Genetic characteristics of CTX-M-type extended-spectrum-ß-lactamase (ESBL)-producing enterobacteriaceae involved in mastitis cases on Japanese dairy farms, 2007 to 2011.

Sixty-five CTX-M-2/15/14 extended-spectrum-ß-lactamase-producing Enterobacteriaceae were isolated from 258,888 mastitic milk samples from Japanese dairy farms between 2007 and 2011. CTX-M-2-producing Klebsiella pneumoniae and CTX-M-15-producing Escherichia coli were the predominant strains isolated. There was no predominant clonal type, and clonal diversity was found even in strains isolated from a single farm.

Prevalence and molecular epidemiological characterization of antimicrobial-resistant Escherichia coli isolates from Japanese black beef cattle.

We investigated the prevalence of antimicrobial-resistant Escherichia coli in Japanese black beef cattle from the three major production regions of Japan. We collected and examined 291 fecal samples from Japanese black beef cattle in Hokkaido, Chubu, and Kyushu. Of the 3,147 E. coli isolates, 1,397 (44.4%) were resistant to one or more antibiotics; these included 553 (39.8%) of 1,388 isolates from Hokkaido, 352 (54.4%) of 647 isolates from Chubu, and 492 (44.2%) of 1,112 isolates from Kyushu. The difference in resistance rates between the three regions was significant. The antibiotics with the highest rates of resistance were oxytetracycline and dihydrostreptomycin (35.8% each), followed by ampicillin (21.4%). Further, E. coli isolates from calves had higher resistance rates than those from growing cattle and mature cattle, and the calf isolates showed high rates of resistance to gentamicin (20.2%), enrofloxacin (9.4%), and ceftiofur (4.2%). In addition, the high degrees of similarity in the genotypes of the isolates and in the resistance patterns on each farm suggest that resistance bacteria and resistance genes were horizontally transferred. Most isolates, in each of the three regions, harbored resistance genes such as blaTEM, strA, strB, aphA1, aphAI-IAB, and catI. In contrast to the isolates from Kyushu, most of which harbored aacC2, tetB, and dfrA12, the isolates from Hokkaido and Chubu harbored a variety of resistance genes. Furthermore, the prevalence of genes for resistance to dihydrostreptomycin, gentamicin, chloramphenicol, and trimethoprim differed significantly between the regions. This is the first large-scale study describing and comparing antimicrobial-resistant bacteria from different regions in Japan. The results will contribute to improving food safety and promoting careful usage of antimicrobial agents.

Prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan.

This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan. We collected bovine colostrum samples from 210 dams from 21 dairy farms in Hokkaido prefecture (Japan) between March and June 2009. L. monocytogenes was detected in samples from 6 (28.6%) of the 21 farms. Of the 210 samples, 16 (7.6%) were positive for L. monocytogenes. We recovered 80 L. monocytogenes isolates; 44 (55%) isolates were classified as serotype 1/2b and 36 (45%) were classified as serotype 4b. The isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole. Pulsed-field gel electrophoresis (PFGE) characterization of the 80 isolates revealed six PFGE types. Two PFGE types corresponded to human listeriosis cases. Most L. monocytogenes isolates possessed virulence-associated genes (actA, hly, iap, inlA, inlC, mpl, plcA, plcB, opuCA, prfA, and clpC). One PFGE type isolate possessed an epidemic clone II marker. Our findings suggest that isolates from bovine colostrum have the potential to cause human and animal listeriosis. This is the first study on the prevalence and characteristics of L. monocytogenes isolated from bovine colostrum obtained from dairy farms. Our results have important implications for improving public health and elucidating the epidemiology of L. monocytogenes in bovine colostrum.

Antimicrobial resistance in fecal Escherichia coli isolated from growing chickens on commercial broiler farms.

To investigate the effects of rearing practices of commercial broiler chickens on the incidence of antimicrobial resistance in commensal Escherichia coli isolates, fecal E. coli isolates obtained in 4 farms were screened for anitimicrobial resistance. Ten E. coli isolates were recovered from each of the fecal samples collected from 10 birds in the farms at the ages of 2 days, 14-17 days, and 47-50 days. In 2 out of the 4 farms, no antimicrobials were used during the rearing period. In the other two farms, following collection of the fecal samples at 14 and 15 days of age, oxytetracycline (OTC), sulfadimethoxine (SDMX), and tylosin were given to birds on one farm and SDMX was used in the other. Isolates resistant to ampicillin and OTC that were obtained from an untreated flock at different sampling times were closely related to each other by pulsed-field gel electrophoresis patterns (PFGE) of XbaI-digested chromosomal DNA. PFGE analysis together with in vitro conjugation experiments suggested that diversity of resistance phenotypes within a clone may be resulted from the acquisition and loss of R-plasmids in an untreated and a treated flock. The numbers of resistance phenotypes observed among fecal isolates increased during the growth of the chickens in all the farms. The results in the present study suggest that persistence of commensal E. coli strains resistant to antimicrobials even in the absence of antimicrobial administration. It is also hypothesized that horizontal transmission of resistance determinants resulted in the emergence of different resistance phenotypes in those farms.

Isolation of Pseudomonas aeruginosa from open ocean and comparison with freshwater, clinical, and animal isolates.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for morbidity and mortality in humans, animals, and plants. This bacterium has been regarded to be widely present in terrestrial and freshwater environments, but not in open ocean environments. Our purpose was to clarify its presence in open ocean, and their genotypic and physiological characteristics were compared with those of isolates from clinical, animal, and freshwater sources. Water samples were collected from freshwater, bays, and offshore environments in Japan. Sixty-two isolates, including 26 from the open ocean, were identified as P. aeruginosa by phenotypic characteristics and the BD Phoenix System. Pulsed-field gel electrophoresis (PFGE) was performed on all strains, together with 21 clinical and 8 animal strains. The results showed that open ocean strains are composed of a few genotypes, which are separated from other strains. Although some clinical isolates made a cluster, other strains tended to mix together. Different antibiotypes were observed among marine isolates that had similar PFGE and serotyping patterns. Some were multidrug-resistant. Laboratory-based microcosm study were carried out to see the responses of P. aeruginosa toward increased NaCl concentrations in deionized water (DW). Marine strains showed better survival with the increase, whereas river and clinical strains were suppressed by the increase. These findings illustrate the potential significance of open ocean as a possible reservoir of P. aeruginosa, and there may be clones unique to this environment. To our knowledge, this is the first report on the presence and characterization of P. aeruginosa in the open ocean.

Antimicrobial resistance in Salmonella isolates from apparently healthy food-producing animal from 2000 to 2003: the first stage of Japanese veterinary antimicrobial resistance monitoring (JVARM).

Antimicrobial susceptibility of 183 Salmonella isolates from apparently healthy food-producing animals obtained during the period from 2000 to 2003 throughout Japan was examined. Of 29 serovars identified, Salmonella Infantis (37.7%) was the most prevalent, followed by S. Typhimurium (19.7%). Salmonella bacteria resistant to dihydrostreptomycin (77.6%) were about 10% more prevalent than those resistant to oxytetracycline (67.8%), though the nation-level veterinary use of tetracycline antibiotics is much greater than that of streptomycin in Japan. In seventeen isolates (9.3%) resistant to nalidixic acid, single point mutations were detected at 84 or 87 in the quinolone resistance-determining region of the gyrA gene.

Antimicrobial resistance types and genes in Salmonella enterica infantis isolates from retail raw chicken meat and broiler chickens on farms.

Salmonella enterica subsp. enterica serotype Infantis isolates from retail raw chicken meat (n = 98) and broiler chickens on farms (n = 70) were examined for antimicrobial susceptibility and antimicrobial resistance genes. A total of 15 antimicrobial resistance types, 14 in meat and 10 in broiler isolates, were identified, and 9 of the 15 types were indistinguishable between meat and broiler isolates. Resistance to both oxytetracycline and dihydrostreptomycin accounted for 94.0% of the resistance types in meat and broiler isolates, and each type harbored aadA1 within 1.0 kb of class 1 integron and tetA. Of nalidixic acid resistance types, point mutations at 87Asp (GAC) to Tyr (TAC) in the quinolone resistance-determining region of gyrA was detected in 10 of 13 meat isolates and at 87Asp to Asn (AAC) in four of seven broiler isolates. These findings suggest that the antimicrobial resistance of Salmonella Infantis in retail chicken meat predominantly originates from broiler chickens.

Extended-spectrum-beta-lactamase-producing Escherichia coli strains isolated from farm animals from 1999 to 2002: report from the Japanese Veterinary Antimicrobial Resistance Monitoring Program.

A nationwide surveillance for antimicrobial susceptibility in Escherichia coli strains isolated from food-producing animals in Japan was conducted from 1999 to 2002. Eighteen cefazolin-resistant E. coli strains were isolated from broilers. Six were CTX-M-type producing, and eight were CMY-2 producing, while eight had mutations at the ampC promoter region.

Antimicrobial susceptibility of Mannheimia haemolytica isolates from cattle in Japan from 2001 to 2002.

A total of 27 clinical isolates of Mannheimia haemolytica from cattle in Japan from 2001 to 2002 were examined for antimicrobial susceptibility to 25 antimicrobial agents. The minimum inhibitory concentrations of 25 different antimicrobials were determined by an agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards. Of the 27 isolates, seven isolates (26.9%) were resistant to at least one of the 25 drugs and resistance rates ranged from 3.7 to 18.5%. Resistance rates to dihydrostreptomycin (18.5%), oxytetracycline (11.1%), and doxycycline (11.1%) were relatively high and those to the remaining drugs were less than 10%.

Epidemiological characterization of Salmonella Typhimurium DT104 prevalent among food-producing animals in the Japanese veterinary antimicrobial resistance monitoring program (1999-2001).

In the course of nationwide investigation on epidemiological characteristics in Salmonella Typhimurium isolates from food-producing animals in Japan between 1999 and 2001, fifty-seven isolates of S. Typhimurium DT104 and 104B obtained from cattle and swine at farm level in Japan between 1999 and 2001 were classified with pulsotype and antimicrobial resistance type. Most of the isolates were resistant to five or more antimicrobials and were genotyped into four groups. The present nationwide investigation shows that at least 11 types of S. Typhimurium related to DT104 are prevalent among food-producing animals across the country.

Rapid detection of quinolone-resistant Salmonella by real time SNP genotyping.

A total of 63 isolates were screened for the gyrA mutation (87Asp-Tyr) in Salmonella enterica serovars using real time PCR. All of the isolates were successfully identified as resistant or susceptible, consistent with the MIC result of the agar dilution method and gyrA sequencing.

Antimicrobial susceptibility of Salmonella isolated from cattle, swine and poultry (2001-2002): report from the Japanese Veterinary Antimicrobial Resistance Monitoring Program.

OBJECTIVES: The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) Program was established in 1999 to examine the susceptibility of bacteria from food-producing animals to antimicrobial agents. This study tested the susceptibility of Salmonella isolates collected during 2001-2002 to 20 antimicrobials. MATERIALS AND METHODS: MICs of antimicrobial agents were determined using the NCCLS agar dilution method, and interpreted according to breakpoints obtained from the bimodal MIC distributions. RESULTS: A total of 82 Salmonella were isolated from food-producing animals and tested for antimicrobial susceptibility. Isolates resistant to ampicillin, dihydrostreptomycin, kanamycin, oxytetracycline, chloramphenicol, bicozamycin, nalidixic acid, oxolinic acid and trimethoprim were obtained from healthy animals and diagnostic sample submissions. Salmonella Dublin was isolated only from cattle and showed resistance to older quinolones. Resistance to ampicillin, dihydrostreptomycin, kanamycin and oxytetracycline was common across all serotypes. Fluoroquinolone-resistant Salmonella Choleraesuis was isolated from swine and was the first Japanese report on this type of resistance in Salmonella from an animal origin. Most Salmonella Typhimurium isolates showed resistance to ampicillin, chloramphenicol, dihydrostreptomycin and oxytetracycline. S. Typhimurium DT104 accounted for 40.7% of S. Typhimurium isolates and was more often multi-drug resistant. Most Salmonella Infantis isolates from poultry showed resistance to dihydrostreptomycin, oxytetracycline, trimethoprim or kanamycin. In Salmonella Enteritidis, the major serotype isolated from food-poisoning in Japan, only resistance to dihydrostreptomycin was observed. CONCLUSIONS: This is the first JVARM report of Salmonella isolates, and continuous investigations at the national level on antimicrobial resistance in Salmonella isolated from food-producing animals will be important in the JVARM Program.