Prevalence of Salmonella in broiler chickens in Kagoshima, Japan in 2009 to 2012 and the relationship between serovars changing and antimicrobial resistance.

BACKGROUND: This study aimed to examine the prevalence, serovars, and antimicrobial resistance of Salmonella isolates from broiler chickens in Kagoshima, Japan. A total of 192 flocks and 3071 samples were collected from broiler chickens at local farms in Kagoshima, Japan from 2009 to 2012. RESULT: Among the tested farms, 49.0% of flocks were positive for Salmonella, and 243 isolates were obtained from 3071 cecal samples (7.9%). All the Salmonella isolates were one of three serovars: S. Infantis (57.6%); (140/243), S. Manhattan (40.3%; 98/243 and S. Schwarzengrund (2.1%; 5/243). The proportion of S. Infantis isolates decreased from 66.0% in 2009 to 50.0% in 2011 but increased to 57.6% in 2012, while the proportion of S. Manhattan isolates significantly increased from 26.4 to 50% from 2009 to 2011, and decreased moderately to 40.9% in 2012. Most of the recovered Salmonella isolates were resistant to three antimicrobials, i.e., streptomycin (95.1%), sulfamethoxazole (91.0%) and oxytetracycline (91.4%). In contrast, all Salmonella strains were susceptible to chloramphenicol. Comparison of this study to previous studies of the antimicrobial susceptibility of Salmonella isolates showed that: the percentage of antibiotic-resistance isolates increased dramatically for two antibiotics, ampicillin (from 22.4 to 55.1%) and cefotaxime (from 9.1 to 52.7%). In contrast, the percentage of ofloxacin-resistant isolates decreased across the three survey periods, from 20.8% in 2004-2006 to 1.6% in the present study period (2009-2012). In addition, S. Infantis exhibited a variety of resistance to antimicrobials examined from sensitive to resistance to eight antimicrobials. Multidrug resistance to more than 6 six antimicrobials was detected in 113 (46.5%) of the isolates, and most of them were S. Manhattan. CONCLUSIONS: There was a marked change in the serovars and antimicrobial resistance profiles of the Salmonella isolates in this study compared to those in previous studies. The percentage of S. Manhattan isolates increased as did the percentages of ampicillin- and cefotaxime-resistant isolates.

Antimicrobial susceptibility of Trueperella pyogenes isolated from cattle and pigs with septicemia in southern Kyushu, Japan.

We investigated the antimicrobial susceptibility of Trueperella pyogenes isolates from cattle and pigs diagnosed with septicemia at slaughter. The strains investigated in this study were found to be susceptible to ampicillin, cefazolin, gentamicin, and levofloxacin. However, 53.3% of the strains were resistant to tetracycline, 49.7% to streptomycin, 13.8% to erythromycin, 13.2% to clindamycin, and 1.2% to oxacillin and kanamycin. We also found that the resistance of pig-derived strains of T. pyogenes to tetracycline, streptomycin, erythromycin, and clindamycin was higher than that of cattle-derived strains, and these strains were resistant to up to four antimicrobial agents used simultaneously. The present results provide new data on the range of the minimum inhibitory concentration (MIC) values of various antimicrobial agents for T. pyogenes isolated from livestock with septicemia. The breakpoint values used in this study are generally considered applicable.

The incidence of Campylobacter contamination levels through Chicken-Sashimi Processing steps in A Small-scale Poultry processing plant applying the External stripping method.

This study aimed to analyze the incidence of Campylobacter in a small-scale chicken meat processing plant producing "chicken-sashimi", and determine the effectiveness of surface burning as a treatment during processing. The most probable number (MPN) method was used to analyze the load of Campylobacter in 48 samples from four different processing steps (de-feathering, chilling, surface burning, and final-products; 12 samples each). We found the highest load of isolated bacteria in chicken skin after de-feathering. Campylobacter was not detected after the surface burning step despite a large load of bacteria present in the cecum content. Campylobacter was absent in the final products. Adequate surface burning can avoid Campylobacter contamination of chicken sashimi in the processing plant by applying the external stripping method.

Decreasing trend of ß-lactam resistance in Salmonella isolates from broiler chickens due to the cessation of ceftiofur in ovo administration.

Ceftiofur, a third-generation cephalosporin antimicrobial, was used in Japanese hatcheries for many years before 2012. We continue to study Salmonella in broilers and their antimicrobial resistance. The current study aimed to express how the cessation of ceftiofur affects the antimicrobial resistance of Salmonella isolated from broiler chickens in Kagoshima Prefecture, Japan in 2017 and 2018. A total of 274 Salmonella isolates were recovered from 1535 cecal content samples obtained from 96 broiler flocks over years 2017 and 2018. Among the S. enterica isolates, the predominant serovars were S. Manhattan (128/274, 46.7%), S. Schwarzengrund (120/274, 43.8%), and S. Infantis (26/274, 9.5%). The isolates showed a high proportion of antimicrobial resistance for oxytetracycline, sulfamethoxazole, and streptomycin. However, the ß-lactam resistance rates were significantly decreased (p < 0.01) in 2017, while no ß-lactam resistant isolates detected in 2018. The highlight of this study was the complete disappearance of ß-lactam resistance in Salmonella isolates from broiler chicken in Kagoshima, Japan.

Detection and characterization of extended-spectrum beta-lactamase (TEM-52)-producing Salmonella serotype Infantis from broilers in Japan.

During 2004 and 2006, multidrug-resistant Salmonella enterica subspecies enterica serovar Infantis (Salmonella Infantis) isolates (n = 120) were recovered from broiler cecal samples collected from a meat-processing plant, and the isolates were examined. The study was conducted to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Salmonella Infantis isolates recovered from broiler chickens and determine the mechanisms of transfer of the resistance traits. Extended-spectrum cephalosporins-resistant Salmonella Infantis isolates producing ESBL TEM-52 were detected. The mutant bla(TEM-52) gene and the wild-type bla(TEM-1) gene that mediated resistance to ampicillin (an extended-spectrum penicillin) and cephalothin (a narrow-spectrum cephalosporin) were located on approximately 50-kb conjugative plasmids among beta-lactam-resistant (n = 29) isolates. The bla(TEM) genes did not cotransfer with aadA1, sul1 (both associated with class 1 integrons), tetA, and dfrA5, signifying a chromosomal location of these non-beta-lactam resistance-encoding genes. This is the first report describing TEM-52-producing S. enterica from food-producing animals in Japan. An emergence of TEM-type ESBL is an important concern to public health because this readily transferable resistance mechanism threatens the value of the third-generation cephalosporins and may reduce the clinical utility of this class of antibiotics against pathogenic Gram-negative bacteria.

Antimicrobial susceptibility of Erysipelothrix rhusiopathiae isolated from pigs in Southern Japan with a modified agar dilution method.

The determination of antimicrobial minimum inhibitory concentration (MIC) in Erysipelothrix rhusiopathiae by using the agar dilution method has not been covered by the Clinical and Laboratory Institute (CLSI). Only the broth microdilution method has been outlined. This report describes a modification of the agar dilution procedure for E. rhusiopathiae using Trypto-soy agar supplemented with 0.1% Tween 80 and incubation in ambient air at 37 degrees C for 24 hr. The MICs of the assay were in agreement with those of the broth microdilution method recommended by the CLSI. Antimicrobial susceptibility test was performed using this method for 149 E. rhusiopathiae isolates from 2 meat processing plants in Kagoshima Prefecture during the period of April 2004 to March 2005. The number of strains resistant to oxytetracycline, erythromycin, lincomycin, ofloxacin and enrofloxacin were 56 (37.6%), 4 (2.7%), 18 (12.1%), 21 (14.1%) and 19 (12.8%), respectively. All strains were susceptible to ampicillin.

Antimicrobial susceptibility of Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis lesions in Japan.

The minimal inhibitory concentrations (MICs) of 23 Treponema phagedenis-like spirochetes isolated from dairy cattle with papillomatous digital dermatitis (PDD) lesions in Japan were investigated by a broth microdilution method using 15 antimicrobial agents. Although all MIC values showed a monomodal distribution, the MICs of the antimicrobial agents for 90% (MIC(90)) of the isolates tested varied among the agents examined. The MIC(90) values for penicillin G, ampicillin, and erythromycin were <0.06 microg/ml. In contrast, the MIC(90) values for kanamycin, streptomycin, rifampicin, sulfamethoxazole, trimethoprim, and colistin were >128 microg/ml. Oxytetracycline, lincomycin, enrofloxacin, chloramphenicol, ceftiofur, and gentamicin showed intermediate values, i.e., 0.5~32 microg/ml. The present study suggested that no isolate had acquired resistance to the antimicrobial agents examined, although they may have natural resistance to some agents. Furthermore, the in vitro antimicrobial susceptibility data would provide helpful information for PDD treatment and the development of a selective medium for isolating the organism effectively.

Characterization of antibiotic resistance and the emergence of AmpC-producing Salmonella Infantis from pigs.

During the period of 2007-2008, a total of 270 pig fecal samples were collected from a meat processing plant located in southern Japan and examined for Salmonella species. A total of 44 Salmonella isolates were recovered, and antimicrobial resistance was detected in serotypes Typhimurium (n=9), Infantis and Choleraesuis (n=2), and Derby, Miyazaki and Schwarzengrund (n=1). Multidrug resistance was seen in serotypes Typhimurium (n=8) and Infantis (n=2). The most commonly observed resistance phenotypes were against streptomycin, oxytetracycline and sulfamethoxazole (100%), ampicillin (90%), chloramphenicol (50%), cephalothin (30%) and cefoxitin, ceftazidime and kanamycin (each 20%). Extended-spectrum cephalosporin-resistant Salmonella Infantis isolates producing plasmid-mediated, bla(CMY-2) gene were detected. These AmpC-producing isolates showed resistance to ampicillin and cephems (cephalothin, cefoxitin and ceftazidime). Resistance transfer experiments showed that transconjugants and transformants coexpressed resistance phenotypes similar to the donor isolates. To the best of our knowledge, this is the first report worldwide describing serovar Infantis from pigs capable of producing AmpC ß-lactamase. Then, we detected the pentadrug-resistance phenotype in Salmonella Typhimurium isolates, which yielded class 1 integron amplicons of 1.0 and 1.2 kb. Genetic fingerprinting analysis by pulsed-field gel electrophoresis and an assay by polymerase chain reaction confirmed the isolates to be Salmonella Typhimurium DT104. In conclusion, the findings of this survey call for the systematic and comprehensive domestic and international surveillance programs to determine the true rates of occurrence of AmpC-producing Salmonella both in the livestock and public health sectors.

α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.

Differences in phenotypic and genetic characteristics of Trueperella pyogenes detected in slaughtered cattle and pigs with septicemia.

We investigated the hemolytic properties, biochemical properties, and possession of virulence factor genes of Trueperella pyogenes isolated from cattle and pigs with septicemia. The porcine strains showed significantly stronger hemolyticity than the bovine strains. In addition, T. pyogenes from cattle and pigs also differed in biochemical properties. Virulence factor genes (nanP, cbpA, fimC, and fimE) were more prevalent in bovine strains, whereas other virulence factor genes (nanH and fimG) were more prevalent in porcine strains. T. pyogenes isolated from pig and cattle with septis cases in Japanese meat inspection showed variability in biochemical and genetic properties. Differences were observed between porcine and bovine strain in term of the hemolytic strength and possession of genes for factors promoting adhesions which are considered pathogenic.

Increased Salmonella Schwarzengrund prevalence and antimicrobial susceptibility of Salmonella enterica isolated from broiler chickens in Kagoshima Prefecture in Japan between 2013 and 2016.

This study aimed to analyze the Salmonella serovars, measure the minimum inhibitory concentration of antimicrobials, and examine the antimicrobial resistance genes of Salmonella isolated from 192 broiler flocks in Kagoshima Prefecture in Japan, from 2013 to 2016. We found that all Salmonella isolates belonged to three serovars: Salmonella Manhattan, S. Infantis, and S. Schwarzengrund. Among them, S. Schwarzengrund prevalence has recently increased annually making the main serovar. Most recovered isolates were highly resistant to streptomycin, sulfamethoxazole, and oxytetracycline. We saw the reduction of third-generation cephalosporin resistance and identified the reason of increased kanamycin resistance to be the increased number of S. Schwazengrund isolates. Among the kanamycin-resistant Salmonella isolates, aphA1 constituted the main resistance gene detected.

16S rRNA Gene Amplicon Sequence Data from Feces of Wild Deer (Cervus nippon) in Japan.

We report 16S rRNA amplicon sequence data from feces of 109 wild deer in Japan. The dominant bacterial taxa in fecal microbiota of wild deer hunted between village and mountainous areas and those living on Miyajima Island and in Nara Park were similar but differed in abundance.

16S rRNA Gene Amplicon Sequence Data from Feces of Five Species of Wild Animals in Japan.

We report 16S rRNA amplicon sequence data from feces from 58 wild boars, 60 feral raccoons, 9 wild Japanese badgers, 21 wild masked palm civets, and 8 wild raccoon dogs in Japan. The predominant bacterial taxa in the fecal microbiota were similar in part but varied among the animal species.

Surveillance of Shiga toxin-producing Escherichia coli and Campylobacter spp. in wild Japanese deer (Cervus nippon) and boar (Sus scrofa).

Increasing game meat consumption in Japan requires the dissemination of safety information regarding the presence of human pathogens in game animals. Health information regarding the suitability of these animals as a meat source is not widely available. In this study, we aimed to evaluate the safety of game meat and detect potential human pathogens in wild deer (Cervus nippon) and boar (Sus scrofa) in Japan. Fecal samples from 305 wild deer and 248 boars of Yamaguchi, Kagoshima, and Tochigi prefectures collected monthly for 2 years were examined for the prevalence of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter spp. STEC was isolated from 51 deer consistently throughout the year and from three boars; O-antigen genotype O146, the expression of stx2b, and eaeA absence (n=33) were the major characteristics of our STEC isolates. Other serotypes included the medically important O157, stx2b or stx2c, and eaeA-positive (n=4) and O26, stx1a, and eaeA-positive strains (n=1). Campylobacter spp. were isolated from 17 deer and 31 boars. Campylobacter hyointestinalis was the most common species isolated from 17 deer and 25 boars, whereas Campylobacter lanienae and Campylobacter coli were isolated from three and two boars, respectively. Seasonal trends for the isolation of these bacteria were not significant. This study demonstrates that wild game animals carry human pathogens; therefore, detailed knowledge of the safe handling of game meat is needed to prevent foodborne infections.

Successful treatment of bacillary hemoglobinuria in Japanese Black cows.

Four pasture-fed Japanese Black cows showed the main clinical symptoms of severe hemoglobinuria at different periods between 2003 and 2007. Hematological analyses at the first consultation revealed severe anemia, and biochemical analyses indicated both severe hemolysis and disruption of hepatic function. Although the first 2 patients died, the hemoglobinuria and general condition of the remaining 2 cows, who were immediately initiated on large doses of antibiotics, improved within 3 days. Clostridium haemolyticum was detected by polymerase chain reaction (PCR) analysis of the blood sample of 1 of the infected cows. Anti-fascioliasis medicine is administered, and since then, no case of hemoglobinuria has been observed. The cows were diagnosed with bacillary hemoglobinuria, and they represent the first few cases in Japan.

Surveillance of antibiotic resistance in Escherichia coli isolated from wild cranes on the Izumi plain in Kagoshima prefecture, Japan.

The prevalence of antibiotic resistance in 376 Escherichia coli (E. coli) isolates from fecal samples of Hooded and White-naped cranes was investigated on the Izumi plain in Kagoshima prefecture, Japan, during winter 2016 and 2017. Resistance to oxytetracycline, ampicillin, and nalidixic acid were observed in 10.9%, 3.1-4.4%, and 2.1-7.7% of isolates, respectively. Since the previous surveillance in 2007, isolation rates of antibiotic-resistant E. coli recovered from wild cranes have remained at significantly low levels compared with those in Japanese livestock. Our results indicate that surveillance of antibiotic-resistant E. coli from wild cranes wintering in the Izumi plain could be a useful strategy to indicate natural environmental pollution by antibiotic-resistant bacteria in the environment.

Antimicrobial susceptibility phenotypes, resistance determinants and DNA fingerprints of Salmonella enterica serotype Typhimurium isolated from bovine in Southern Japan.

A longitudinal study was conducted in cattle to determine the antimicrobial resistance phenotypes, integron elements, resistance genes and pulsed-field gel electrophoresis fingerprints among Salmonella enterica serotype Typhimurium isolates. A total of 33 strains were isolated and categorised into Groups A, B and C during the period 1989-2004. Thirty-one strains (93.9%) showed resistance to ampicillin (A) encoded by bla(OXA-1), bla(TEM) and bla(PSE-1) genes; 84.8% showed resistance to chloramphenicol (C) encoded by floR and catA1; 97.0% were resistant both to streptomycin (S) and sulfamethoxazole (Su), the former encoded by aadA1 and aadA2; 100% were resistant to oxytetracycline (T) encoded by tetA, tetB and tetG; and 42.4% were resistant to kanamycin (Km) encoded by aphA1-Iab. Multidrug resistance types observed were ACSSuT-Km (n=13), ACSSuT (n=15), ASSuT (n=3) and SSuT (n=2). Class 1 integrons ranging from 1.0 kb to 1.9 kb were detected from 54.5% of isolates (18/33). Integrons were not detected initially (1989-1992), then during the 1993-1996 interval a high frequency of 1.0 kb and 1.2kb amplicons were detected and during 2000-2004 the amplicon size increased to 1.7 kb and 1.9 kb. We report evidence of additional integration of resistance gene cassettes as shown by integrons with increased size. Finally, group B strains showed banding patterns indistinguishable from S. Typhimurium DT104 reference strain, indicating that the DT104 lineage existed on the island since 1993.

Absence of correlation between karyotype profiles of Trypanosoma congolense and resistance to isometamidium chloride.

Chromosome profiles of 10 Trypanosoma (T.) congolense populations with different isometamidium sensitivities were compared using the pulsed field gel electrophoresis technique. The aim was to elucidate whether there was a karyotype pattern specific to eight isometamidium resistant phenotypes. Analysis of the profiles indicated that all populations displayed several discrete bands at the region of small, intermediate and large chromosomes. The highest similarity was observed between two isolates originating from Burkina Faso, indicating that they had the same genetic origin. Other eight strains exhibited different patterns in terms of chromosome size and numbers such that there was no characteristic karyotype pattern that was established specifically to identify resistant populations and discriminate them from the sensitive ones. This study has revealed that isometamidium resistance is not correlated to karyotype profile in T. congolense.

Molecular epidemiology of antimicrobial resistance among Salmonella enterica serovar Infantis from poultry in Kagoshima, Japan.

Antimicrobial susceptibility and resistance genes of 135 strains of Salmonella enterica serovar Infantis isolated from poultry in Kagoshima were examined. One strain (0.7%) was resistant to ampicillin (A), 97% to streptomycin (S), 95.6% to sulphamethoxazole (Su), 96.3% to oxytetracycline (T), 11.1% to kanamycin (Km) and 36.3% to ofloxacin (O). Multiresistant phenotypes identified were ASSuT-Km, SSuT-Km, SSuT-O and SSuT. Class 1 integrons were detected in 94.8% of isolates. Approximately 89% of oxytetracycline-resistant strains carried the tetA gene and all of the 131 streptomycin-resistant isolates carried the aadA1a gene. Forty-percent of kanamycin-resistant isolates carried the aphA1-Iab gene. All isolates were susceptible to chloramphenicol. Recognition of TEM-type beta-lactamase in a S. Infantis strain from chickens is a recent rare finding in Japan.

Analysis of Campylobacter spp. contamination in broilers from the farm to the final meat cuts by using restriction fragment length polymorphism of the polymerase chain reaction products.

We investigated the genotype diversity and dynamics of Campylobacter jejuni and Campylobacter coli in six commercial broiler farms during rearing and abattoir processing. In total, 223 C. jejuni and 36 C. coli strains isolated (on farm, transportation crates, carcasses after defeathering, and chicken wing meat at the end of the processing line) were subtyped by PCR-RFLP based on flagellin (fla typing) gene. Eleven (C. jejuni) and four (C. coli) different RFLP patterns were found. Multiple C. jejuni genotypes were identified in five out of six farms (except Farm 5). Furthermore, a clear tendency for dominance of particular genotypes was observed in almost all farms except Farm 3. Although diverse C. jejuni genotypes were isolated on the farms and transport crates, they were not detected in chicken wing cuts at the end of the processing line. We also observed varied distribution of types in different sampling stages both at the farm level and the processing environment. However, the interpretation of such fluctuations is precarious as new types occurred on some occasions, particularly during processing. Our results show that chicken wing meat contamination resulted mainly from farm strain carryover, and that the carcasses were probably contaminated during processing. In addition, the new strain types observed were isolated more frequently after defeathering as compared to other processing steps. Therefore, this stage, in addition to evisceration, is one of the critical control points in the processing line to prevent cross-contamination and for controlling the spread of campylobacters.