Migma Dorji Tamang

Molecular Characterization of Extended-Spectrum-β-Lactamase-Producing and Plasmid-Mediated AmpC β-Lactamase-Producing Escherichia coli Isolated from Stray Dogs in South Korea

ABSTRACT A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried blaCTX-M genes. The most common CTX-M types were CTX-M-14 (n = 5) and CTX-M-24 (n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry blaCMY-2 genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to blaTEM-1, two isolates carried blaDHA-1, and one of them cocarried blaCMY-2. Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of blaCTX-M and blaCMY-2 genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of blaCMY-2 was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of blaDHA-1 in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea.

Plasmid-Mediated Quinolone Resistance Determinants qnrA, qnrB, and qnrS among Clinical Isolates of Enterobacteriaceae in a Korean Hospital

ABSTRACT Screening of 368 consecutive nonreplicate clinical isolates of Enterobacteriaceae resistant to nalidixic acid and at least one extended-spectrum β-lactam revealed the presence of qnrA, qnrB, and qnrS determinants, and identified novel qnrB variants, in Citrobacter freundii isolates. This study also revealed, for the first time, the linkage of qnrB, armA, and extended-spectrum and/or AmpC-type β-lactamase genes on large conjugative plasmids.

Prevalence and Molecular Characterization of CTX-M β-Lactamase–Producing Escherichia coli Isolated from Healthy Swine and Cattle

The purpose of this study was to determine the prevalence and characteristics of CTX-M β-lactamases in Escherichia coli among healthy swine and cattle in Korea. A total of 1212 fecal samples obtained from healthy pigs (n=558) and cattle (n=654) were screened for CTX-M-type extended spectrum β-lactamase (ESBL)-producing E. coli isolates. One hundred and twenty-one E. coli that produced ESBL were subjected to phenotypic and genotypic characterization. A high number (120/558, 21.5%) of swine fecal samples showed the presence of CTX-M β-lactamase-producing E. coli compared to cattle samples (1/654, 0.2%). The most predominant CTX-M-type identified was CTX-M-14 (n=82), followed by CTX-M-15 (n=16). Isolates producing CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 were also identified. Overall, the bla(TEM-1) gene was associated with CTX-M β-lactamase in 55 E. coli isolates. Transfer of bla(CTX-M) gene was demonstrated from 76 out of 121 bla(CTX-M)-positive E. coli isolates to the recipient E. coli J53 by conjugation. Plasmid DNA isolation from the transconjugants revealed a large (90-120 Kb) conjugative plasmid. ISEcp1 and IS903 were detected upstream and downstream of bla(CTX-M) genes in 117 and 91 E. coli isolates, respectively. Our results demonstrated that a combination of clonal expansion and horizontal transmission is spreading bla(CTX-M) genes among swine E. coli. The horizontal dissemination of bla(CTX-M) genes among E. coli was mostly mediated by IncF or IncI1-Iγ plasmids. To the best of our knowledge, this study represents the first report of CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 β-lactamases in bacterial isolates from food animals in Korea. This study revealed that the CTX-M β-lactamase-producing E. coli are widely disseminated among healthy pigs but very rare in cattle in Korea. Increasing prevalence of bla(CTX-M) genes in intestinal E. coli of food animals is a matter of concern and should be carefully monitored.

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle, Farm Workers, and the Farm Environment

ABSTRACT A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla CTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The bla CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla CTX-M-14 gene was also detected in an E. coli isolate from a farmers hand. Transfer of the bla CTX-M gene from 60 bla CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.

Emergence of 16S rRNA methylase gene armA and cocarriage of blaIMP-1 in Pseudomonas aeruginosa isolates from South Korea

Of the 100 multidrug-resistant Pseudomonas aeruginosa isolates from a Korean hospital, 14 isolates that were resistant to all aminoglycosides tested carried 16S rRNA methylase gene armA. Fourteen armA-positive isolates were classified into 8 pulsotypes. Seven armA-positive isolates cocarried bla(IMP-1). This study is the first report of occurrence of armA in P. aeruginosa.

Prevalence and characterization of Salmonella in pigs from conventional and organic farms and first report of S. serovar 1,4,[5],12:i:- from Korea.

This study compared the prevalence of Salmonella spp. and their antimicrobial susceptibilities in pigs from conventional and organic farms during 2012-2013 in Korea and characterized them by molecular methods. Altogether, 100 nontyphoid Salmonella were isolated: 47 from 1324 pigs (3.5%) from conventional farms and 53 from 641 pigs (8.3%) from organic farms. The most frequent serovar was Typhimurium (49%) followed by Panama (24%), 1,4,[5],12:i:- (5%), and Virchow (5%). Overall, the isolates were most often resistant to tetracycline (75%) followed by ampicillin (66%), streptomycin (57%), and gentamicin (44%). The prevalence of antimicrobial resistance, multi-drug resistance phenotype, and resistance to tetracycline, ampicillin, and gentamicin were significantly higher in swine Salmonella from conventional farms than those from organic farms. The most common resistance pattern was ampicillin-gentamicin-tetracycline (n=16). All eight ceftiofur-resistant Salmonella identified produced CTX-M-15. Overall, decreased susceptibility to ciprofloxacin was observed in 39 isolates. Among them, a single isolate was positive for qnrS1 gene. An insertion sequence ISEcp1 was detected upstream of blaCTX-M gene in all isolates. The spread of blaCTX-M-15 gene was attributed to combination of clonal expansion and horizontal dissemination mediated by IncHI2 plasmid. Multilocus variable number of tandem repeats analysis demonstrated clonal dissemination of S. Typhimurium and S. 1,4,[5],12:i:- strains in pigs. To our knowledge, this is the first report of blaCTX-M-15 gene in S. Virchow from pigs and qnrS1 gene in S. Rissen from animals. This study also reports the first occurrence of Salmonella serovar 1,4,[5],12:i:- from Korea and CTX-M-15 producing Salmonella from pigs in Korea.

Detection of novel oxazolidinone and phenicol resistance gene optrA in enterococcal isolates from food animals and animal carcasses

Altogether 7720 Enterococcus faecalis and 3939 E. faecium isolated from food animals and animal carcasses during 2003-2014 in Korea were investigated to determine if linezolid-resistant (LR) enterococci (≥8μg/ml) are present. Overall, 12 E. faecalis and 27 E. faecium recovered from chickens (n=32), pigs (n=6), and cattle (n=1) were resistant to linezolid and were further characterized using molecular methods Most LR isolates were also resistant to chloramphenicol (97.44%) and florfenicol (92.31%). Molecular analysis showed no mutations in the 23S ribosomal RNA and in the ribosomal protein L3. The optrA gene was found in 89.74% of the LR enterococci, including 12 E. faecalis and 23 E. faecium isolates. Among them, 30 optrA-positive isolates co-carried phenicol exporter gene fexA. Seven LR E. faecium isolates had Asn130Lys mutations in the ribosomal protein L4, of which six also carried optrA gene. None of the isolates carried the mutliresistance gene cfr. Transfer of optrA gene was observed in 16 of the 35 optrA-positive isolates by conjugation. Pulsed-field gel electrophoresis demonstrated that the vast majority of Enterococcus strains carrying optrA gene were genetically heterogeneous. Multi-locus sequence typing revealed eight novel Sequence types among E. faecalis and E. faecium strains. To our knowledge, this is the first report of optrA gene in isolates from cattle and animal carcasses. This is also the first report of optrA gene in Korea. Active surveillance of optrA in enterococci is urgently warranted.

Molecular Basis of Resistance to Selected Antimicrobial Agents in the Emerging Zoonotic Pathogen Streptococcus suis

ABSTRACT Characterization of 227 Streptococcus suis strains isolated from pigs during 2010 to 2013 showed high levels of resistance to clindamycin (95.6%), tilmicosin (94.7%), tylosin (93.8%), oxytetracycline (89.4%), chlortetracycline (86.8%), tiamulin (72.7%), neomycin (70.0%), enrofloxacin (56.4%), penicillin (56.4%), ceftiofur (55.9%), and gentamicin (55.1%). Resistance to tetracyclines, macrolides, aminoglycosides, and fluoroquinolone was attributed to the tet gene, erm(B), erm(C), mph(C), and mef(A) and/or mef(E) genes, aph(3′)-IIIa and aac(6′)-Ie-aph(2″)-Ia genes, and single point mutations in the quinolone resistance-determining region of ParC and GyrA, respectively.

Antimicrobial susceptibility and virulence characteristics of Salmonella enterica Typhimurium isolates from healthy and diseased pigs in Korea.

This study compared the antimicrobial susceptibility and prevalence of virulence genes in Salmonella enterica Typhimurium isolated from healthy and diseased pigs in Korea. A total of 456 Salmonella Typhimurium isolated from healthy (n = 238) and diseased (n = 218) pigs between 1998 and 2011 were investigated. In total, 93.4% of the Salmonella Typhimurium isolates were resistant to at least one antimicrobial agent tested. The isolates were most often resistant to tetracycline (85.7%), followed by streptomycin (83.6%), nalidixic acid (67.3%), ampicillin (49.3%), chloramphenicol (42.8%), and gentamicin (37.1%). Moreover, multidrug resistance phenotype and resistance to ampicillin, florfenicol, gentamicin, nalidixic acid, neomycin, streptomycin, and tetracycline were significantly higher (P < 0.01) among Salmonella Typhimurium isolates from the diseased pigs compared with those from the healthy pigs. The most common resistance pattern observed in both groups of isolates was streptomycin-tetracycline. Overall, more than 96% of the isolates tested possessed invA, spiA, msgA, sipB, prgH, spaN, tolC, lpfC, sifA, sitC, and sopB virulence genes. The prevalence of orgA, pagC, and iroN were 50.2, 74.1, and 91.0%, respectively, whereas isolates carrying cdtB (1.5%), pefA (7.0%), and spvB (14.9%) were identified much less frequently. Furthermore, the prevalence of invA, lpfC, orgA, pagC, and iroN was significantly higher (P < 0.01) among the isolates from the diseased pigs than in isolates from the healthy pigs. Our results demonstrated that, among diseased pigs, there was significantly higher resistance to some antimicrobials and greater prevalence of some virulence genes than in healthy pigs, indicating the role these factors play in pathogenesis. Multidrug-resistant Salmonella isolates that carry virulence-associated genes are potentially more dangerous and constitute a public health concern. Thus, continuous surveillance of antimicrobial resistance and virulence characteristics in Salmonella is essential.

Characterization of plasmids encoding CTX-M β-lactamase and their addiction systems in Escherichia coli isolates from animals

This study was focused on characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from chickens and CTX-M associated plasmid addiction systems (PASs) in E. coli from animals using molecular methods. In total, E. coli from nine (9.0%) of the 100 chicken samples examined produced CTX-M type ESBL namely CTX-M-14 (n=4), CTX-M-15 (n=4), and CTX-M-1 (n=1). All of them harbored an additional blaTEM-1 gene. Transfer of blaCTX-M gene was observed in eight out of the nine blaCTX-M-positive isolates by conjugation. Plasmid profiling of blaCTX-M-positive transconjugants revealed a high-molecular weight (95-165 kb) plasmid. Pulsed-field gel electrophoresis showed that most CTX-M-producing chicken isolates were genetically diverse. Furthermore, investigation of 92 conjugation-positive E. coli strains carrying blaCTX-M genes from pigs (n=76), chickens (n=8), and dogs (n=8) identified 230 PASs in the parental strains and 118 in their transconjugants. Among them, hok-sok, pemKI, and pndAC were the most frequently represented PASs in both the parental strains and the transconjugants. Moreover, the hok-sok and pemKI systems were strongly associated to IncF plasmids and the pndAC system to IncI1-Iγ plasmids. Our results suggest that the rapid spread of CTX-M genes in E. coli isolates among the animals could be attributed to the presence of multiple PASs in the CTX-M plasmids. To our knowledge, this is the first report of characterization of CTX-M associated PASs in E. coli isolates from pigs, chickens, and dogs. In addition, CTX-M-1 was detected for the first time in Korea.