A total of 318 Escherichia coli isolates obtained from different food-producing animals affected with colibacillosis between 2001 and 2006 were subjected to phylogenetic analysis: 72 bovine isolates, 89 poultry isolates and 157 porcine isolates. Overall, the phylogenetic group A was predominant in isolates from cattle (36/72, 50%) and pigs (101/157, 64.3%) whereas groups A (44/89, 49.4%) and D (40/89, 44.9%) were predominant in isolates from poultry. In addition, group B2 was not found among diseased food-producing animals except for a poultry isolate. Thus, the phylogenetic group distribution of E. coli from diseased animals was different by animal species. Among the 318 isolates, cefazolin resistance (minimum inhibitory concentrations: ≥32 μg/ml) was found in six bovine isolates, 29 poultry isolates and three porcine isolates. Of them, 11 isolates (nine from poultry and two from cattle) produced extended spectrum β-lactamase (ESBL). The two bovine isolates produced blaCTX-M-2, while the nine poultry isolates produced blaCTX-M-25 (4), blaSHV-2 (3), blaCTX-M-15 (1) and blaCTX-M-2 (1). Thus, our results showed that several types of ESBL were identified and three types of β-lactamase (SHV-2, CTX-M-25 and CTX-M-15) were observed for the first time in E. coli from diseased 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-β-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.

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 β-lactamase genes. β-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.

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.

ABSTRACT. A total of 250 fecal content samples were collected from 25 farrow-to-finish pig farms and examined for the prevalence of Clostridium difficile by using ethanol treatment followed by plating onto selective media-cycloserine-cefoxitin-mannitol agar-for the isolation of Clostridium difficile. Two specimens (0.8%, 95% confidential interval: 0-2.9%) were positive for C. difficile. One isolate was only positive for toxin B, and the other isolate was negative for both toxins A and B. Thus, prevalence of Clostridium difficile was found to be low among finishing pigs in Japan.