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

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

The phylogenetic grouping of E. coli for the classification of extraintestinal pathogenic strains (groups B2 and D) and commensal strains (groups A and B1) in humans [2] has been applied to the characterization of E. coli strains from poultry. Previous studies have shown that group A and group D were predominant in APEC in Japan [3] and the United States [4]. On the other hand, Jakobsen et al. reported that only group A (approximately 40%, nontypeable was counted as group A as was done in this study) was the dominant phylogenetic group among E. coli from healthy broilers (n = 138) [5]. Thus, the proportion of the phylogenetic groups might be different between APEC and E. coli from healthy broilers.
Some virulence genes are frequently found in APEC and assumed to be related to avain colibacillosis. iutA and iroN are iron transporter-encoding genes linking with growth in iron-poor environment. cvaC, iss, and ompT are associated with serum and/or complement resistance linking to systemic infection. tsh is associated with pathogenic process. hlyF is associated with toxin [6][7][8]. However, the pathogenesis and the role of virulence genes in avian colibacillosis have been obscure. Therefore, prevalence of these genes was analyzed as the virulence-associated genes in E. coli isolates from healthy broilers in this study, to compare with that of APEC in Japan.
Molecular analyses, such as multi-locus sequence typing (MLST) of E. coli [9,10], may provide additional epidemiological information on E. coli isolates from healthy broilers when used in combination with phylogenetic grouping or virulence profiling.
We have revealed previously that the resistance rates to ampicillin and enrofloxacin of APEC strains were higher than those of E. coli isolated from healthy broilers, suggesting that the antimicrobial resistance profile of APEC was different from that of E. coli isolated from healthy broilers. Furthermore, several studies have shown that phylogenetic groups were related to the antimicrobial resistance of E. coli isolates of human origin [5,[11][12][13].
The aim of our study was to investigate possible associations of E. coli isolates from fecal samples of healthy broilers with APEC in Japan using phylogenetic grouping, virulence-associated gene profiling, MLST, and antimicrobial resistance profiling.

Bacteria
Ninety-six E. coli isolates were collected from 53 fecal samples from healthy broilers in 22 prefectures across Japan under the Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) in 2009. Fresh fecal samples were collected from two or three healthy broilers at the different farms in each prefecture as previously described [14]. E. coli was isolated from the fecal samples using desoxycholate-hydrogen sulfate-lactose agar (Eiken Co., Ltd., Japan). Candidate colonies were identified biochemically using a commercially available kit (API20E, bioMe'rieux, Marcy-l'Etoile, France). The isolates were then stored in 10% skimmed milk (Wako Pure Chemical Industries, Ltd., Japan) at −80°C until use.

Statistical analysis
Differences in the prevalence of virulence-associated genes and the antimicrobial resistance rate between phylogenetic groups were analyzed using Fisher's exact test, disregarding group B2 because only a single isolate was classified into this group. A value of P < 0.05 (two-sided) was considered statistically significant.
A difference in the resistance rates between APEC strains of the previous study [10] and E. coli isolated from healthy broilers of this study was observed. The resistance rates against ampicillin (P = 0.00002), kanamycin (P = 0.006), and oxytetracycline (P = 0.005) of E. coli isolated from healthy broilers were significantly lower than those of APEC [10]. Our previous study showed that the resistance rates against ampicillin and enrofloxacin were higher in APEC strains than in isolates from healthy broilers [10]. Although the higher resistance rate to enrofloxacin was not observed in this study, the 1116.7-kg enrofloxacin was used for the treatment of broilers in 2009 (National Veterinary Assay Laboratory, 2009). In Japan, oxytetracycline has been widely used for the treatment of bacterial infection in poultry, ampicillin and kanamycin as well as enrofloxacin have been approved for the treatment of avian colibacillosis.

Conclusion
We demonstrated that the proportion of the phylogenetic groups in E. coli isolated from healthy broilers is different from that in APEC strains in Japan. On the other hand, even though group D strains accounted for a minor portion of E. coli isolates from healthy broilers, group D strains frequently possessed virulence-associated genes. Thus, E. coli isolates from the intestines of healthy broilers pose a potential risk of causing avian colibacillosis.