Characterization of Actinobacillus pleuropneumoniae isolated from pigs in Japan using whole genome sequencing.

We conducted whole-genome sequencing to investigate the serotypes, the presence of virulence and antimicrobial resistance genes, and the genetic relationships among isolates of Actinobacillus. pleuropneumoniae derived from diseased pigs. Serotype 2 (71.2%) was the most common, but the prevalence of serotypes 6 (13.6%) and 15 (6.8%) increased. Existing vaccines are considered ineffective on the isolates belonging to serotypes 6 and 15. The phylogenetic tree based on core genome single nucleotide polymorphisms showed that the isolates were clustered by serotype. Of the isolates, 62.5% did not have an antimicrobial resistance gene, including a florfenicol resistance gene, but 32.2% had a tetracycline resistance gene. The antimicrobial resistant phenotype and genotype were almost identical. The plasmid-derived contigs harbored resistance genes of aminoglycosides, tetracyclines, ß-lactams, phenicols, or sulfonamides. It has been suggested that isolates with different genetic properties from vaccine strains are circulating; however, antimicrobial resistance may not be widespread.

Evaluation of the relationship between the minimum inhibitory concentration of ceftiofur and third-generation cephalosporins in Escherichia coli isolates from food-producing animals.

To enable future comparison of the antimicrobial susceptibility data between bacteria obtained from animals and humans, it is necessary to compare the relationships between minimum inhibitory concentrations (MICs) of veterinary and human medicine. We evaluated the relationship between the MIC of ceftiofur (CTF) and the MICs of other third-generation cephalosporins (TGCs): cefotaxime (CTX), cefpodoxime (CPDX), and ceftazidime (CAZ), determined by the broth microdilution method using 118 cefazolin-resistant Escherichia coli isolates from food-producing animals. Using the Clinical and Laboratory Standards Institute criteria, very major classification errors were observed only in CAZ (17.8%, 21 of 118); major and minor errors were observed in all TGCs (CTX: 0.8% [1 of 118] and 9.3% [11 of 118]; CPDX: 9.3% [11 of 118] and 6.8% [8 of 118]; CAZ: 2.5% [3 of 118] and 9.3% [11 of 118], respectively). The Spearman correlation coefficients between the MICs of CTF and CTX, CPDX, and CAZ were 0.765, 0.731, and 0.306, respectively. The sensitivity and specificity values were 100.0% and 81.8% for CTX, 99.0% and 27.3% for CPDX, and 76.0% and 86.4% for CAZ compared with CTF. The C-statistic was 0.978 for CTF and CTX, 0.953 for CPDX, and 0.798 for CAZ. For the TGCs evaluated in our study, testing for CTX susceptibility results showed the highest correlation with the results given when testing for CTF susceptibility.

Pathogenic characterization of Erysipelothrix rhusiopathiae Met-203 type SpaA strains from chronic and subacute swine erysipelas in Japan.

To characterize the Erysipelothrix rhusiopathiae Met-203 type surface protective antigen (Spa) A strains causing swine erysipelas in Japan, the nucleotide sequence of the hypervariable region of the spaA gene was determined in 80 E. rhusiopathiae (serotype 1a) isolates collected from pigs with chronic and subacute swine erysipelas in 14 prefectures in 2008-2014. In this study, 14 (17.5%) isolates were Met-203 type SpaA strains. We confirmed the pathogenicity of a Met-203 type SpaA strain in specific-pathogen-free pigs. In this experiment, the two challenged pigs displayed arthritis, urticaria and other clinical signs, but recovered within 10 days. Our results reveal the existence of the E. rhusiopathiae Met-203 type strains that have been causing chronic erysipelas in Japan.

Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.

Molecular Typing of Fluoroquinolone-Resistant Campylobacter jejuni Isolated from Broilers in Japan Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis.

Fluoroquinolone-resistant Campylobacter jejuni isolates from broilers in Japan were characterized using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) in order to elucidate the genetic relationship between these strains. Forty-three of the isolates were classified into 20 sequence types and were clustered into 21 PFGE types with 70% similarity. The most dominant clonal complex (CC) was CC-21 (41.9%). Diverse PFGE patterns were observed within the same CC, but the combined analysis of PFGE type and CC revealed that the strains with the same combination were isolated from the same district or neighboring districts. On the other hand, strains with the same combination pattern were also isolated from geographically distant districts. Our results elucidate two possible reasons for the prevalence of fluoroquinolone-resistant C. jejuni among broiler farms: (1) the resistant C. jejuni is clonally disseminated within the limited area, and (2) susceptible C. jejuni acquired fluoroquinolone resistance during the use of fluoroquinolone on the farms.

Decreased Resistance to Broad-Spectrum Cephalosporin in Escherichia coli from Healthy Broilers at Farms in Japan After Voluntary Withdrawal of Ceftiofur.

The high prevalence of broad-spectrum cephalosporin (BSC) resistance in Escherichia coli isolates from healthy broilers at farms is a source of grave concern in Japan. In an effort to solve this problem, the off-label use of ceftiofur (CTF) at hatcheries was voluntarily withdrawn around March 2012. The objective of this study was to assess the effect of the voluntary withdrawal on the prevalence of BSC resistance in E. coli from healthy broilers at farms. A total of 693 E. coli isolates collected from 362 fecal samples of healthy broilers at farms between 2010 and 2013 were examined to determine their antimicrobial resistance profiles and ß-lactamase genes. ß-Lactamase genes were characterized by polymerase chain reaction and sequencing. BSC resistance was detected in 84 of the 693 E. coli isolates (12.1%) from healthy broilers between 2010 and 2013. The percentage of BSC-resistant E. coli isolates was significantly decreased: from 16.4% (32/195) in 2010 and 16.8% (27/161) in 2011 to 9.2% (19/206) in 2012 and 4.6% (6/131) in 2013 (2010 versus 2012: p=0.024, 2010 versus 2013: p=0.001, 2011 versus 2012: p=0.038, and 2011 versus 2013: p=0.001). Regarding ß-lactamase genes, 58 of the 84 BSC-resistant E. coli isolates (69.0%) harbored blaCMY-2. The prevalence of BSC resistance in E. coli isolated from healthy broilers at farms was markedly decreased within a year after the voluntary withdrawal from CTF use at hatcheries. This indicates that BSC resistance in E. coli isolates from broilers could be controlled by restricting the use of CTF at the hatchery level.

Phylogenetic grouping, epidemiological typing, analysis of virulence genes, and antimicrobial susceptibility of Escherichia coli isolated from healthy broilers in Japan.

BACKGROUND: The aim of our study was to investigate the possible etiology of avian colibacillosis by examining Escherichia coli isolates from fecal samples of healthy broilers. FINDINGS: Seventy-eight E. coli isolates from fecal samples of healthy broilers in Japan were subjected to analysis of phylogenetic background, virulence-associated gene profiling, multi-locus sequence typing (MLST), and antimicrobial resistance profiling. Phylogenetic analysis demonstrated that 35 of the 78 isolates belonged to group A, 28 to group B1, one to group B2, and 14 to group D. Virulence-associated genes iutA, iss, cvaC, tsh, iroN, ompT, and hlyF were found in 23 isolates (29.5%), 16 isolates (20.5%), nine isolates (11.5%), five isolates (6.4%), 19 isolates (24.4%), 23 isolates (29.5%), and 22 isolates (28.2%) respectively. Although the genetic diversity of group D isolates was revealed by MLST, the group D isolates harbored iutA (10 isolates, 71.4%), iss (6 isolates, 42.9%), cvaC (5 isolates, 35.7%), tsh (3 isolates, 21.4%), hlyF (9 isolates, 64.3%), iroN (7 isolates, 50.0%), and ompT (9 isolates, 64.3%). CONCLUSIONS: Our results indicated that E. coli isolates inhabiting the intestines of healthy broilers pose a potential risk of causing avian colibacillosis.

Control of the development and prevalence of antimicrobial resistance in bacteria of food animal origin in Japan: a new approach for risk management of antimicrobial veterinary medicinal products in Japan.

Antimicrobial agents are essential for controlling bacterial disease in food-producing animals and contribute to the stable production of safe animal products. The use of antimicrobial agents in these animals affects the emergence and prevalence of antimicrobial resistance in bacteria isolated from animals and animal products. As disease-causing bacteria are often transferred from food-producing animals to humans, the food chain is considered a route of transmission for the resistant bacteria and/or resistance genes. The Food Safety Commission of Japan (FSC) has been assessing the risk posed to human health by the transmission of antimicrobial-resistant bacteria from livestock products via the food chain. In addition to the FSC's risk assessments, the Japanese Ministry of Agriculture, Forestry and Fisheries has developed risk-management guidelines to determine feasible risk-management options for the use of antimicrobial veterinary medicinal products during farming practices. This report includes information on risk assessment and novel approaches for risk management of antimicrobial veterinary medicinal products for mitigating the risk of development and prevalence of antimicrobial resistance in bacteria originating from food-producing animals in Japan.

Detection of aac(6')-Ib-cr in avian pathogenic Escherichia coli isolates in Japan.

We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in avian pathogenic Escherichia coli (APEC) strains in Japan. A total of 117 APEC strains collected between 2004 and 2007 were examined for PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA and oqxAB) by polymerase chain reaction. None of the APEC strains carried qnrA, qnrB, qnrC, qnrD, qnrS, qepA or oqxAB, but one of the isolates was identified as an AAC (6')-Ib-cr producer. Phylogenetic grouping, multi-locus sequence typing and serotyping showed that this isolate belonged to phylogenetic group A, sequence type 167 and untypable serogroup. To our knowledge, this is the first report of the aac (6')-Ib-cr gene in bacteria from food-producing animals in Japan.

Diversity of plasmid replicons encoding the bla(CMY-2) gene in broad-spectrum cephalosporin-resistant Escherichia coli from livestock animals in Japan.

Broad-spectrum cephalosporin (BSC) resistance has increased in Escherichia coli isolates from broiler chickens in Japan since 2004. The purpose of this study was to understand the epidemiology of BSC-resistant E. coli in livestock animals. Among 3274 E. coli isolates from 1767 feces of apparently healthy animals on 1767 farms between 2004 and 2009, 118 ceftiofur (CTF)-resistant isolates (CTF MIC ≥4 µg/mL) were identified on 74 farms. After elimination of apparently clonal isolates from a single animal, 75 selected CTF-resistant isolates (62 isolates from 61 broiler chickens, 10 isolates from 10 layer chickens, two isolates from two cows, and one isolate from a pig) were characterized. The bla(CMY-2) gene was most frequently detected in 50 isolates, followed by bla(CTX-M) (CTX-M-2: six isolates; CTX-M-14: four isolates; CTX-M-25: two isolates; CTX-M-1: one isolate) and bla(SHV) (SHV-12: seven isolates; SHV-2, SHV-2a, SHV-5: one isolate each). In particular, 42 of 62 broiler chicken isolates harbored bla(CMY-2). Pulsed-field gel electrophoresis analyses using XbaI revealed divergent profiles among the BSC-resistant isolates. The incompatibility groups of bla(CMY-2) plasmids from 34 of the 42 broiler chicken isolates belonged to IncIγ (10 isolates), IncA/C (nine isolates), IncB/O (seven isolates) and IncI1 (six isolates), or were nontypeable (two isolates). Co-transmission of resistance to non-ß-lactam antibiotics was observed in transconjugants with IncA/C plasmids, but not with IncI1, IncIγ, and IncB/O plasmids except for one isolate with IncB/O. Our findings suggest that the bla(CMY-2) gene is a key player in BSC-resistant E. coli isolates and that coselection is unlikely to be associated with the abundance of bla(CMY-2) plasmids, except for IncA/C plasmids.

Antimicrobial resistance in Shiga toxin-producing Escherichia coli O157 and O26 isolates from beef cattle.

This study was conducted to determine the prevalence of antimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) O157 (n = 241) and O26 (n = 11) isolated from beef cattle and to characterize their antimicrobial resistance profiles. Resistance to dihydrostreptomycin was detected most frequently (STEC O157, 9.5%; STEC O26, 54.5%), followed by resistance to oxytetracycline (7.9%; 45.5%) and ampicillin (5.4%; 36.4%). Resistance to one or more antimicrobial agents was detected in 13.3% (32/241) of the STEC O157 isolates and 54.5% (6/11) of the STEC O26 isolates. The antimicrobial resistance rate in the STEC O26 isolates was significantly higher than that in the STEC O157 isolates (P = 0.002, Fisher's exact test). The antimicrobial resistance rate in the STEC O157 isolates possessing both stx(1) and stx(2) genes was 26.3% (15/57), while that in the isolates possessing stx(2c) gene alone was 3.9% (3/77). These findings suggest that the antimicrobial resistance in STEC O157 is associated with serogroups and the Shiga toxin genotype.

Antimicrobial susceptibility of Escherichia coli isolates from wild mice in a forest of a natural park in Hokkaido, Japan.

To reveal the antimicrobial susceptibilities of Escherichia coli isolates from wild mice, 81 E. coli isolates were obtained from 109 voles (Clethrionomys spp.), 52 large Japanese field mice (Apodemus speciosus) and 19 small Japanese field mice (A. argenteus) captured in a forest of a natural park in Hokkaido, Japan. Seventy-eight of the 81 E. coli isolates were susceptible to all 10 antimicrobial agents tested. One E. coli isolate was resistant to ampicillin, dihydrostreptomycin, kanamycin, chloramphenicol and oxytetracycline. Two isolates were resistant to oxytetracycline. A low prevalence of antimicrobial resistance was maintained among wild mice that inhabited the forest.

Molecular typing and antimicrobial resistance of Salmonella enterica subspecies enterica serovar Choleraesuis isolates from diseased pigs in Japan.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates derived from diseased pigs in Japan during 2001 and 2005 were analyzed for biotype, based on H(2)S production and dulcitol fermentation, pulsed-field gel electrophoresis (PFGE) profile, and antimicrobial resistance profile. S. Choleraesuis biotype Choleraesuis (biotype Choleraesuis) was classified into one genotype, while varietas Kunzendorf (var. Kunzendorf) was classified into two genotypes. The isolates of var. Kunzendorf belonging to one genotype were isolated in a limited area of Japan. Variation in the antimicrobial resistance pattern was observed in isolates of both biotypes Choleraesuis and var. Kunzendorf. We have also shown that the PFGE profile was associated with the biotype and isolation region of each isolate.

Conjugative transposition of Tn916 and detection of Tn916-Like transposon in Erysipelothrix rhusiopathiae.

In order to investigate the origin of tetracycline resistance in Erysipelothrix rhusiopathiae, conjugative transpositions of Tn916 were tested. The frequency of transfer between strains of E. rhusiopathiae was about 10-fold higher than that between Enterococcus faecalis and E. rhusiopathiae. In addition, detection of a Tn916-like transposon was performed by PCR assay and DNA sequencing in E. rhusiopathiae field isolates. Of 49 tetracycline-resistant isolates, 38 (77.6 %) carried a Tn916-like transposon, while 11 (22.4 %) carried tet(M) only. These results suggested that Tn916-like transposon may be widely present in the E. rhsuiopathiae field isolates resistant to tetracycline.

National monitoring for antimicrobial resistance among indicator bacteria isolated from food-producing animals in Japan.

The antimicrobial susceptibilities of 2,205 isolates of Escherichia coli and 1,181 isolates of Enterococcus faecalis (n=610) and E. faecium (n=571) from apparently healthy cattle, pigs and broiler and layer chickens collected from 2000 to 2003 were examined using an agar dilution method. Overall, the isolates from cattle and layer chickens showed a lower incidence of resistance to almost all antimicrobials studied compared with those from pigs and broiler chickens. Fluoroquinolone resistance was found at a low level in isolates of E. coli from four animal species and in E. faecalis from pigs and broiler and layer chickens. Resistance to cephalosporin was identified in isolates of E. coli from broiler chickens in 2000-2002 and from four animal species in 2003. Incidence of antimicrobial resistance in the bacteria did not vary from year to year during the investigation period.

National surveillance of Salmonella enterica in food-producing animals in Japan.

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 beta-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.

Etiological and biological characteristics of Erysipelothrix rhusiopathiae isolated between 1994 and 2001 from pigs with swine erysipelas in Japan.

We investigated 66 Erysipelothrix rhusiopathiae strains isolated from pigs affected with swine erysipelas in Japan from 1994 to 2001 for serotype, pathogenicity towards mice, protection in vaccinated mice and antimicrobial susceptibility. Most of the isolates (84.8%) were serotype 1 or 2. For the first time, strains belonging to serotype 21 were isolated from cases of septicemia. Fifty isolates (75.8%) were highly virulent, 12 isolates (18.2%) were weakly virulent and 4 isolates were avirulent strains. All the mice vaccinated with the Koganei 65-0.15 vaccine strain survived challenge exposure with 50 highly virulent isolates. Six isolates (9.1%) grew on TPB-T80 agar containing 0.02% of acriflavine, and this was identical to the growth of the vaccine strain. Forty-seven isolates (71.2%) were resistant to oxytetracycline. The number of strains resistant to oxytetracycline among field isolates increased rapidly each year. Tylosin-resistant strains were also isolated (6.1%). These results suggest that certain characteristics, particularly antimicrobial susceptibility of E. rhusiopathiae isolates, change yearly in the field. Therefore, further investigation of the characteristics of E. rhusiopathiae field isolates is necessary.

Antimicrobial susceptibilities, serogroups, and molecular characterization of avian pathogenic Escherichia coli isolates in Japan.

In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83 --> Leu, Asp87 --> Asn) and ParC (Ser80 --> Ile, Glu84 --> Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan.

Farm-level impact of therapeutic antimicrobial use on antimicrobial-resistant populations of Escherichia coli isolates from pigs.

We investigated the farm-level impact of the use of several different antimicrobial agents on the population of antimicrobial-resistant commensal bacteria of animal origin to appropriately assess the release risk of resistance. This study was carried out based on the results of a survey on the history of antimicrobial drug use in 297 pig farms and antimicrobial susceptibility testing for the 545 Escherichia coli isolates (one or two isolates/pig/farm). A comparative analysis with the nonexposed herd revealed that ampicillin (ABPC) resistance in E. coli increased in the herds that were exposed to penicillin (relative risk [RR], 1.75) and penicillin-streptomycin (RR, 2.28); dihydrostreptomycin (DSM) resistance, in the penicillin-streptomycin-exposed herd (RR, 1.75); and trimethoprim (TMP) resistance in the methoprim-sulfonamide-exposed herd (RR, 2.10). On the other hand, ABPC and DSM resistances increased in the tetracycline-exposed herd (RR, 1.66 and 1.58, respectively); TMP resistance, in the penicillin-exposed herd (RR, 1.77); and oxytetracycline and kanamycin resistances, in the penicillin-streptomycin-exposed herd (RR, 1.28 and 2.22, respectively). These results demonstrated that the development of cross-resistance and coresistance, imposed by the therapeutic use of the antimicrobials studied, contributed the farm-level prevalence of antimicrobial-resistant E. coli and that the influence of coselection was characteristic to individual antimicrobial agents used.

Phage type and antimicrobial susceptibility of Salmonella enterica serovar Enteritidis from food-producing animals in Japan between 1976 and 2004.

A total of 56 isolates of Salmonella enterica serovar Enteritidis, including 38 isolates from poultry, 16 from cattle and two from pigs, collected between 1976 and 2004, were subjected to bacteriophage typing and antimicrobial susceptibility testing. Phage type (PT) 8 was predominant in bovine isolates, whereas PT1 and PT4 were predominant in poultry isolates. Resistance was found for 8 of 11 antimicrobials tested, at the following rates: 46.4% for dihydrostreptomycin followed by ampicillin and oxytetracycline (both 8.9%).