An insight into newly emerging avian pathogenic E. coli serogroups, biofilm formation, ESBLs and integron detection and in vivo pathogenicity in chicken

Bacterial diseases alone or in combination with other pathogens lead to significant economic losses in poultry globally including India. One of these diseases is avian colibacillosis which is caused by avian pathogenic Escherichia coli (APEC). The present study sought to isolate and characterize using in vivo and in vitro assays E. coli recovered from poultry diagnosed with colibacillosis. A total of 55 E. coli isolates were recovered from tissues of 55 broiler flocks affected with colibacillosis by using standard microbiological techniques, Vitek 2 Compact system and polymerase chain reaction. Out of 55 E. coli isolates, 50 (90.9 %) were characterized as APEC by multiplex PCR using a set of five virulence genes. On serotyping, 16 (32 %) APEC isolates were serogrouped as O26 followed by O98 (28 %), O120 (14 %), O11 (12 %), O135 (8 %) and O17 (4 %). The antimicrobial susceptibility testing of E. coli isolates revealed high antibiotic resistance against imipenem, tetracycline, ciprofloxacin and levofloxacin (96 % each). Interestingly all the 50 suspect APEC isolates were found to be multiple drug resistant (MDR) and the antimicrobial profiling indicated that these isolates could be classified into 38 resistotypes. Moreover, 10 (20 %) isolates were ESBL producers as per phenotypic characterization using combined disk diffusion test. On genotypic characterization of ESBLs, 31 (62 %) isolates were found positive for the blaTEM gene, whereas, 34 (68 %) isolates carried intI1 gene. On assessment for biofilm formation at 72 h incubation, thirteen (26 %) isolates were found to be strong biofilm producers, whereas nine (18 %) and twentyeight (56 %) isolates were moderate and weak biofilm producers, respectively. Later, the LD50 of one MDR and strong biofilm producing isolate (APEC-P02) was calculated by in vivo oral challenge study in day old broiler chicks. The findings of this study demonstrated that LD50 of APEC-P02 isolate was 1.12 x 108 CFU/ml. The unexpectedly high prevalence of O11, O126, O98, O120 and O135 isolates suggest that there may be emergent serogroups causing colibacillosis in India. The current oral challenge study seems to be the first of its kind in India to estimate the LD50 of a multidrug resistant biofilm producer APEC isolate in day-old chicks.