In this study, we investigated the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals.
Electrophoresis, Gel, Pulsed-Field ; Hospitals ; Humans ; Klebsiella pneumoniae/*classification/enzymology/genetics/isolation & purification ; Sequence Analysis, DNA ; beta-Lactamases/*biosynthesis ; Mycobacteria, Atypical/*drug effects/genetics/isolation & purification

BACKGROUND: Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM. METHODS: One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene. RESULTS: In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively. CONCLUSIONS: With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.
Bacterial Proteins/genetics ; Chaperonins/genetics ; Computer Systems ; DNA, Bacterial/chemistry ; Mycobacteria, Atypical/genetics/*isolation & purification ; Nucleic Acid Denaturation ; Polymerase Chain Reaction/*methods ; RNA, Ribosomal, 16S/genetics