In this study, we isolated 62 (54.38%) E. coli isolates from 114 different samples, which were confirmed by selective isolation on MLA and EMB, conventional PCR and Vitek2. Using a set of five virulence genes as described above, 75.8% isolates were characterized as APEC. The maximum APEC detection was from tissue lesions (100%) followed by environmental samples from the poultry house (73.5%) and cloacal swabs of affected birds (60%). The lower isolation of APEC from cloacal swabs of birds might be due to the use of antimicrobials in the feed and water of these farms (Hasan et al. 2011). Awawdeh (2018), reported that all the E. coli isolates from tissue lesions of affected birds (100%) were APEC, however, reporting higher (70%) APEC isolate from the feces of affected birds compared to this study (60%). The APEC isolates obtained from the environment of the diseased flocks (73.5%) are higher than that obtained by Hussein et al. 2013 (approx. 30%) which might be due to the fact that the authors have limited the environment sampling only to litter and drinker swabs.
The prevalence of the five virulence genes was found to be significantly higher among APEC isolates. The maximum prevalence (100% each) was observed in the case of four genes viz., hlyF, ompT, iroN, iutA, and 95.7 % for iss gene. The hlyF gene is involved in the production of outer membrane vesicles which act as a channel to deliver virulence factors into the host (Murase et al. 2016), the ompT gene encodes the episomal outer membrane protease that cleaves colicins, iroN and iutA encodes for an iron acquisition system for bacteria and the iss gene encodes for a protein which inhibits the deposition of membrane attack complex (MAC) to provide serum resistance (Dziva and Stevens 2008). Among non-APEC isolates, the prevalence of hlyF and ompT was 26.7% each and iroN was 6.7%. The iutA and iss were not amplified from any of non-APEC isolates, suggesting their highly significant association with APEC isolates. Geographical variations in the prevalence of these genes have been reported. For example, prevalence of hlyF ranged from 24%-100%, ompT from 2.2%-100%, iroN from 84%-100%, iutA from 69–82% and iss 80–100% among APEC isolates (Johnson et al. 2008; De Oliveira et al. 2015; Awawdeh 2018; Subedi et al. 2018; Kazibwe et al. 2020). This study along with others suggests that the five genes used in the study are significantly associated to APEC isolates compared to non-APEC isolates (Johnson et al., 2008; Awawdeh 2018; Kazibwe et al. 2020). The high prevalence of these five genes which encode for various virulence traits suggests the pathogenic nature of APEC and these virulence genes (VGs) may be involved at different stages of infection in poultry birds. The frequency of APEC related VGs among E. coli strains varied and the virulence gene profile may be influenced by several factors such as geographical location, season, bird immune status, variation in sampling sites, different husbandry, and vaccination protocols (Johnson et al. 2008; Wang et al. 2016).
APEC biofilms in poultry settings have been reported as responsible for failure of disinfectants, failure of antimicrobials, and persistent E. coli infection of birds at the farm (Maharjan et al. 2016; Branco et al. 2016). We found in the present study that APEC (n = 30) and non-APEC (n = 6) isolates have the potential to form biofilms in vitro. Our biofilm results are close to the findings of Skyberg et al. (2007) and Wang et al. (2016) who observed 55.2% and 56.6% APEC isolates as moderate to strong biofilm producers. However, a lower numbers of biofilm producers were reported by Dou et al. (2015) and Oosterik et al. (2014), who found 36.2% and 44% APEC isolates as biofilm producers, respectively. This variation might be due to the different inoculating media used in these studies along with different incubation temperatures as adhesion of medium components such as amino acids or lipids to the abiotic surface may stimulate cell adherence and mucus-derived proteins may elicit bacterial cell agglutination as reported in previous studies using commensal and laboratory E. coli strains (Orndorff et al. 2004). However, our study along with previous investigations revealed that APEC isolates can form biofilm in vitro and thus can be one of the reasons of constant occurrence of colibacillosis in poultry farms.
Biofilm associated genes viz., crl, csgA, fimH, luxS, and papC were detected with high frequency among APEC and non-APEC in this study. The csgA was found to have a nearly similar distribution among APEC and non-APEC isolates as reported previously (Maluta et al. 2014; Cunha et al. 2017). The significant association of fimH with VGs used for APEC characterization thus suggests that this gene can be used as a molecular marker for APEC characterization along with other VGs used for characterization (Schwartz et al. 2013).
The ubiquity of biofilm associated genes among APEC and non-APEC (Fig. 10) does not mean that these genes are not important for pathogenesis (Wang et al. 2016). All these biofilms associated genes have previously been reported among ExPEC strains of human and canine origin, which again suggests a possible linkage of APEC and ExPEC strains. Out of 30 moderate-strong biofilm producer APEC, two isolates were positive for the five genes, 17 isolates were positive for four genes, nine isolates were having three genes each, and two isolates were having two genes. Wang et al. (2016) also reported a high prevalence of biofilm associated genes among biofilm producing APEC isolates. However, it cannot be concluded that these biofilm associated genes are responsible for biofilm formation among these isolates as 14 APEC isolates, despite having more than three such genes, were either not able to produce any biofilm or only produce weak biofilms. This finding thus suggests that the mere presence of biofilm associated genes might not be attributed to biofilm formation, rather it is the level of expression of biofilm related genes, which might be temporally or environmentally regulated which should be assessed (Cunha et al. 2017; Rodrigues et al. 2018). Thus, quantification of the expression of biofilm related genes by real-time PCR or expression of curli and cellulose by using phenotypic methods is needed.
A total of 47 APEC isolates were tested for antimicrobial susceptibility to 18 antibiotics of GN AST 65 cards. ). A total of 43 isolates (91.5%) were resistant to at least four antimicrobials. The higher resistance to antimicrobials is suggestive of rampant use of antibiotics in the commercial broiler industry as also concluded in the survey by Grakh et al. (2020A total of 22 MDR phenotypes and the most common form of antibiotic resistance were to tetracycline, enrofloxacin, marbofloxacin, sulphamethoxazole/trimethoprim, ampicillin, and piperacillin (R type: TEMStAP). In the present study, the significantly higher association of ESBL with imipenem, gentamicin, and nitrofurantoin resistant isolates is interesting as these are not beta-lactam antibiotics. Such an association of ESBL with the above said antibiotics is also previously reported in human medicine (Rawat and Nair 2010; Al-Zarouni et al. 2012). The plasmids carrying ESBL genes also carry genes for resistance to other antimicrobial agents such as aminoglycosides, trimethoprim, sulphonamides, tetracyclines and chloramphenicol and thus confer co-resistance to these antibiotics indicating that a very broad antibiotic resistance extending to multiple antibiotic classes has become a frequent characteristic of ESBL producing E. coli isolates (Rawat and Nair 2010). Leverstein-Van Hall et al. (2011), revealed that ExPEC strains isolated from human patients shared the same ESBL genes and plasmids as carried by E. coli isolates cultured from retail chicken meat and colibacillosis affected birds, indicating that they can be easily transferrable among E. coli strains including human strains. Further high resistance against antibiotics was there in strong – moderate biofilm producing isolates as compared with weak or non-biofilm producing isolates. For instance, the six isolates which were resistant to gentamicin were strong biofilm producers. Previous investigations have suggested that biofilms confer resistance to bacteria by providing a platform for the exchange of resistance genes/plasmids (Romling and Balsalobre 2012; Wang et al. 2016).
Using chi-square test, a significant association of antimicrobial resistance was found with two virulence genes viz., iss and csgA. Significantly higher prevalence of csgA gene was found among tetracycline resistant and sulphamethoxazole/trimethoprim resistant isolates. And significantly higher prevalence of iss gene was found among isolates that were resistant to ampicillin and sulphamethoxazole/trimethoprim. Similar association of biofilm related genes with antimicrobial resistance was previously reported (Awawdeh 2018), and needs further studies to derive any conclusions. To the best of our knowledge, this is the first elaborative study on APEC characterization and biofilm formation in India. The constant occurrence of avian colibacillosis caused by APEC is a challenge to the poultry industry and leads to economic losses to stakeholders. The biofilm formation ability of APEC might be responsible for the constant occurrence of APEC and avian colibacillosis at these farm and antibiotic resistance of these isolates. Our study thus emphasizes the endemicity of biofilm producing and multiple drug resistant APEC infection in broiler chickens and their environment in Haryana. Further association and characterization of APEC and their comparison with ExPEC of human origin, might reveal their zoonotic potential, which may help to design prevention and control measures at poultry farms.