Erratum to: Draft Genome Sequences of a Unique t324-ST541-V Methicillin-Resistant Staphylococcus aureus Strain from a Pig.

This erratum is being published to correct the errors of the words in the section of introduction and title. The words of 't324'(left column, line 20, 21; right column, line 4, 14) in page 799 and title should be corrected as 't034'.

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.

Genome Sequence of a Unique t2247-ST692-III Livestock-Associated Methicillin-Resistant Staphylococcus aureus Strain from Chicken Carcass.

We report the draft genome sequence of a novel livestock-associated t2247-ST692-III methicillin-resistant Staphylococcus aureus strain designated K12S0375, which was isolated from a chicken carcass in South Korea. The K12S0375 strain contains uncommon genes, including antimicrobial resistance genes (tetL and tetS) and leukotoxin (lukED), and the genomic distance indicates a single lineage in a genome-based phylogenetic tree compared with 459 S. aureus genome sequences. This genome sequence will contribute to understanding epidemiological and genomic features of the ST692 lineage, including antimicrobial resistance and virulence genes.

Draft Genome Sequences of a Unique t324-ST541-V Methicillin-Resistant Staphylococcus aureus Strain from a Pig.

Methicillin-resistant Staphylococcus aureus (MRSA), the major causative agent of nosocomial infection, has also been reported from non-human sources. A sequence type (ST) 541 MRSA isolate designated K12PJN53 was isolated from a healthy pig in 2012. The genome of K12PJN53 consists of 44 contiguous sequences (contigs), totalling 2,880,108 bases with 32.88% GC content. Among the annotated contigs, 14, 17, and 18 contained genes related to antimicrobial resistance, adherence, and toxin genes, respectively. The genomic distance of strain K12PJN53 was close to the ST398 strains. This is the first report of the draft genome sequence of a novel livestock-associated MRSA ST541 strain.

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.

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

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.

Identification of livestock-associated methicillin-resistant Staphylococcus aureus isolates in Korea and molecular comparison between isolates from animal carcasses and slaughterhouse workers.

This study was undertaken to screen methicillin-resistant Staphylococcus aureus (MRSA) in animal carcasses and slaughterhouse workers and characterize MRSA isolates identified during 2010-2012 in Korea. A total of 830 (16.4%) S. aureus and 65 (1.3%) MRSA were isolated from 9669 carcass samples. MRSA was more frequently detected in chicken carcasses (1.2%) than in cattle (0.3%) and pig carcasses (0.6%). The prevalence of MRSA in workers was 6.9% (4/58) in chicken slaughterhouse workers, but no MRSA was detected in pig and cattle slaughterhouse workers (0/41). Two different lineages of MRSA were identified (i.e., human-associated type [ST5, ST59, and ST72] and livestock-associated [LA] type [ST398, ST541, and ST692]); only LA MRSA was observed in chicken carcasses, whereas both types were found in cattle and pig carcasses and workers. All human-associated MRSA isolates carried enterotoxin and/or leukotoxin genes, whereas LA MRSA types did not carry these genes, except ST692 type. However, all LA MRSA isolates were multiresistant, whereas human-associated types were susceptible or resistant to fewer than two antimicrobials except ST5. Furthermore, one or more resistance genes were attributed for resistance to aminoglycosides (aac(6')-Ie-aph(2″), ant(4')-Ia, and aph(3')-IIIa), tetracycline [tet(K), tet(L), tet(M), and tet(S)], macrolide-lincosamide-streptogramin B (ermA, ermB, ermC, and ermT), lincosamide [lnu(B)], phenicol-lincosamide-oxazolidinone-pleuromutilin-streptogramin A (cfr), chloramphenicol (fexA), and fusidic acid [fus(C)]. To our knowledge, this is the first report of tet(S) gene in MRSA isolates and first detection of a unique (ST692) type of MRSA in occupational workers. Detection of new types of human-associated and LA MRSA with multiple resistance and virulence genes in food animal products constitutes a potential threat to public health.

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

This study was focused on characterization of extended-spectrum ß-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.

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.

Molecular characterization of CTX-M ß-lactamase and associated addiction systems in Escherichia coli circulating among cattle, farm workers, and the farm environment.

A total of 84 extended-spectrum ß-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 blaCTX-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 blaCTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A blaCTX-M-14 gene was also detected in an E. coli isolate from a farmer's hand. Transfer of the blaCTX-M gene from 60 blaCTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from blaCTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) blaCTX-M genes possessed an insertion sequence, ISEcp1, upstream of the blaCTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The blaCTX-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 blaCTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying blaCTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the blaCTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.

Prevalence and molecular characterization of CTX-M ß-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 ß-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 ß-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 ß-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 ß-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 ß-lactamases in bacterial isolates from food animals in Korea. This study revealed that the CTX-M ß-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.

Prevalence of plasmid-mediated quinolone resistance determinants among Escherichia coli isolated from food animals in Korea.

The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in Escherichia coli isolated from food-producing animals and to characterize the PMQR-positive isolates. A total of 365 E. coli isolates which were either nalidixic acid resistant and ciprofloxacin susceptible (NAL(R)-CIP(S); n=185), or nalidixic acid and ciprofloxacin resistant (NAL(R)-CIP(R); n=180) were assessed for the presence of PMQR determinants by polymerase chain reaction. PMQR-positive isolates were further characterized by mutation analysis within the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE, phylogenetic group analysis, and pulsed-field gel electrophoresis (PFGE). Fourteen NAL(R)-CIP(S) (n=8) and NAL(R)-CIP(R) (n=6) E. coli isolates were positive for PMQR genes. Among them, qnrB4, qnrS1, and aac(6')-Ib-cr genes were detected in two (0.5%), eight (2.2%), and four (1.1%) isolates, respectively. None of the isolates harbored qnrA, qnrC, qnrD, and qepA genes. All but one PMQR-positive isolates harbored one or more point mutations in the QRDR of gyrA, and five of these isolates had additional mutations in the parC gene. Furthermore, one isolate each had additional substitutions in gyrB and parE genes, respectively. The most prevalent mutation was Ser83-Leu within the QRDR of gyrA. Phylogenetic analysis identified three major phylogenetic lineages, with phylogroups A (n=7) and D (n=4) being the most common phylogroups. None of the isolates belonged to virulent phylogroup B2. PFGE demonstrated that a combination of clonal and horizontal gene transmission is disseminating PMQR genes among the veterinary E. coli isolates in Korea. To our knowledge, this is the first report of occurrence of qnrB, qnrS, and aac(6')-Ib-cr genes in E. coli isolated from food-producing animals in Korea. Isolation of PMQR genes from food animals is a matter of concern since they could be transmitted to humans via food animals.

Molecular characterization of extended-spectrum-ß-lactamase-producing and plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolated from stray dogs in South Korea.

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 ß-lactamase (ESBL) and AmpC ß-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried bla(CTX-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 ß-lactamase-producing isolates were found to carry bla(CMY-2) genes. TEM-1 was associated with CTX-M and CMY-2 ß-lactamases in 4 and 15 isolates, respectively. In addition to bla(TEM-1), two isolates carried bla(DHA-1), and one of them cocarried bla(CMY-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 ß-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 bla(CTX-M) and bla(CMY-2) genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of bla(CMY-2) was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of bla(DHA-1) in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 ß-lactamase-producing E. coli isolates from dogs in the Republic of Korea.

Prevalence and mechanisms of quinolone resistance among selected nontyphoid Salmonella isolated from food animals and humans in Korea.

The aim of this study was to investigate the prevalence and mechanism of quinolone resistance among selected nontyphoid Salmonella (NTS) isolates. A total of 1279 NTS isolated from food animals (n=692) and humans (n=587) between 1995 and 2009 were investigated by serotyping, antimicrobial susceptibility testing, screening for plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6')-Ib-cr, and qepA and mutations in the quinolone resistance-determining region (QRDR) of gyrA and parC by PCR, and DNA sequencing. Three hundred thirty (47.7%) of 692 animal isolates and 177 (30.2%) of 587 human isolates were resistant to nalidixic acid. Most animal (94.8%, 313/330) and human (99.4%, 176/177) NTS exhibited decreased ciprofloxacin susceptibility (minimum inhibitory concentration [MIC]: 0.125-2 mg/L). None of them carried qnr or qepA gene. However, aac(6')-Ib was identified in six animal isolates, of which four carried aac(6')-Ib-cr gene. Based on antimicrobial resistance profile, year of isolation, MIC for quinolones and fluoroquinolones, and isolation frequency of serotype, 114 animal and 83 human isolates were tested for QRDR mutations. All contained a single mutation within the QRDR of gyrA at either codon 87 or 83, and 41 of them contained an additional mutation in parC. The most prevalent mutation was Asp87-Tyr (n=107), followed by Asp87-Gly (n=28), Asp87-Asn (n=26), Ser83-Tyr (n=22), and Ser83-Phe (n=14). Point mutations in parC were observed outside the QRDR, which included 40 isolates with Thr57-Ser substitution and 1 Salmonella Typhimurium with a novel Glu51-Lys substitution. In conclusion, a point mutation within the QRDR of gyrA was primarily responsible for quinolone resistance and reduced susceptibility to fluoroquinolones in NTS in Korea. To our knowledge, this is the first report of occurrence of aac(6')-Ib-cr gene among NTS in Korea. The spread of NTS carrying aac(6')-Ib-cr is of serious concern and should be carefully monitored.

Emergence of extended-spectrum beta-lactamase (CTX-M-15 and CTX-M-14)-producing nontyphoid Salmonella with reduced susceptibility to ciprofloxacin among food animals and humans in Korea.

Twenty of 1,279 nontyphoid Salmonella strains isolated from food animals and humans produced CTX-M-type extended-spectrum ß-lactamase. All expressed CTX-M-15, except two which coexpressed CTX-M-14 and TEM-1. Insertion sequence ISEcp1 was identified upstream of bla(CTX-M) genes. The bla(CTX-M-15) and bla(CTX-M-14) genes were disseminated by large conjugative IncFIIs and IncI1-Iγ plasmids, respectively.

Antimicrobial susceptibility of Staphylococcus aureus and characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Korea.

A total of 402 Staphylococcus aureus isolates from bovine mastitis milk collected during 2003-2009 in Korea were tested for susceptibility to 20 antimicrobial agents. All S. aureus isolates were susceptible to 11 of 20 antimicrobials tested; no resistance was observed against pirlimycin, telithromycin, novobiocin, penicillin/novobiocin, quinupristin/dalfopristin, clindamycin, rifampin, ciprofloxacin, trimethprim/sulfamethoxazol, vancomycin, and linezolid. Over 66% of the S. aureus isolates were resistant to penicillin. Resistance was also seen for gentamicin (11.9%), erythromycin (7.7%), methicillin (oxacillin and cefoxitin, 6.2%), and tetracycline (4.2%). No noticeable change was observed in penicillin, gentamicin, and erythromycin resistance over the 7-year period. Tetracycline resistance appeared to decrease consistently, whereas methicillin resistance was observed from 2005. About 2.7% (11/402) were resistant to three or more antimicrobials. Genotyping of the 17 methicillin-resistant S. aureus (MRSA) isolated from each cow revealed two staphylococcal cassette chromosome mec (SCCmec) types (IV and IVa), three spa types (t286, t324, and untypable), and two sequence types (ST1 and ST72). Eleven of 17 (64.7%) MRSA strains belonged to SCCmec IVa-t324-ST72. The rest of strains belonged to SCCmec IVa-t286-ST1 (n = 3) and SCCmec IV-untypable-ST72 (n = 3). None of the MRSA carried the Panton-Valentine leukocidin gene. These characteristics are the same as those found in community-acquired (CA) MRSA strains prevalent in humans in Korea. Three pulsed-field gel electrophoresis types (A-C) were observed among the 17 MRSA strains examined, and 14 strains belonged to the same pulsed-field gel electrophoresis pattern regardless of their geographical origin and year of isolation. The results of this study provide evidence of CA-MRSA infection in dairy cattle for the first time in Korea.