Mototaka Hiki

Phylogenetic groups and cephalosporin resistance genes of Escherichia coli from diseased food-producing animals in Japan

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.

Diversity of Plasmid Replicons Encoding the blaCMY-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-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.

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 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.

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.

Effect of antimicrobial exposure on AcrAB expression in Salmonella enterica subspecies enterica serovar Choleraesuis

Understanding the impact of antimicrobial use on the emergence of resistant bacteria is imperative to prevent its emergence. For instance, activation of the AcrAB efflux pumps is responsible for the emergence of antimicrobial-resistant Salmonella strains. Here, we examined the expression levels of acrB and its multiple regulator genes (RamA, SoxS, MarA, and Rob) in 17 field isolates of S. Choleraesuis by using quantitative PCR methods. The expression of acrB increased in eight of the field isolates (P < 0.05). The expression of acrB was associated with that of ramA in one isolate, soxS in one isolate, and both these genes in six isolates. Thereafter, to examine the effect of selected antimicrobials (enrofloxacin, ampicillin, oxytetracycline, kanamycin, and spectinomycin) on the expression of acrB and its regulator genes, mutants derived from five isolates of S. Choleraesuis were selected by culture on antimicrobial-containing plates. The expression of acrB and ramA was higher in the mutants selected using enrofloxacin (3.3–6.3- and 24.5–37.7-fold, respectively), ampicillin (1.8–7.7- and 16.1–55.9-fold, respectively), oxytetracycline (1.7–3.3- and 3.2–31.1-fold, respectively), and kanamycin (1.6–2.2- and 5.6–26.4-fold, respectively), which are AcrAB substrates, than in each of the parental strains (P < 0.05). In contrast, in AcrAB substrate-selected mutants, the expression of soxS, marA, and rob remained similar to that in parental strains. Of the four antimicrobials, the level of ramA expression was significantly higher in the enrofloxacin- and ampicillin-selected mutants than in the oxytetracycline- and kanamycin-selected mutants (P < 0.05), whereas the expression levels of acrB and multiple regulator genes in spectinomycin-selected mutants were similar to those in each parental strain. These data suggest that exposure to antimicrobials that are AcrAB substrates enhance the activation of the AcrAB efflux pump via RamA, but not via SoxS, MarA, or Rob in S. Choleraesuis.

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

BackgroundThe aim of our study was to investigate the possible etiology of avian colibacillosis by examining Escherichia coli isolates from fecal samples of healthy broilers.FindingsSeventy-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%).ConclusionsOur results indicated that E. coli isolates inhabiting the intestines of healthy broilers pose a potential risk of causing avian colibacillosis.

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

ABSTRACT 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.

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.