Pathogenic and phylogenetic characteristics of non-O157 Shiga toxin-producing Escherichia coli isolates from retail meats in South Korea

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.

Comparison of 3 Phenotypic-detection Methods for Identifying Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Strains / 대한진단검사의학회지

BACKGROUND: Plasmid-mediated AmpC beta-lactamases (PABLs) have been detected in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. PABLs may be difficult to detect and might interfere in the therapeutic and infection-control processes. Although several PABL-detection methods based on phenotypes have been reported, the Clinical and Laboratory Standards Institute currently does not recommend a routine detection method for PABLs. The aim of this study is to compare the performances of 3 phenotypic PABL detection methods. METHODS: Total 276 non-duplicated clinical isolates of E. coli (N=97), K. pneumoniae (N=136), and P. mirabilis (N=43) were collected from 14 hospitals in Korea between April and June 2007 in a non-consecutive and non-random manner. Multiplex PCR was performed to detect the PABL genes. Further, 3 phenotypic detection methods-cephamycin-Hodge test, Tris-EDTA (TE) disk test, and combination-disk test with 3-aminophenylboronic acid (BA)-were performed using cefoxitin and cefotetan disks. RESULTS: PABL genes were detected by multiplex PCR in 122/276 isolates, including 14/97 E. coli, 105/136 K. pneumoniae, and 3/43 P. mirabilis isolates. The combination-disk test with BA showed higher sensitivity (98.4%), specificity (92.2%), and efficiency (96.3%) than the cephamycin-Hodge (76.2%, 96.1%, and 88.6%, respectively) and the TE-disk (80.3%, 91.6%, and 87.9%, respectively) tests. CONCLUSIONS: The combination-disk test with BA is a simple, efficient, and interpretable test that can be applicable in clinical laboratories involved in the detection of PABLs in clinical isolates of E. coli, K. pneumoniae, and P. mirabilis.

Antimicrobial Resistance of Clinically Important Bacteria Isolated from 12 Hospitals in Korea in 2005 and 2006 / 대한임상미생물학회지

BACKGROUND: Emergence and spread of antimicrobial resistant bacteria make it difficult to treat infections. A rapid increase in antimicrobial-resistant bacteria has become a serious problem in many countries including Korea, and it is important to perform a nationwide study of antimicrobial resistance to obtain some basic data that will help solve these problems. The aim of this study was to determine the nationwide prevalence of resistance among frequently isolated bacterial pathogens in 2005 and 2006 in Korea. METHODS: We collected routine susceptibility data for medically important bacterial pathogens from 12 university and general hospital laboratories in Korea from April to September in 2005 and from January to June in 2006. Collected data was analyzed by patient group. RESULTS: The proportions of methicillin-resistant Staphylococcus aureus (MRSA) were 65% in 2005 and 72% in 2006, respectively. The resistance rates of Enterococcus faecium to vancomycin were 29% in 2005 and 24% in 2006. The non-susceptible rates of Streptococcus pneumoniae to penicillin were 68% in 2005 and 74% in 2006. The resistant rates of Escherichia coli and Klebsiella pneumoniae to the 3rd generation cephalosporin were 10~12% and 25~39%, respectively, in 2005 and 11~15% and 30~34% in 2006. In Citrobacter freundii, Enterobacter cloacae and Serratia marcescens, the resistance rates to 3rd generation cephalosporin were 23~31%, 32~34%, and 17~27%, respectively, in 2005 and 21~37%, 37~43%, and 13~31% in 2006. The resistance rates to imipenem and meropenem were 21% and 18%, respectively, in Pseudomonas aeruginosa and 18% and 25% in Acinetobacter baumannii in 2005; 29% and 20% in P. aeruginosa and 18% and 23% in A. baumannii in 2006. Cotrimoxazole and levofloxacin resistance rates of Stenotrophomonas maltophilia were 5% and 13%, respectively, in 2005 and 3% and 7% in 2006. There were no isolates resistant to 3rd generation cephalosporin and fluoroquinolone among non-typhoidal Salmonella in 2005. CONCLUSION: Antimicrobial resistance of medically important bacteria is still a serious problem in Korea. To manage the problem, a continuous nationwide surveillance and diversified investigation and effort have become more important.

Prevalence and Genetic Relatedness of Vancomycin-resistant Enterococci Isolated from Livestock and Humans after Ban of Avoparcin in Korea / 감염과화학요법

BACKGROUND: Avoparcin, cross-resistance with vancomycin, was added as feed-additive since 1970s and was prohibited in 1997 in Korea. After avoparcin was banned we examined prevalence and genetic relatedness of VRE in enterococci isolated from livestock and humans. MATERIALS AND METHODS: Using enrichment broth and 6 microgram/mL vancomycin-containing enterococcosel selective agar, vancomycin-resistant enterococci (VRE) were isolated from fecal sample of 255 pigs of 8 farms, 431 chickens of 9 farms, and 328 humans (Food industry employee and Institution cafeteria employee) of 5 public health centers, and 100 raw chicken meats from April to June 2003. Antimicrobial susceptibility was examined by disk diffusion and minimum inhibitory concentrations (MICs), and E-test. Species identification and genotyping were done by multiplex PCR method. Pulsed-field gel electrophoresis (PFGE) of vanA-type VRE isolates was performed by CHEF-Mapper system. RESULTS: 19 isolates from 255 pigs, 122 isolates from 431 chickens, 19 isolates from 100 raw chicken meat, and 7 isolates from 328 humans were resistant to vancomycin. Of the 167 VRE isolates, vanA gene was detected in 141 isolates; 1 isolate (0.4%) in pigs, 121 isolates (28.1%) in chickens, 18 isolates (18.0%) in raw chicken meat, and 1 isolate (0.3%) in humans. Resistant rates of streptomycin, tetracycline, and erythromycin were over 60% in vanA-type E. faecium isolated from poultry. PFGE analysis resulted in two major patterns, F and P types. Also PFGE pattern of 1 VRE from human was identical to that of 1 VRE from poultry. CONCLUSION: Despite the high prevalence of vanA-type VRE in poultry farms, VRE isolation rate in human was relatively low. This result suggests that the possibility of VRE transmission from poultry to human is low but that possibility may be not ruled out. In PFGE analysis showing 51.5% identical in 2 PFGE patterns, the dissemination of VRE isolates in poultry may be transmitted vertically and horizontally.

Prevalence and Genetic Relatedness of Vancomycin-resistant Enterococci Isolated from Livestock and Humans after Ban of Avoparcin in Korea / 감염과화학요법

BACKGROUND: Avoparcin, cross-resistance with vancomycin, was added as feed-additive since 1970s and was prohibited in 1997 in Korea. After avoparcin was banned we examined prevalence and genetic relatedness of VRE in enterococci isolated from livestock and humans. MATERIALS AND METHODS: Using enrichment broth and 6 microgram/mL vancomycin-containing enterococcosel selective agar, vancomycin-resistant enterococci (VRE) were isolated from fecal sample of 255 pigs of 8 farms, 431 chickens of 9 farms, and 328 humans (Food industry employee and Institution cafeteria employee) of 5 public health centers, and 100 raw chicken meats from April to June 2003. Antimicrobial susceptibility was examined by disk diffusion and minimum inhibitory concentrations (MICs), and E-test. Species identification and genotyping were done by multiplex PCR method. Pulsed-field gel electrophoresis (PFGE) of vanA-type VRE isolates was performed by CHEF-Mapper system. RESULTS: 19 isolates from 255 pigs, 122 isolates from 431 chickens, 19 isolates from 100 raw chicken meat, and 7 isolates from 328 humans were resistant to vancomycin. Of the 167 VRE isolates, vanA gene was detected in 141 isolates; 1 isolate (0.4%) in pigs, 121 isolates (28.1%) in chickens, 18 isolates (18.0%) in raw chicken meat, and 1 isolate (0.3%) in humans. Resistant rates of streptomycin, tetracycline, and erythromycin were over 60% in vanA-type E. faecium isolated from poultry. PFGE analysis resulted in two major patterns, F and P types. Also PFGE pattern of 1 VRE from human was identical to that of 1 VRE from poultry. CONCLUSION: Despite the high prevalence of vanA-type VRE in poultry farms, VRE isolation rate in human was relatively low. This result suggests that the possibility of VRE transmission from poultry to human is low but that possibility may be not ruled out. In PFGE analysis showing 51.5% identical in 2 PFGE patterns, the dissemination of VRE isolates in poultry may be transmitted vertically and horizontally.

Emergence of CTX-M-12 and A Novel CTX-M Type Extended-Spectrum beta-Lactamaseproducing Klebsiella pneumoniae / 대한진단검사의학회지

BACKGROUND: The aims of this study were to survey the nation-wide susceptibilities of Klebsiella pneumoniae isolates against ceftazidime and cefotaxime and to determine the prevalence of class A extended-spectrum beta-lactamases (ESBLs). METHODS: During the period of February to July 2004, K. pneumoniae isolates intermediate or resistant to ceftazidime and/or cefotaxime were collected from 12 hospitals in Korea. Antimicrobial susceptibilities were determined by the disk diffusion and the agar dilution methods and ESBL-production was by double-disk synergy test. Ceftazidime or cefotaxime-resistance determinants of the ESBLproducers were transfered to Escherichia coli J53 by transconjugation. Searches for class A ESBL genes were performed by PCR amplication. RESULTS: Among 212 clinical K. pneumoniae isolates, 172 (81%) isolates showed positive results in double-disk synergy test; the most prevalent ESBL was SHV-12 (n=104). Genes encoding ESBLs including SHV-2 (n=6), SHV-2a (n=17), CTX-M-3 (n=18), CTX-M-9 (n=6), CTX-M-12 (n=1), CTX-M- 14 (n=27), CTX-M-15 (n=3), and a novel CTX-M-type beta-lactamases were also detected. CONCLUSIONS: It is concluded that diversity of ESBLs in K. pneumoniae isolates are increasing in Korea. CTX-M-12 has never been reported in Asia, and a novel CTX-M-type ESBL has emerged.

Emergence of CTX-M-12, PER-1 and OXA-30 beta-Lactamase-Producing Klebsiella pneumoniae / 대한임상미생물학회지

BACKGROUND: The aim of this study was to determine a nation-wide prevalence of Ambler class A and D extended-spectrum-lactamases (ESBL) in Klebsiella pneumoniae isolates in Korea. METHODS: During the period of April to May 2005, 189 isolates of K.pneumoniae were collected from 11 Korean hospitals. Antimicrobial susceptibilities to ceftazidime and cefotaxime were tested by the disk diffusion method, and ESBL production was determined by double-disk synergy test. Determinants of ceftazidime or cefotaxime-resistance were transferred to Escherichia coli J53 (azide-resistant) by transconjugation. Genotypes of class A and D ESBL genes were determined by PCR amplification and sequencing. RESULTS: One hundred-sixty isolates of K.pneumoniae showed positive results in double-disk synergy test. The most prevalent ESBL was SHV-12 (n=148). Also detected were genes encoding ESBLs including TEM-52 (n=1), SHV-2a (n=2), CTX-M-3 (n=15), CTX-M-9 (n=6), CTX-M-12 (n=2), CTX-M-14 (n=9), CTX-M-15 (n=1), PER-1 (n=1), GES-5 (n=3), and OXA-30 (n=2) beta-lactamases. CONCLUSION: With the emergence of CTX-M-12, PER-1, and OXA-30 beta-lactamases, the ESBLs in K.pneumoniae isolates are becoming more diverse in Korea.

Dissemination of CTX-M Type Extended-Spectrum beta-Lactamases and Emergence of CTX-M-12 in Escherichia coli / 대한진단검사의학회지

BACKGROUND: Clinical isolates of Escherichia coli were evaluated to determine the prevalence and genotypes of Ambler class A extended-spectrum beta -lactamases (ESBLs). METHODS: Clinical isolates of E. coli were collected from 12 hospitals from February through July, 2004. Antimicrobial susceptibility was tested by disk diffusion and agar dilution methods, and ESBLproduction was determined by double-disk synergy test. TEM, SHV, CTX-M, PER-1, VEB, IBC, GES, and TLA type ESBL genes were detected by PCR amplifications, and the PCR products were subjected to direct sequencing. RESULTS: The double-disk synergy test was positive in 90.9% (149 in 164) of the ceftazidime- or cefotaxime-resistant E. coli isolates. The most prevalent types of Ambler class A ESBLs in E. coliisolates were CTX-M-15 (n=53). CTX-M-14 (n=24), CTX-M-3 (n=9), CTX-M-9 (n=3), CTX-M-12 (n=3), SHV-2a (n=1), SHV-12 (n=5) and TEM-52 (n=3) were also found. CTX-M-12 ESBL had never been reported before in Korea. CONCLUSIONS: CTX-M type ESBL-producing E. coli isolates are spreading and CTX-M-12 is emerging in Korea.