified IS26-ΔTn6296 and Tn1403-like–ΔTn6296 as the two mobile genetic elements that mediated blaKPC-2 transmission. blaKPC-2 plasmid curing in 28 strains restored CAZ-AVI susceptibility, suggesting that blaKPC-2 was the mediator of CAZ-AVI resistance. Furthermore, the blaKPC-2 copy number was found to correlate with KPC expression and, therefore, CAZ-AVI resistance. Taken together, our results suggest that KPC-PA is becoming a clinical threat and that using CAZ-AVI to treat this specific pathogen should be done with caution. IMPORTANCE Previous research has reported several cases of KPC-PA strains and three KPC-encoding P. aeruginosa plasmid types in China. However, the prevalence and clinical significance of KPC-PA are not available. In addition, the susceptibility of the strains to CAZ-AVI remains unknown. Samples in this study were collected from seven tertiary hospitals prior to CAZ-AVI clinical approval in China. Therefore, our results represent a retrospective study establishing the baseline efficacy of the novel β-lactam/β-lactamase combination agent for treating KPC-PA infections. The observed correlation between the blaKPC copy number and CAZ-AVI resistance suggests that close monitoring of the susceptibility of the strain during treatment is required. It would also be beneficial to screen for the blaKPC gene in CRPA strains for antimicrobial surveillance purposes.

Treatment strategies of infection by carbapenem-resistant Klebsiella pneumoniae (CRKP) are limited. Fosfomycin, a broad-spectrum antibiotic, has attracted renewed interest in combination therapy to fight K. pneumoniae infections. However, reports on fosfomycin-resistant K. pneumoniae are increasing. Among the 57 CRKP strains, 40 (70.2%) were resistant to fosfomycin. Thus, whole-genome sequencing and bioinformatics analysis were conducted to reveal molecular characteristics of fosfomycin-resistant K. pneumoniae. Twenty-three isolates coharbored fosAkp and fosA3, with K. pneumoniae carbapenemase (KPC)-producing ST11-KL64-wzi64-O2 (n = 13) and ST11-KL47-wzi209-OL101 (n = 8), the predominating clonal groups, while fosA3 was not detected in isolates carrying class B carbapenemase genes. Twenty-two (out of 26) ST11-KL64 strains were positive for rmpA2, of which 12 carried fosA3. Four of the 23 fosA3-positive isolates could successfully transfer their fosfomycin-resistant determinants to Escherichia coli J53AziR. All four strains belonged to ST11-KL47 with the same pulsed-field gel electrophoresis profile, and their transconjugants acquired fosfomycin, carbapenem, and aminoglycoside resistance. A 127-kb conjugative pCT-KPC-like hybrid plasmid (pJNKPN52_KPC_fosA) coharboring fosA3, blaKPC–2, blaCTX–M–65, blaSHV–12, rmtB, and blaTEM–1 was identified. ST11-KL64 and ST11-KL47 K. pneumoniae, with higher resistance and virulence, should be critically monitored to prevent the future dissemination of resistance.

The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales is a public health concern. KPC-encoding blaKPC is predominantly spread by strains of a particular phylogenetic lineage, clonal group 258, but can also be spread by horizontal transfer of blaKPC-carrying plasmids. Here, we report the transfer of a blaKPC-2-harboring plasmid via mobilization from K. pneumoniae to Citrobacter freundii complex and Morganella morganii strains in a single patient. We performed draft whole-genome sequencing to analyze 20 carbapenemase-producing Enterobacterales strains (15 of K. pneumoniae, two of C. freundii complex, and three of M. morganii) and all K. pneumoniae strains using MiSeq and/or MinION isolated from a patient who was hospitalized in New York and Montreal before returning to Japan. All strains harbored blaKPC-2-containing Tn4401a. The 15 K. pneumoniae strains each belonged to sequence type 258 and harbored a Tn4401a-carrying multireplicon-type plasmid, IncN and IncR (IncN+R). Three of these K. pneumoniae strains also possessed a Tn4401a-carrying ColRNAI plasmid, suggesting that Tn4401a underwent interplasmid transposition. Of these three ColRNAI plasmids, two and one were identical to plasmids harbored by two Citrobacter europaeus and three M. morganii strains, respectively. The Tn4401a-carrying ColRNAI plasmids were each 23,753 bp long and incapable of conjugal transfer via their own genes alone, but they mobilized during the conjugal transfer of Tn4401a-carrying IncN+R plasmids in K. pneumoniae. Interplasmid transposition of Tn4401a from an IncN+R plasmid to a ColRNAI plasmid in K. pneumoniae and mobilization of Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii. IMPORTANCE Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, blaKPC. We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze blaKPC-2-carrying mobile genetic elements (MGEs) between the blaKPC-2-harboring K. pneumoniae, Citrobacter europaeus, and Morganella morganii strains isolated from a single patient. blaKPC-2 was contained within an MGE, Tn4401a. WGS of blaKPC-2-carrying K. pneumoniae, C. europaeus, and M. morganii strains isolated from one patient revealed that Tn4401a-carrying ColRNAI plasmids were generated by plasmid-to-plasmid transfer of Tn4401a from a multireplicon-type IncN and IncR (IncN+R) plasmid in K. pneumoniae strains. Tn4401a-carrying ColRNAI plasmids were incapable of conjugal transfer in C. europaeus and M. morganii but mobilized from K. pneumoniae to a recipient Escherichia coli strain during the conjugal transfer of Tn4401a-carrying IncN+R plasmid. Therefore, Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii.

The emergence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) was regarded as an emerging threat in clinical settings. Here, we investigated the prevalence of CRKP strains among inpatients in a new hospital over 1 year since its inception with various techniques, and carried out a WGS-based phylogenetic study to dissect the genomic background of these isolates. The genomes of three representative blaNDM–1-positive strains and the plasmids of four blaKPC–2-positive strains were selected for Nanopore long-read sequencing to resolve the complicated MDR structures. Thirty-five CRKP strains were identified from 193 K. pneumoniae isolates, among which 30 strains (85.7%) harbored blaKPC–2, whereas the remaining five strains (14.3%) were positive for blaNDM–1. The antimicrobial resistance profiles of blaNDM–1-positive isolates were narrower than that of blaKPC–2-positive isolates. Five isolates including two blaNDM–1-positive isolates and three blaKPC–2-positive strains could successfully transfer the carbapenem resistance phenotype by conjugation. All CRKP strains were categorized into six known multilocus sequence types, with ST11 being the most prevalent type. Phylogenetic analysis demonstrated that the clonal spread of ST11 blaKPC–2-positive isolates and local polyclonal spread of blaNDM–1-positive isolates have existed in the hospital. The blaNDM–1 gene was located on IncX3, IncFIB/IncHI1B, and IncHI5-like plasmids, of which IncFIB/IncHI1B plasmid has a novel structure. By contrast, all ST11 isolates shared the similar blaKPC–2-bearing plasmid backbone, and 11 of them possessed pLVPK-like plasmids. In addition, in silico virulome analysis, Galleria mellonella larvae infection assay, and siderophore secretion revealed the hypervirulence potential of most blaKPC–2-positive strains. Given that these isolates also had remarkable environmental adaptability, targeted measures should be implemented to prevent the grave consequences caused by hv-CRKP strains in nosocomial settings.

Objectives The Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. Methods A KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. Results Klebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. Conclusion pKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Purpose 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: NCT03950544

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with an increase in the frequency of infections caused by multidrug resistant (MDR) and extensively drug resistant (XDR) strains, limiting the available therapeutic options. The most troublesome resistance is the acquisition and production of carbapenemases such as Verona integron-encoded metallo-β-lactamases (VIM), the most frequent and widespread, and the Klebsiella pneumoniae carbapenemases (KPC), which has continuously spread in the last decade. Its dissemination is linked to their location on mobile genetic elements (MGEs). In Colombia, VIM and KPC have been increasing in its frequency showing major successful dissemination. In this article, we molecularly characterized and analyzed the genetic context of blaVIM and blaKPC in carbapenem-resistant P. aeruginosa (CRPA) isolates from infected and colonized patients in two tertiary-care hospitals, one in Medellín and the other in a municipality close to Medellín, both areas with high carbapenemase endemicity in Colombia (2013–2015). Using whole-genome sequencing (WGS), we identified a remarkable variety of genetic backgrounds in these MDR P. aeruginosa isolates carrying blaKPC–2 and blaVIM–2. There were a diversity of class 1 integron and variations in the gene cassettes associated to blaVIM–2, as well as a possible event of spread of blaKPC–2 mediated by a plasmid that contained part of Tn4401b in one infection case. The dissemination of blaVIM–2 and blaKPC–2 in P. aeruginosa in this area in Colombia has been strongly influenced by successful international clones, carrying these genes and additional determinants of resistance on MGEs, accompanied by gene rearrangement under an antimicrobial selection pressure. These findings emphasize the need to implement control strategies based on rational antibiotic use.

Objectives The resistance rate of Klebsiella pneumoniae to commonly used antibiotics has been increasing rapidly, causing serious concern among clinicians and microbiologists. Resistance to carbapenems in K. pneumoniae is increasing dramatically in Chinese hospitals. Here we report the genome sequence of an NDM-1 and KPC-2 co-producing K. pneumoniae (CRKP380) isolated from a bloodstream infection in Hangzhou, China. Methods The whole genome sequence of strain CRKP380 was determined using an Illumina NovaSeq 6000 platform. Antimicrobial resistance genes (ARGs) were identified using the BacWGSTdb server. The phylogenetic relationship between strain CRKP380 and other K. pneumoniae strains isolated from Hangzhou currently deposited in the NCBI GenBank database was analysed using a core genome-based single nucleotide polymorphism strategy. Results Klebsiella pneumoniae CRKP380 was resistant to all antibiotics tested except tigecycline and colistin. The genome sequence of K. pneumoniae CRKP380 consisted of 75 contigs comprising 5 590 460 bp. According to the Pasteur multilocus sequence typing (MLST) scheme, CRKP380 belongs to ST11. Eight ARGs were identified in CRKP380, including two carbapenemase genes (blaKPC-2 and blaNDM-1). Ten phylogenetically-related K. pneumoniae strains from Hangzhou were identified with identical ARGs and the same capsular serotype KL105, but none of these strains carried the blaNDM gene. Conclusion Here we report the genome sequence of a K. pneumoniae ST11 clinical strain co-carrying blaNDM-1 and blaKPC-2 from Hangzhou, China. The genome sequence of CRKP380 can be used as a reference sequence for future comparative genomic analysis, including the acquisition and mobilisation of carbapenem resistance genes.

Min Xu,1,* Yiqi Fu,2,* Yunhui Fang,3 Hao Xu,3 Haishen Kong,3 Yanchao Liu,1 Yu Chen,1,3 Lanjuan Li3 1Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Center of Clinical Laboratory, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 2Department of Respiratory Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China; 3State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China *These authors contributed equally to this work Background: Klebsiella pneumoniae has been the leading causative pathogen for adult bacterial meningitis in several Asian countries. The clinical and microbiological characteristics of K.pneumoniae meningitis in mainland China are still unknown. Materials and methods: The clinical data of patients with K. pneumoniae meningitis from January 2011 to July 2017 in a tertiary hospital were retrospectively evaluated. The isolates were tested for antibiotic-resistance genes, virulence-associated genes, and molecular subtypes. Hypervirulent K. pneumoniae (hvKP) was defined as the presence of pLVPK-like virulence plasmid. Results: During the study period, a total of 48 patients with meningitis caused by K. pneumoniae were identified, accounting for 21.2% (48/226) of Gram-negative bacilli meningitis. Of the 44 available isolates, 65.9% (29/44) were carbapenem resistant, and all except one harbored blaKPC-2. K64 was the most common serotype (n=13), followed by K47 (n=11) and K1 (n=5). The pLVPK-related genetic loci were found in about half of isolates (iutA: 56.8%, iucA: 56.8%, rmpA2:50.0%, rmpA: 43.2%, and iroN: 40.9%). Twenty-two strains carrying pLVPK-derived virulence plasmid were defined as hvKP. Notably, the coexistence of blaKPC-2-encoding plasmid and the pLVPK-derived virulence plasmid was detected in 15 strains (34.1%, 15/44), suggesting K. pneumoniae carbapenemase-2 (KPC-2)-producing hvKP. The proportion of KPC-2-producing hvKP by year increased remarkably from 0% (2011) to 71.4% (2017). Of the 15 KPC-2-producing hvKP strains, 80.0% (12/15) were assigned to sequence type 11 and 2 strains (13.3%) belonged to clonal complex 23. Most of the patients infected with KPC-2-producing hvKP had preceding postneurosurgical state (93.3%, 14/15) and severe pneumonia (73.3%, 11/15). All the cases (100%, 15/15) had fatal outcome. Conclusion: The high prevalence and mortality of K. pneumoniae, especially KPC-2-producing hvKP meningitis, in China should be of concern. The implementation of epidemiological surveillance and identification of an effective clinical treatment are paramount. Keywords: meningitis, hypervirulent K. pneumonia, blaKPC-2, rmpA2, pLVPK-like virulence plasmid

The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the blaKPC-2 from the blaKPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several blaKPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions.

Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of bla KPC dissemination have been reported in Colombia, a country with high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of bla KPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of bla KPC-2 using both short and long read sequencing. We found that spread of bla KPC-2 among Enterobacterales in the participating hospitals was due to intra and inter-species horizontal gene transfer (HGT) mediated by promiscuous plasmids associated to transposable elements that was originated from a multi-species outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit (ICU). The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene "epidemic" was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp, Escherichia coli, Enterobacter spp and Citrobacter spp. Of note, mcr-9 was found to co-exist with bla KPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of bla KPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections and resistance to carbapenems is now emerging due to the acquisition of carbapenemase. 45 carbapenem-resistant E. coli isolates were collected in a hospital. The resistance mechanisms, plasmid profiles and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of blaKPC-2 was analyzed to trace the potential dissemination of blaKPC-2.45 carbapenem-resistant E. coli ST131strains are closely related. Initially prevalent only in the ward E02, ST131 subsequently spread to other wards and multiple lineages evolved independently, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first E. coli ST131 isolation. The blaKPC-2 positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition of transmissible plasmids pE0272_KPC and pE0171_KPC carrying blaKPC-2 by E. coli ST131. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. E. coli ST131 has become a multidrug resistant epidemic clone that can cause hospital-acquired respiratory infections resulting in mortality. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections. The emergence of blaKPC in E. coli ST131 may herald the beginning of the high prevalence of carbapenem resistance in Chinese communities and hospitals.

Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) is a major cause of infections in transplanted patients and has been associated with high mortality rates in this group. There is a lack of information about the Brazilian structure population of CP-Kp isolated from transplanted patients. By whole-genome sequencing (WGS), we analyzed phylogeny, resistome, virulome of CP-Kp isolates, and the structure of plasmids encoding blaKPC-2 and blaNDM-1 genes. One K. pneumoniae isolated from each selected transplanted patient colonized or infected by CP-Kp over a 16-month period in a hospital complex in Porto Alegre (Brazil) was submitted for WGS. The total number of strains sequenced was 80. The hospital complex in Porto Alegre comprised seven different hospitals. High-resolution SNP typing, core genome multilocus sequence typing (cgMLST), resistance and virulence genes inference, and plasmid reconstruction were performed in 80 CP-Kp. The mortality rate of CP-Kp colonized or infected transplanted inpatients was 21.3% (17/80). Four CP-Kp epidemic clones were described: ST11/KPC-2, ST16/KPC-2, and ST15/NDM-1, all responsible for interhospital outbreaks; and ST437/KPC-2 affecting a single hospital. The average number of acquired resistance and virulence genes was 9 (range = 2-14) and 27 (range = 6-36), respectively. Two plasmids carrying the blaKPC-2 were constructed and belonged to IncN and IncM types. Additionally, an IncFIB plasmid carrying the blaNDM-1 was described. We detected intrahospital and interhospital spread of mobile structures and international K. pneumoniae clones as ST11, ST16, and ST15 among transplanted patients, which carry a significant range of acquired resistance and virulence genes and keep spreading across the world. This work was supported by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, and Spanish Network for Research in Infectious Diseases (REIPI RD16CIII/0004/0002), and co-financed by the European Regional Development Fund ERDF “A way to achieve Europe,” Operative Program Intelligent Growth 2014–2020. This work was also supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001. Sí

Daofeng Qu,1,2 Yang Shen,2 Lingfei Hu,1 Xiaoyuan Jiang,1 Zhe Yin,1 Bo Gao,1 Yuee Zhao,1 Wenhui Yang,1 Huiying Yang,1 Jianzhong Han,2 Dongsheng Zhou1 1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; 2School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China Background: IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described.Methods: Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1).Results: The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary IncFIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as blaKPC, tetA, dfrA, and qnr).Conclusion: In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number. Keywords: multi-replicon plasmids, multi-drug resistance, blaKPC-2, mobile elements

Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids., Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16). IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

Modified carbapenem inactivation method (mCIM) testing was currently recommended by Clinical and Laboratory Standards Institute (CLSI) for detection of carbapenemase among Enterobacteriaceae clinical isolates. In this study, a panel of 145 clinical strains were collected for evaluating the mCIM for detection of carbapenemase. Antimicrobial susceptibility testing were performed by microbroth dilution and the results were interpreted according to CLSI guidelines. All strains were resistant to ertapenem with high MIC50 and MIC90 (64 mg/L –>128 mg/L). For blaNDM-1-positive or blaOXA-232-positive strains, the zone of inhibition of meropenem were all 6 mm despite the incubation time of 6 h, 18 h or 24 h. For 6 h, the zone of meropenem inhibition for most of carbapenemase-positive isolates were meet the positive criteria 6–15 mm. However, for carbapenemase-negative isolates, the zone of meropenem inhibition were 16–18 mm after 6 h incubation which should be considered indeterminate for standard incubating time such as 18 h or 24 h. After incubating for 18 h or 24 h, the zone of meropenem inhibition were 22–25 mm for carbapenemase-negative isolates and meet the negative criteria. Our study indicate mCIM is a simple and effective method to identify the carbapenemases producers among Enterobacteriaceae clinical isolates.

Background Epidemiology of fosfomycin susceptibility and the plasmid-mediated fosfomycinase genes of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Taiwan remain unclear. Methods 642 CRKP clinical isolates were collected from a nation-wide surveillance study (16 hospitals) in Taiwan in 2012–2013. Antimicrobial susceptibilities were determined. PFGE and MLST determined the clonal relatedness. Carbapenemases and fosfomycinases genes were detected by PCR, and their flanking regions were determined by PCR and sequencing. Synergistic activity of meropenem with fosfomycin was examined by the checkerboard method. Results In total, 36.4% (234/642) of CRKP isolates in Taiwan were resistant to fosfomycin. Among 234 fosfomycin-resistant CRKP isolates, PFGE analysis revealed 81 pulsotypes. Pulsotype XXIII (n = 63) was predominant and belonged to ST11. 71 had carbapnemases (65 bla KPC-2 -positive, 1 bla VIM-1 -positive and 5 bla IMP-8 -positive) and 62 had fosfomycinases (35 fosA3 -positive and 27 foskp96 -positive). Only 18.5% (5/27) of foskp96 -positive isolates carried foskp96 and bla KPC-2 , while 71.4% (25/35) of fosA3 -positive isolates contained fosA3 and bla KPC-2 . There were five types of flanking sequences for fosA3 , and 85.7% (30/35) of fosA3 genes were flanked by IS 26 , suggesting possible horizontal gene transfer. Synergistic effect of fosfomycin and meropenem was observed in all 25 randomly selected pulsotype XXIII strains (100%; 25/25), even those containing fosfomycinase (48%, 12/25) or carbapnemase (96%, 24/25). Conclusions A clone (pulsotype XXIII, ST11) has been found to be prevailing among fosfomycin-resistant CRKP in Taiwan. According to the in vitro data, the combination of fosfomycin and meropenem is a potentially alternative choice.

The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE) - mediated hospital infections in China prompted a need to investigate the genetic basis of emergence of such strains. A nationwide survey was conducted in China covering a total of 1105 CRE strains collected from 25 geographical locales with results showing that acquisition of two carbapenemase genes, blaKPC-2 and blaNDM, was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32% respectively), among which several major strain types, such as ST11 of K. pneumoniae and ST131/ST167 of E. coli, were identified, suggesting that dissemination of specific resistant clones is mainly responsible for emergence of new CRE strains. Prevalence of the fosA3 gene which mediates fosfomycin resistance, was high, while the colistin resistance determinant mcr-1 was rarely present in these isolates. Consistently, the majority of the blaNDM-bearing plasmids recoverable from the test strains belonged to IncX3, which contained a common core structure, blaNDM-blaMBL-trpF. Likewise, the core structure of ISKpn27-blaKPC-2-ISKpn2 was observed among plasmids harboring the blaKPC-2 gene, although they were genetically more divergent. In conclusion, the increasing prevalence of CRE strains in China is attributed to dissemination of conservative mobile elements carrying blaNDM or blaKPC-2 on conjugative and non-conjugative plasmids., Highlights • Over 90% of CRE strains in China harbored the blaNDM or blaKPC-2 gene. • The predominant types of carbapenem-resistant K. pneumoniae and E. coli were ST11 and ST131/ST167 respectively. • Conservative mobile elements carrying blaNDM and blaKPC-2 contributed to transmission of carbapenemase genes in CRE. Large scale surveillance of CRE was rarely reported worldwide except for a recent survey reported in European countries. A nationwide survey was conducted in this study to depict the epidemiological features of CRE strains in China and the types of resistance elements that they harbored. We showed that the increasing prevalence of CRE strains in China was due to dissemination of major resistant clones that harbor several major types of blaNDM or blaKPC-2 – bearing conjugative plasmids, and that infections caused by such strains remain treatable by various non-β-lactam antibiotics, including colistin.

The emergence of blaKPC-2 and blaNDM-1 producing Klebsiella pneumoniae represents a great problem in many Egyptian hospitals. One hundred and twenty-six K. pneumoniae isolates from patients admitted to Assiut University Hospital were identified by an API20E kit. Carbapenemase-producing K. pneumoniae (CPKP) was detected by the modified carbapenem inactivation method (mCIM), the EDTA-modified carbapenem inactivation method (eCIM), and an E-test. Based on the polymerase chain reaction, all isolates were negative for bla-VIM-1 and bla-IMP-1, fifteen of these isolates were positive for both blaKPC-2 and blaNDM-1, two isolates were positive for blaKPC-2 only, and twenty-eight isolates were positive for bla-NDM-1 only. Although one isolate was positive for the string test, all CPKP isolates were negative for capsular genes. Only 71.1% of CPKP transferred their plasmids to their corresponding transconjugants (E. coli J53). The resistance patterns of the clinical isolates and their transconjugates were similar, except for 12 isolates, which showed differences with their transconjugates in the resistance profile of four antibiotics. Molecular typing of the plasmids based on replicon typing showed that Inc FIIK and FII plasmids predominated in isolates and their transconjugants carrying blaKPC-2 and/or blaNDM-1. Conjugative Inc FII plasmids play an important role in the spread of CPKP, and their recognition is essential to limit their spread.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) Klebsiella pneumoniae resistant to beta-lactams are present in hospitals worldwide and reduce treatment alternatives. In this study, 40 K. pneumoniae isolates were characterized. All of them were resistant to beta-lactam antibiotics and classified as multidrug resistant. Among these, eight isolates were resistant to carbapenems and six of which had the gene blaKPC-2, encoding the enzyme Klebsiella pneumoniae carbapenemase (KPC). The blaTEM and blaCTX-M genes were found in 80% of the samples. The 40 isolates were grouped into 12 pulses (A to L) according to the genetic similarity determined by the DNA macro-restriction followed by pulsed-field gel electrophoresis. A total of 20 isolates were classified as pulsotype A, which was subdivided into 13 subtypes (A1 to A13), with subtype A1 being the most prevalent. Representatives of pulsotype A belong to the ST11, clonal complex CC258. In this study, we identified four new STs: ST2540, ST2308, ST2258, and ST2307. AMKP5 and AMKP2, belonging to subtypes A7 and A8, respectively, are resistant to carbapenems but did not present the blaKPC gene. Sequencing of the AMKP5 genome indicated mutations in the C-terminal region of the OmpK36 porin which may alter the entry of carbapenems into the cell. Genome sequencing of the AMKP10 isolate identified the plasmid pAMKP10, with 48.454 bp belonging to the IncX5 incompatibility group. In this plasmid, the blaKPC-2 gene was found in a non-Tn4401 genetic element (NTEKPC) containing tnpA-ISEc63-like / tnpR-Tn3 / tnpA-ISKpn27 / blaKPC-2 / ΔISKpn6, based on Tn1722. All isolates with the blaKPC-2 gene had the same plasmid. Conjugation of this plasmid to Escherichia coli J53 occurred from ST11 isolates with low conjugation frequencies (8.23 x 10-12 to 1.31 x 10-7) resulting in resistance to carbapenems. Finally, the outbreak of carbapenem-resistant K. pneumoniae resistant of this Amazonian hospital involved the transfer of the NTEKPC-containing pAMKP10 between two lineages (ST11 and ST2302) but was largely due to the clonal dissemination of the ST11 endemic lineage. Klebsiella pneumoniae resistentes aos beta-lactâmicos estão presentes em hospitais do mundo todo e reduzem as alternativas de tratamento. Nesse estudo, 40 isolados de K. pneumoniae foram caracterizados. Todos foram classificados como multirresistentes e com resistência aos antibióticos beta-lactâmicos. Dentre estes, oito amostras são resistentes aos carbapenêmicos e seis delas têm o gene blaKPC-2, que codifica a enzima Klebsiella pneumoniae carbapenemase (KPC). Os genes blaTEM e blaCTX-M foram encontrados em 80% das amostras. Os 40 isolados foram agrupados em 12 pulsotipos (A a L) de acordo com a similaridade genética determinada pela macrorrestrição do DNA seguida de eletroforese em gel de campos pulsados. Um total de 20 isolados foram classificados como pulsotipo A, o qual foi subdividido em 13 subtipos (A1 a A13), sendo o subtipo A1 o prevalente. Representantes do pulsotipo A pertencem ao ST11, complexo clonal CC258. Neste estudo, quatro novos STs foram identificados: ST2540, ST2308, ST2258 e ST2307. AMKP5 e AMKP2, dos subtipos A7 e A8, respectivamente, são resistentes aos carbapenêmicos, mas não apresentaram o gene blaKPC. O sequenciamento do genoma da AMKP5 indicou mutações na região C-terminal da porina OmpK36 que pode alterar a entrada dos carbapenêmicos na célula. O sequenciamento do genoma da amostra AMKP10 identificou o plasmídeo pAMKP10, com 48,454 pb pertencente ao grupo de incompatibilidade IncX5. Neste plasmídeo, o gene blaKPC foi encontrado em um ambiente genético diferente do Tn4401 (NTEKPC do inglês, non-Tn4401 genetic element) contendo tnpA-ISEc63-like/tnpR-Tn3/tnpA-ISKpn27/blaKPC-2/ΔISKpn6, baseado no Tn1722. Todos os isolados com o gene blaKPC-2 apresentaram o mesmo plasmídeo. A conjugação deste plasmídeo para Escherichia coli J53 ocorreu a partir dos isolados ST11 com baixas frequências de conjugação (de 8,23 x 10-12 a 1,31 x 10-7) resultando em resistência aos carbapenêmicos. Por fim, observou-se que o surto de K. pneumoniae resistentes aos carbapenêmicos deste hospital Amazonense envolveu a transferência do pAMKP10 contendo NTEKPC entre duas linhagens (ST11 e ST2302), mas ocorreu em grande parte devido à disseminação clonal da linhagem endêmica ST11. FAPEAM: 24868.484.14650.07112014-35526 FAPESP: 13/07600-l3 CNPq: 131314/2018-1

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn 4401b :: bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn 4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Introducción: El clon de Pseudomonas aeruginosa con tipo de secuencia 235 (ST235) es uno de los clones con resistencia a múltiples antibióticos y de mayor diseminación mundial. Este clon tiene una extraordinaria capacidad de adquirir elementos genéticos móviles, lo cual está asociado a la adquisición de genes de resistencia. Objetivo: Comparar la estructura genética de las plataformas de movilización de genes de resistencia del clon de Pseudomonas aeruginosa ST235, causantes de infecciones en Colombia respecto a los clones reportados a nivel mundial. Materiales y Métodos: Se analizaron aislamientos de Pseudomonas aeruginosa causantes de infecciones en cinco UCI de cinco ciudades de Colombia. Se evaluó su perfil de resistencia y la presencia de 32 determinantes de resistencia asociados a los principales antibióticos de uso clínico. La relación genética se determinó por PFGE y MLST. De estos, se seleccionó un aislamiento representativo del clon ST235 (24pae112), se secuenció su genoma por PacBio-RS-II, se identificaron y analizaron sus plataformas de movilización de genes de resistencia y se comparó con 44 genomas ST235 reportados. Resultados: De los 58 aislamientos analizados, 9(15,5%) presentaron un perfil de multirresistencia y 27(46,5%) fueron resistentes a carbapenémicos, de los cuales 6(10,3%) se asociaron a blaVIM y 4(6,9%) a blaKPC-2, estos últimos pertenecientes al ST235. El tamaño del genoma de 24pae112 (ST235) fue de 7'097.241pb, en el cual se identificaron 12(0,2%) genes de resistencia, y múltiples EGM que incluyeron: cinco transposones Tn6162-like con aadB, dos Tn402-like, uno con aadA1 y otro con blaOXA-2, y dos Tn4401b cada uno con blaKPC-2, siete islas genómicas y seis profagos. Conclusión: La resistencia en el clon de Pseudomonas aeruginosa ST235 está dada principalmente por la ganancia de múltiples genes de resistencia a través de la adquisición de diferentes plataformas genéticas (transposones, integrones, profagos e islas genómicas), algunas de ellas en un modelo de “russian doll”. Este fenómeno podría ser un factor clave en su alta capacidad de diseminación y persistencia. Abstract Introduction: Pseudomonas aeruginosa clone with sequence type 235 (ST235) is one of the clones with resistance to multiple antibiotics and of greater worldwide dissemination. This clone has an extraordinary ability to acquire mobile genetic elements (MGE), which is associated with the acquisition of resistance genes. Objective: Compare the genetic structure of the resistance gene mobilization platforms of the Pseudomonas aeruginosa ST235 clone, which cause infections in Colombia with respect to the clones reported worldwide. Materials and Methods: Pseudomonas aeruginosa isolates causing infections in five ICUs from five cities in Colombia. Its resistance profile and the presence of 32 resistance determinants associated to the main antibiotics of clinical use were evaluated. The genetic relationship was determined by PFGE and MLST. Of these, a representative isolate of the ST235 clone (24pae112) was selected, its genome was sequenced by PacBio-RS-II, it’s resistance gene mobilization platforms were identified and analyzed and compared with 44 reported ST235 genomes. Results: Of the 58 isolates analyzed, 9(15.5%) presented a multiresistance profile and 27(46.5%) were carbapenem-resistant, of which 6(10.3%) were associated with blaVIM and 4(6.9%) to blaKPC-2, the latter belonging to ST235. The genome size of 24pae112 (ST235) was 7'097.241pb, in which 12(0.2%) resistance genes were identified, and multiple MGEs included: five Tn6162-like transposons with aadB, two Tn402-like, One with aadA1 and one with blaOXA-2, and two Tn4401b each with blaKPC-2, seven genomic islands and six prophages. Conclusion: Resistance in the Pseudomonas aeruginosa ST235 clone is mainly due to the gain of multiple resistance genes through the acquisition of different genetic platforms (transposons, integrones, prophages and genomic islands), some of them in a model of "russian doll ". This phenomenon could be a key factor in its high capacity for dissemination and persistence. Maestría

The present work describes the abrupt emergence of Klebsiella pneumoniae carbapenemase (KPC) and characterizes the first 79 KPC-producing enterobacteria from Argentina (isolated from 2006 to 2010). The emergence of bla(KPC-2) was characterized by two patterns of dispersion: the first was the sporadic occurrence in diverse enterobacteria from distant geographical regions, harbouring plasmids of different incompatibility groups and bla(KPC-2) in an unusual genetic environment flanked by ISKpn8-Δbla(TEM-1) and ISKpn6-like. bla(KPC-2) was associated with IncL/M transferable plasmids; the second was the abrupt clonal spread of K. pneumoniae ST258 harbouring bla(KPC-2) in Tn4401a.

Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.

INTRODUCTION : Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing isolates pose a major worldwide public health problem today. METHODS : A carbapenem-resistant Proteus mirabilis clinical isolate was investigated for plasmid profiles and the occurrence of β-lactamase genes. RESULTS : The isolate exhibited resistance to ertapenem and imipenem and was susceptible to meropenem, polymyxin, and tigecycline. Five plasmids were identified in this isolate. DNA sequencing analysis revealed the presence of bla KPC-2 and bla TEM-1 genes. An additional PCR using plasmid DNA confirmed that bla KPC-2 was present in one of these plasmids. Conclusions: We report the detection of bla KPC-2 in P. mirabilis in Brazil for the first time. This finding highlights the continuous transfer of bla KPC between bacterial genera, which presents a serious challenge to the prevention of infection by multidrug-resistant bacteria.