Genome dynamics and molecular infection epidemiology of multi-drug resistant Helicobacter pullorum isolates obtained from broiler and country chickens in India

Some of the life-threatening, foodborne, and zoonotic infections are transmitted through poultry birds. Inappropriate and indiscriminate use of antimicrobials in livestock industry has led to increased prevalence of multidrug resistant bacteria of epidemic potentials. Herein, we present the functional molecular epidemiological analysis entailing phenotypic as well as whole genome sequence based characterization of eleven H. pullorum isolates from broiler and free range chickens sampled from retail wet markets of Hyderabad city in India. Antimicrobial susceptibility tests revealed all isolates to be resistant to multiple antibiotic classes such as fluoroquinolones, cephalosporins, sulfonamides and macrolides. The isolates were also found to be extended-spectrum β-lactamase (ESBL) producers and were even resistant to clavulanic acid. Whole genome sequencing and comparative genomic analysis of these isolates revealed the presence of five to six well characterized antimicrobial resistance genes including those encoding Resistance- Nodulation- Division (RND) efflux pump(s). Phylogenetic analysis when combined with pan-genome analysis revealed a remarkable degree of genetic diversity among the isolates of free range chickens; in contrast, a high degree of genetic similarity was observed among broiler chicken isolates. Comparative genomic analysis of all publicly available H. pullorum genomes, including our isolates ( n=16 ), together with the genomes of 17 other Helicobacter species revealed a high number (8560) of H. pullorum specific protein encoding genes with an average of 535 such genes per isolate. In silico virulence screening identified 182 important virulence genes and also revealed high strain specific gene content in isolates of free range chickens (average 34) as compared to broiler chicken isolates. A significant prevalence of prophages (ranging from 1-9) and abundance of genomic islands (0-4) was observed in free range and broiler chicken isolates. Taken together, these observations provide a significant baseline data for functional molecular infection epidemiology of non-pyloric Helicobacters such as H. pullorum by unraveling their evolution in chickens and the possible zoonotic transmission to humans. Importance Globally, the poultry industry is expanding with an ever growing consumer base for chicken meat. Given this, food associated transmission of multidrug resistant bacteria represents an important healthcare issue. Our study involves a critical baseline approach directed at genome sequence based epidemiology and transmission dynamics of H. pullorum , a poultry pathogen having established zoonotic acumen. We believe our studies would facilitate development of surveillance systems that ensure safety of food for humans, and guide public health policies related to the use of antibiotics in animal feed in countries such as India. We sequenced 11 new genomes of H. pullorum as a part of this study. These genomes would provide much value addition to the ongoing comparative genomic studies of Helicobacters.