Among 5938 clinical Shigella spp. isolates, two S. flexneri strains were isolated as those resistant to fluoroquinolones based on the MICs of the following antibiotics: ciprofloxacin, norfloxacin, ofloxacin, sparfloxacin and levofloxacin. S. flexneri 021787 had three substitutions, one in GyrA (Ser83Leu) and two in ParC (Ser80Ile and Arg91Gln). S. flexneri 021895 had four substitutions, two in GyrA (Ser83Leu and Asp87Gly) and two in ParC (Ser80Ile and Arg91Gln). The increased susceptibility of S. flexneri 021787 and S. flexneri 021895 to ciprofloxacin, norfloxacin and ofloxacin in the presence of the uncoupler carbonyl cyamide-m-chlorophenyldrazone implied that energy-dependent active efflux pumps contributed to the resistance against fluoroquinolones. Both S. flexneri 021787 and S. flexneri 021895 were also induced to express tolC (encoding a resistance-nodulation-division transporter), mdfA (encoding a major facilitator superfamily transporter), and ydhE (encoding a multidrug and toxic compound extrusion transporter) in the presence of ciprofloxacin. Thus, these results indicated that chromosome-mediated fluoroquinolone resistance of S. flexneri 021787 and S. flexneri 021895 resulted from the combination of target site mutations and enhanced expression of genes encoding efflux pumps.

A multiplex PCR method has been developed to classify extended spectrum β-lactamase (ESBL) and plasmidmediated AmpC β-lactamase (PABL). This method consists of the use of two four-multiplex PCRs for the detection of TEM, OXA, SHV, CTX-M, CMY, and DHA type β-lactamases. We have compared findings from the use of conventional detection methods with that of this newly developed typing method. In testing for 73 ESBLproducing and PABL-producing isolates, 100% of the isolates were correctly identified as previously characterized types and, 44 types of β-lactamases were additionally identified from 33 isolates. This assay not only reduces the time for classification but also increases the accuracy for detection.

Campylobacter jejuni is one of the major causative pathogens of outbreaks or sporadic cases of diarrhoeal diseases worldwide. In this study, we compared the phenotypic and genetic characteristics of C. jejuni isolates of human and food-producing animal origins in Korea and examined the genetic relatedness between these two groups of isolates. Regardless of isolation source, all C. jejuni isolates harboured four virulence genes, cadF, cdtB, ciaB and racR, whereas the wlaN and virB11 genes were more frequently observed in human isolates. Antimicrobial susceptibility testing showed that the majority of C. jejuni isolates displayed high-level resistance to fluoroquinolone (95.2%) or tetracycline (76.2%) antibiotics, and 12.4% of isolates exhibited multidrug resistance (more than three classes of antibiotics tested). Pulsed-field gel electrophoresis (PFGE) of all Campylobacter isolates revealed 51 different SmaI-PFGE patterns and six major clusters containing both human and animal isolates. These results indicate that genetically diverse strains of C. jejuni with antimicrobial drug-resistance and virulence properties have prevailed in Incheon. Nevertheless, some particular populations continue to circulate within the community, providing the evidence for an epidemiological link of C. jejuni infections between humans and food-producing animals. Therefore, the continued monitoring and surveillance of C. jejuni isolates of human and food-producing animal origins are required for public health and food safety.

Repeated injection of triamcinolone contaminated with NTM from the clinic environment may have caused this post-injection outbreak.

ObjectivesA multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.MethodsSpecific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.ResultsSpecific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 104 colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.ConclusionThis assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.

Two molecular epidemiologic methods, IS restriction fragment length polymorphism (IS-RFLP) and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR), are used worldwide in studies of (MTB). Conversely, because of its poor resolution, pulsed-field gel electrophoresis (PFGE) is not widely used for MTB. In this study, we improved the 24-locus MIRU-VNTR and PFGE protocols and compared the effectiveness of these approaches for the molecular typing of MTB using 75 clinical isolates obtained from a cohort investigation of high-risk populations infected with MTB. The 24-locus MIRU-VNTR method demonstrated superior discriminatory ability, followed by PFGE and IS-RFLP. Next, we analyzed six isolates with clear epidemiologic connections; that is, isolates from patients who attended the same school. IS-RFLP and PFGE identified these samples as the same type. By contrast, according to MIRU-VNTR, two isolates differed from four other isolates at one locus each; one isolate was identified as Mtub29 and the other as QUB-26. In summary, the 24-locus MIRU-VNTR assay was the most useful molecular typing method among the three methods investigated due to its discriminatory power, short time required, and availability as an epidemiologic investigation tool. PFGE was the second-best method. Compared with the other loci assessed in the 24-locus MIRU-VNTR assay, the Mtub29 and QUB-26 loci appeared to exhibit greater variability during transmission.

ObjectivesMycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods.MethodsAntibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed.ResultsAll the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR.ConclusionThe high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types.

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