Temporal shifts in the predominant carbapenemase gene types among carbapenemase-producing Klebsiella pneumoniae isolated in Bangkok, Thailand, during 2013-2016.

Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice.Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48-like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited.Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand.Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013-2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships.Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014-2015 was bla NDM-1, whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1. Other carbapenemase gene variants, such as bla NDM-4, bla NDM-5, bla OXA-48, bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181, emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years.Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.

A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-ß-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae Clones and Spreading Plasmids.

Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome. We analyzed 371 Enterobacteriaceae isolates carrying blaNDM-1 and 114 Enterobacteriaceae isolates carrying blaNDM-5 obtained from clinical samples of 473 patients in 11 representative hospitals located in six provinces in Thailand between 2012 and 2017. The complete structures of plasmids carrying blaNDM and chromosomal phylogeny were determined by combining Southern blotting hybridization analysis and our previously performed whole-genome short-read sequencing data. Dissemination of the blaNDM-5 gene among the Enterobacteriaceae isolates in Thailand was mainly owing to the nationwide clonal spread of Escherichia coli ST410 and regional clonal spreads of Escherichia coli ST361 and ST405. Analysis of blaNDM-1-carrying isolates revealed nationwide dissemination of two specific plasmids and nationwide clonal dissemination of Klebsiella pneumoniae ST16 accompanied with regional disseminations of three distinctive K. pneumoniae clones (ST231, ST14, and ST147) with different plasmids. Dissemination of CRE carrying blaNDM in Thailand is mainly based on nationwide clonal expansions of E. coli ST410 carrying blaNDM-5 and K. pneumoniae ST16 carrying blaNDM-1, nationwide dissemination of two distinctive plasmids carrying blaNDM-1, and accumulation of clonal expansions in regional areas. Although the overuse of antibiotics can promote CRE dissemination, the limited variety of transmitters highlights the importance of preventing horizontal dissemination among patients.

PCR-Dipstick-Oriented Surveillance and Characterization of mcr-1- and Carbapenemase-Carrying Enterobacteriaceae in a Thai Hospital.

Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types-IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (<2 h) and robust sensitivity (100%)/specificity (~99%) of PCR-dipstick show that this specimen-direct screening method could aid in implementing infection control measures at the earliest to control the dissemination of MCRPEn and CPE.

Genomic Characterization of Carbapenemase-Producing Klebsiella pneumoniae with Chromosomally Carried blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring.

Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae from Bangkok, Thailand, and Their Detection by the Carba NP and Modified Carbapenem Inactivation Method Tests.

AIM: The purpose of the study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant Enterobacteriaceae and evaluate the Carba NP and modified carbapenem inactivation method (mCIM) tests in their detection. MATERIALS AND METHODS: A total of 287 nonduplicated Enterobacteriaceae isolates, which were at least resistant to one of the carbapenems, were identified and detected for carbapenemase genes by multiplex PCR covering blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like. All positive genes were then sequenced. These isolates were phenotypically tested for the production of carbapenemases by mCIM and Carba NP tests to evaluate the efficacy of these methods. RESULTS: Seven species of carbapenem-resistant isolates mainly Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae were detected. Of these isolates, three families of carbapenemase genes, including blaNDM (blaNDM-1, -4, -5, -9), blaOXA (blaOXA-48, -181, -232), and blaIMP-14, were found. Of these, 223 (77.70%) carried at least one of the carbapenemase genes. The blaNDM was detected in 160/223 (71.75%) isolates, of which 153/160 (95.63%) were the blaNDM-1. Three types of the blaOXA-48-like group, blaOXA-48, blaOXA-181, and blaOXA-232, were found, 91/104 (87.5%) harbored the blaOXA-232. In addition, 25.11% (56/223) of the carbapenemase-producing isolates harbored a combination of blaNDM and blaOXA-48-like. Phenotypic detection methods, mCIM and Carba NP, showed 100% sensitivity and specificity to blaNDM, blaIMP-14, and blaOXA-48, while the mCIM was positive in all blaOXA-181 and blaOXA-232 isolates, only 12.5% (1/8) and 28.95% (11/38), respectively, were detected by the Carba NP test. CONCLUSIONS: This study revealed a unique prevalence of carbapenemase genes in Bangkok, Thailand, as well as demonstrated the efficacy and limitation of phenotypic detection methods of carbapenemase in the area where blaNDM-1 and blaOXA-232 were predominant.

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens.

A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.