Molecular characterization of NDM-1-producing Klebsiella pneumoniae ST29, ST347, ST1224, and ST2558 causing sepsis in neonates in a tertiary care hospital of North-East India.

Geographical differences can manifest in different spectra of microorganisms and patterns of antibiotic resistance. Considering this, Enterobacteriacae isolated from septicemic neonates from a tertiary care centre in Agartala, India were studied with focus on carbapenem resistance. Two hundred non-duplicate Enterobacteriaceae, of which 12 NDM-1-producing Klebsiella pneumoniae were recovered. Antibiotic susceptibility tests and detection of ESBLs and carbapenemases were performed for all Enterobacteriaceae. For NDM-1-producing isolates, plasmid-mediated quinolone resistance genes, addiction systems, genetic environment of blaNDM-1 and virulence genes was investigated by PCR. Bacterial clonal relatedness was established using REP-PCR, PFGE, and multi-locus sequence typing (MLST). Transferability of blaNDM-1 was tested by conjugation and transconjugants were characterized. K. pneumoniae was the primary organism causing sepsis in neonates. Resistance to different antimicrobials was high except for aminoglycosides and carbapenems. blaCTX-M was present in all isolates. All carbapenem-resistant isolates harboured blaNDM-1 as the only carbapenemase. blaCTX-M-15 and qnrS1 were detected in all NDM-1-producing isolates. Plasmid analysis of transconjugants revealed that blaNDM-1 along with blaCTX-M-15, qnrS1, qnrB1, aac(6')-Ib, aac(6')-Ib-cr and ccdAB or vagCD addiction systems were carried on large IncFIIK conjugative plasmids of varied sizes. blaNDM-1 was associated with ISAba125 or ISEc33 element at its 5'-end. In addition, isolates also harboured wabG, uge, fimH, mrkD, and entB virulence genes. The NDM-1-producing K. pneumoniae belonged to four distinct clones and were distributed in 4 STs (ST347, ST29, ST2558, and ST1224), of which ST347 was predominant. The association of blaNDM-1 with diverse STs in K. pneumoniae from neonates indicates the promiscuity of the gene and its widespread dissemination.

Mukherjee S ，Bhattacharjee A ，Naha S ，Majumdar T ，Debbarma SK ，Kaur H ，Dutta S ，Basu S ... -  《-》

Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital.

Emergence of multi-drug resistance, especially carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health. The aim of this study was to characterize CRKP isolates from infants admitted to the Neonatal Intensive Care Unit (NICU) to find the clonal outbreak of New Delhi metallo-β-lactamase (NDM) producers. In this study 17 CRKP isolates were analysed. Antimicrobial susceptibility of the isolates was determined by the disc diffusion and micro-dilution method. Carba-NP test and double-disk synergy test (DDST) were performed for the detection of carbapenemase and metallo-β-lactamase-producing K. pneumoniae. Antibiotic-resistant markers were detected by polymerase chain reaction (PCR) followed by sequencing. Clonal relatedness of the isolates was checked by multi-locus sequence typing. Conjugation experiments were performed to determine the transferability of the plasmids. All 17 CKRP isolates were found to carry blaNDM (13 blaNDM-1, 1 blaNDM-4 and 3 blaNDM-5), seven isolates carried blaOXA-48, 13 isolates had blaCTX-M-15, seven isolates carried blaCMY-1 and five isolates were found to carry blaSHV-1 on conjugative plasmids of different types (IncFIA, IncFIB, IncFIIAs, IncFIC, IncA/C, IncF, IncK, IncX, IncW and IncY). Of six different sequence types (STs) identified, ST3344 was detected as a novel ST in two K. pneumoniae isolates. Genetic environment analysis revealed ISAba125 and bleomycin genes flanking to all blaNDM variants. This is the first report of novel ST3344 in two NDM-1-producing K. pneumoniae isolates from neonates admitted to the NICU of a North Indian Hospital. This study is provides understanding of the genetic features of this newly emerged strain type.

Ahmad N ，Ali SM ，Khan AU 《-》

Occurrence of OXA-48 and NDM-1 carbapenemase-producing Klebsiella pneumoniae in a Moroccan university hospital in Casablanca, Morocco.

The purpose of this investigation was to determine the prevalence and the characteristics of carbapenemase-producing Klebsiellapneumoniae isolates recovered from various clinical specimens in the university hospital of Casablanca, in Morocco. We conducted a prospective study on a total of 166 K. pneumoniae isolates collected from June to August 2011. The strains suspected to carry carbapenemase showed reduced susceptibility to imipenem or ertapenem. The PCR and a sequencing strategy were used to identify carbapenemases, expended spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamases, plasmid mediated quinolone resistance and aminoglycoside resistance determinants. The clonal relationships between isolates were analyzed by pulsed field gel electrophoresis (PFGE). Among the 166 K. pneumoniae isolates studied, 11 (6%) were carbapenemases producers, 9 of which harbored blaOXA-48 and 2 were positive for blaNDM-1. All carbapenemase-producing K. pneumoniae were also ESBL producers and the blaCTX-M-15 was the most frequent ESBL gene detected (n=9), blaCTX-M-28 and blaSHV-28 were also encountered in one isolate each. The K.pneumoniae isolates carried also non-ESBL genes blaTEM-1 (n=9), blaSHV-1 (n=8) and blaOXA-1 (n=3). Five isolates harbored qnr genes, qnrS1 (n=3) and qnrB1 (n=2) variants. Six isolates were positive for aac(6')-Ib-cr gene and two for aac(3)-II gene. The class 1 integron was detected in five isolates. PFGE has revealed the presence of a clonal dissemination in our hospital. The results of conjugation experiments indicated that blaOXA-48+blaCTX-M-15, blaOXA-48+blaCTX-M-28, blaNDM-1+blaCTX-M-15+blaTEM-1+blaOXA-1+qnrS1+aac(6')-Ib-cr and blaNDM-1+blaCTX-M-15+blaTEM-1+qnrB1+aac(6')-Ib-cr genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight.

Barguigua A ，Zerouali K ，Katfy K ，El Otmani F ，Timinouni M ，Elmdaghri N ... -  《-》

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece.

Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-β-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ∼95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region.

Voulgari E ，Gartzonika C ，Vrioni G ，Politi L ，Priavali E ，Levidiotou-Stefanou S ，Tsakris A ... -  《-》

Multicentre investigation of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Bulgarian hospitals - Interregional spread of ST11 NDM-1-producing K. pneumoniae.

The aim of this study was to investigate the mechanisms of beta-lactam-resistance and the clonal relatedness of carbapenem-nonsusceptible Klebsiella pneumoniae and Escherichia coli isolates, collected consecutively in eight centers in five Bulgarian cities from November 2014 to March 2018. Carbapenemase-producing enterobacteria were detected in all but one centers. Overall, 104 K. pneumoniae and one E. coli were analysed. Antimicrobial susceptibility and beta-lactamases were analysed. Conjugation experiments, plasmid fingerprinting and replicon typing, as well as MLST and ERIC-PCR were carried out. KPC-2 (51%) and NDM-1 (47%) were the main carbapenemases identified. KPC-2 producing K. pneumoniae were classified into 10 MLST-types. The four dominating MLST-types ST29, ST15, ST336 and ST902 comprised 79% of the KPC-2 producers. All but one of the NDM-1 producing isolates belonged to the MLST-type ST11 and were found in seven centers. Furthermore, single K. pneumoniae isolates producing VIM-1 (ST147) and OXA-48 (ST15) were identified. In addition to the carbapenemases, the ESBLs CTX-M-15, CTX-M-3, and SHV-12 as well as AmpC enzyme CMY-4 were found. The FIIAs-replicon-type was found in all KPC-2 producers while the A/C-replicons dominated in NDM-1 producing isolates. The single NDM-1 producing E. coli was determined as MLST-Type ST10 (Warwick scheme). The interregional clonal expansion of NDM-1 producing ST11 K. pneumoniae and the dissemination of blaKPC-2 carrying plasmids were responsible for the spread of carbapenemase-producing K. pneumoniae in Bulgaria. Our findings highlight the urgency to prevent dissemination of these highly transmissible and dangerous lineages.

Markovska R ，Stoeva T ，Boyanova L ，Stankova P ，Schneider I ，Keuleyan E ，Mihova K ，Murdjeva M ，Sredkova M ，Lesseva M ，Nedelcheva G ，Petrova A ，Ivanova D ，Lazarova G ，Kaneva R ，Mitov I ... -  《-》