Chronological Change of Resistance to ß-Lactams in Salmonella enterica serovar Infantis Isolated from Broilers in Japan.

Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the ß-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis) isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3%) isolates recovered, 33 (35.5%) isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC), conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum ß-lactamases (ESBLs). In addition to ESC-resistance, eight (8.6%) isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC ß-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the bla TEM-20 and bla CMY-2 genes were associated with IncP plasmids, bla TEM-52 was linked with a non-typable plasmid and bla CTX-M-25 was carried by an IncA/C plasmid. Non-ß-lactam resistance to streptomycin, sulfamethoxazole, and oxytetracycline encoded by the aadA1, sul1, and tet(A) genes, respectively, was found in 86 (92.5%) isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9%) and 11 (11.8%) isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC ß-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying bla TEM-52 remarkably increased and S. Infantis strains harboring bla CMY-2, bla TEM-20, or bla CTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.

Prevalence and epidemiological relationship of CMY-2 AmpC ß-lactamase and CTX-M extended-spectrum ß-lactamase-producing Escherichia coli isolates from broiler farms in Japan.

To evaluate the prevalence of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae in broiler chickens, 41 rectal samples taken from 4 commercial farms were examined. Desoxycholate hydrogen sulfide lactose agars, supplemented with either 4 µg/ml cefotaxime or 16 µg/ml ceftazidime, were used to screen ESC-resistant bacteria. ESC-resistant bacteria were isolated from all samples. Of the 164 ESC-resistant bacteria (included 4 isolates per a sample), 163 were Escherichia coli, while 1 isolate was identified as Enterobacter cloacae. Extended-spectrum ß-lactamase (ESBL) genes and plasmid-mediated AmpC ß-lactamase genes in the isolates were determined by PCR and sequencing. One AmpC ß-lactamase gene, bla(CMY-2) (66%), and 4 ESBL genes, bla(CTX-M-1) (26%), bla(CTX-M-55) (10%), bla(SHV-5) (4%) and bla(CTX-M-2) (3%), were detected in the E. coli isolates. The epidemiological relationship of the CMY-2 and CTX-M ß-lactamase-producing isolates among the farms was analyzed by pulsed-field gel electrophoresis using the XbaI restriction enzyme. Forty-one (Y1-Y41) and 14 (X1-X14) clusters were found in the CMY-2 and CTX-M-carrying E. coli isolates, respectively. Some clusters included isolates derived from more than 1 farm, indicating some cross-contamination of clonal strains and spread of CMY-2 AmpC ß-lactamase or CTX-M ESBL among the farms.

Emergence of Salmonella enterica serovar infantis harboring IncI1 plasmid with bla(CTX-M-14) in a broiler farm in Japan.

Cefotaxime (CTX)-resistant and -susceptible Salmonella enterica serovar Infantis isolates obtained from broilers raised on a farm in January 2010 in Japan were characterized to establish their resistance determinants. The CTX-resistant isolates produced CTX-M-14 extended-spectrum ß-lactamase and harbored 2 distinct plasmid of approximately 140- and 95-kb, whereas the CTX-susceptible isolates harbored one 140-kb plasmid. The 95-kb plasmids were replicon typed as IncI1 carrying the bla(CTX-M-14) gene, while the 140-kb plasmids were IncP and harbored the aphA1, aadA1, tetA, and sul1 genes. Genetic fingerprinting by pulsed-field gel electrophoresis revealed similar macrorestriction profiles amongst CTX-resistant and susceptible isolates, suggesting a clonal relationship. The presence of CTX-resistant S. Infantis on a broiler farm has occurred through the acquisition of IncI1 resistance plasmid.

Effect of cooled and chlorinated chiller water on Campylobacter and coliform counts on broiler carcasses during chilling at a middle-size poultry processing plant.

To evaluate the effect of cooled and chlorinated chill water for Campylobacter and coliforms at a middle-size processing plant which was considered to be difficult for eliminate pathogenic bacteria on carcasses, following three conditions were examined; keeping temperature at < 20, < 10 and < 10°C, and chlorine concentration at < 50, < 50 and 50 to 70 ppm during processing in experiment 1, 2 and 3 respectively. Fifteen prechill and 15 postchill carcasses were examined in each experiment. In lower temperature of experiment 2, decreasing rate (%) of coliforms was significantly higher (P<0.01) than that in experiment 1. In higher chlorination of experiment 3, no Campylobacter was detected from all postchill carcasses.

Re-emergence of multi-drug resistant Salmonella enterica serovar Stanley from cattle.

During 2009, Salmonella enterica subspecies enterica serovar Stanley isolates were recovered from cattle diagnostic specimens in southern Japan, and the isolates were examined to characterize the genetic determinants involved in this new pathogenicity that associated with mortality in cattle. All the isolates were multi-drug resistance exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, and kanamycin (ACSSuT-Km) encoded by blaTEM, catA, aadA1, sul1, tet(A), and aphA1 genes, respectively. Class 1 integrons of 1.5-kb size were detected in all MDR isolates. The isolates harboured easily transferable plasmids of ca. 210-kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. XbaI-digested PFGE patterns generated two related clusters implicated in the dissemination of multi-drug resistance amongst Salmonella Stanley isolates. An emergence of multi-drug resistant Salmonella Stanley amongst food-producing animals, including cattle is a threat to human health, as resistant isolates may be transmitted to humans through the food chain.

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.

Genetic analysis of multidrug-resistant Salmonella enterica serovars Stanley and Typhimurium from cattle.

During 2005-2008, a longitudinal study was conducted in southern Japan to detect and characterize multidrug-resistant Salmonella enterica serovars recovered from cattle diagnostic specimens. Determination of antimicrobial resistance phenotypes and genotypes, identification of Salmonella genomic island 1 (SGI1), detection of virulence genes, plasmid analysis, conjugal transfer experiments, and sequencing of class 1 integrons were conducted. Multidrug-resistant Salmonella detected were serovars Stanley, Typhimurium, and O4:d. Salmonella Stanley isolates exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, trimethoprim, and kanamycin (ACSSuT+) encoded by bla(TEM), catA, aadA2, tetA, sul1, dfrA12, and aphA1 genes, respectively. Sequencing analysis revealed that aadA2 and dfrA12 were integrated as gene cassettes within the class 1 integrons of 1.5kb size. Importantly, the isolates harboured easily transferable plasmids of ca. 210kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. Moreover, Salmonella Typhimurium DT104 isolates with typical SGI1 were detected and presented ACSSuT+ resistance phenotype encoded by bla(PSE-1) and bla(TEM); floR; aadA1; sul1; and tetA and tetG, respectively. Salmonella Typhimurium isolates carried plasmids of variable sizes ranging from 3.5 to 100 kb with DT104 isolates harbouring plasmids of ca. 90 kb. Salmonella serovar O4:d had ACSSuT+ resistance phenotype mediated by bla(TEM), catA, aadA1, sul1, tetA, and aphA1 genes. A virulence gene invA was found in all multidrug-resistant Salmonella Typhimurium, Stanley and O4:d clinical isolates. In conclusion, this is the first report describing the occurrence of multidrug-resistant Salmonella Stanley from bovine species. The emergence of Salmonella Stanley isolates exhibiting plasmid-encoded high-level multidrug resistance is an important health concern because this new pathogenecity was associated with mortality in cattle.

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.

Genetic analysis of multi-drug resistance and the clonal dissemination of beta-lactam resistance in Salmonella Infantis isolated from broilers.

An epidemiologic study was conducted to investigate the incidence and characterize the antimicrobial resistance determinants, analyzing plasmid profiles, and establishing the genetic relationship among beta-lactam-resistant isolates of Salmonella Infantis from broilers in Southern Japan. A total of 120 isolates were recovered from 56 flocks belonging to 44 holdings during 2004-2006. The percentages of resistance were as follows: ampicillin (24%), cephalothin (23%), cefoxitin (0%), ceftazidime (11%), cefotaxime (11%), chloramphenicol (0%), kanamycin (7.5%), ofloxacin (20%), oxytetracycline, streptomycin and sulfamethoxazole (100%) and trimethoprim (75%). The incidence of bla(TEM)-encoded beta-lactam resistance in 2004-2006 was significantly higher than in 1998-2003 (P<0.001). BlnI-digested PFGE patterns generated two related clusters implicated in the dissemination of beta-lactam resistance. Two types of plasmid profiles were observed and two plasmids of ca. 50 and 180-kb size were carried by beta-lactam-resistant isolates. Streptomycin resistance was conferred by aadA1 (n=116), aadA1-aadA2 (n=1), and aadA1-strA-strB (n=3). Resistances to kanamycin, oxytetracycline, sulfamethoxazole and trimethoprim were conferred by aphA1 (n=9, 100%), tetA (n=120, 100%) sul1 (n=120, 100%) and dfrA5 (n=90, 100%), respectively. Two types of class 1 integrons were detected: 1.0 kb (n=120) and, 1.0/1.5 kb (n=3). Integrons of 1.0/1.5 kb were found in isolates with the aadA1-strA-strB gene combination. For the first time, all S. Infantis isolates showed resistance to at least three classes of antimicrobial agents; and the intestinal tract of healthy poultry was a reservoir of the extended-spectrum cephalosporin-resistant isolates of serovar Infantis.

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.

Lysine decarboxylase-negative Salmonella enterica serovar enteritidis: antibiotic susceptibility, phage and PFGE typing.

One hundred twenty Salmonella Enteritidis isolates collected from 1992 to 2005 in Nagasaki prefecture (65 isolates from 40 outbreak cases, 44 from sporadic diarrhea patients, and 11 from chicken-related products) were investigated by their antibiotic susceptibility profiles, phage typing, and pulsed-field gel electrophoresis (PFGE) typing. Out of them, 18 were identified as lysine decarboxylase (LDC)-negative isolates, and 15 showed resistance toward streptomycin. Based on the PFGE typing, the isolates were classified into five clusters by UPGMA clustering method. Three LDC-negative isolates belonged to cluster A and were of phage type (PT) 4 and isolated between 2000 and 2004. Other 15 LDC-negative isolates belonged to cluster E. They were PT1, reacted but did not conform (RDNC), or untypable and were isolated between 2001 and 2004. LDC-negative isolates of the cluster A differed from LDC-negative isolates of the cluster E in antibiotic susceptibility profiles, phage typing, and PFGE typing. LDC-negative isolates of the cluster E were isolated after 2001 in Nagasaki prefecture.

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.

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.

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.

Acquisition of quinolone resistance and point mutation of the gyrA gene in Campylobacter jejuni isolated from broilers and in vitro-induced resistant strains.

A dramatic rise in the number of resistant Campylobacter to quinolones has been documented in human patients and domestic animals. In this study, the mechanism of acquisition of quinolone resistance was studied by detecting point mutations in the gyrA gene of Campylobacter strains obtained from broilers and strains with in vitro-induced resistance. The minimal inhibitory concentrations (MICs) of norfloxacin (NFLX) and ofloxacin (OFLX) for the strains that had no point mutation were slightly increased from the source strain (Campylobacter jejuni ATCC 33560). The MICs of nalidixic acid (NA), NFLX, and OFLX for the strains that had the point mutation at Thr-86 were 100 or 200 microg/ml, 50 microg/ml, and 25 microg/ml, respectively. The MIC of NA for the strain that had a point mutation at Asp-90 higher than those for the strains that had the point mutation at Thr-86, but the MICs of NFLX and OFLX were relatively lower than those for the strains that had point mutation at Thr-86. These findings suggest that the degree of antimicrobial resistance against NA, NFLX, and OFLX in the in vitro-induced C. jejuni strains was associated with the location of the point mutation in gyrA. On the other hand, a point mutation in all seven resistant strains isolated from broilers was located only at Thr-86, while the MICs of the three quinolones varied in each wild strain. This suggests that another mechanism might also be involved in the acquisition of quinolone resistance in C. jejuni wild strains.