PCR-based ORF typing of Klebsiella pneumoniae for rapid identification of global clones and transmission events.

AIMS: Klebsiella pneumoniae is a major cause of healthcare-associated infections. In this study, we aimed to develop a rapid and simple genotyping method that can characterize strains causing nosocomial infections. METHODS AND RESULTS: The PCR-based open reading frame (ORF) typing (POT) method consists of two multiplex PCR reactions that were designed to detect 25 ORFs specific to bacterial genetic lineages, species, antimicrobial-resistant genes (blaCTX-M group-1 , blaCTX-M group-9 , blaIMP and blaKPC ), a capsular K1-specific gene and a virulence factor gene (rmpA/A2). The electrophoresis results are then digitized. A total of 192 strains (136 clinical and 8 reference strains of K. pneumoniae, 33 clinical and 1 reference strains of K. variicola and 14 clinical strains of K. quasipneumoniae) were classified into 95, 26 and 11 POT values, respectively. The distribution patterns of ORFs among K. pneumoniae correlated well with multilocus sequence typing (MLST). Furthermore, closely related species could be distinguished and key antimicrobial resistance and hypervirulence genes were identified as part of POT. CONCLUSIONS: The POT method was developed and validated for K. pneumoniae. In comparison to MLST, the POT method is a rapid and easy genotyping method for monitoring transmission events by K. pneumoniae in clinical microbiology laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: The POT method supplies clear and informative molecular typing results for K. pneumoniae. The method would facilitate molecular epidemiological analysis in infection control and hospital epidemiology investigations.

Molecular Epidemiology of Enterobacter cloacae Complex Isolates with Reduced Carbapenem Susceptibility Recovered by Blood Culture.

The Enterobacter cloacae complex (ECC) is one of the most common causes of bacteremia and leads to poor clinical outcomes. The aim of this study was to clarify the antimicrobial susceptibility profiles and genetic backgrounds of non-carbapenemase-producing reduced-carbapenem-susceptible (RCS) ECC blood isolates in Japan using agar dilution antimicrobial susceptibility testing, whole-genome sequencing, and quantitative polymerase chain reaction for ampC, ompC, and ompF transcripts. Forty-two ECC blood isolates were categorized into RCS and carbapenem-susceptible groups based on the minimum inhibitory concentration of imipenem. The RCS ECC blood isolates belonged to distinct species and sequence types and produced varying class C ß-lactamases. The E. roggenkampii, E. asburiae, and E. bugandensis isolates belonged only to the RCS group. Some E. hormaechei ssp. steigerwaltii isolates from the RCS group exhibited AmpC overexpression caused by amino acid substitutions in AmpD and AmpR along with ompF downregulation. These findings suggest that non-carbapenemase-producing RCS ECC blood isolates are genetically diverse.

Characterization of blaCTX-M-27/F1:A2:B20 Plasmids Harbored by Escherichia coli Sequence Type 131 Sublineage C1/H30R Isolates Spreading among Elderly Japanese in Nonacute-Care Settings.

We characterized 29 blaCTX-M-27-harboring plasmids of Escherichia coli sequence type 131 (ST131) sublineage C1/H30R isolates from healthy individuals and long-term-care facility (LTCF) residents. Most (27/29) plasmids were of the FIA, FIB, and FII multireplicon type with the same plasmid multilocus sequence typing (pMLST). Several plasmids (7/23) from LTCF residents harbored only blaCTX-M-27 as the resistance gene; however, their fundamental structures were very similar to those of previously isolated blaCTX-M-27/F1:A2:B20 plasmids, suggesting their prevalence as a newly arising public health concern.