Molecular epidemiology of bacterial poultry diseases in Bangladesh and analysis of their zoonotic transmission

Abstract

Poultry diseases caused by numerous zoonotic bacterial pathogens have become increasingly prevalent in recent decades, posing threat to public health. This present study analyzed the molecular epidemiology of poultry pathogens in Bangladesh, their virulence and antibiotic resistance pattern to provide an insight into the origins and patterns of avian-borne zoonotic bacterial infections in Bangladesh. The thesis is made up of four quantitative investigations (Study I-IV) to examine the most commonly circulating MDR poultry pathogens: Escherichia coli, Escherichia fergusonii, Pasteurella multocida, Klebsiella pneumoniae, Staphylococcus, Micrococcus from various poultry samples (e.g., droppings, egg surface swab, feeding water, feed, handler's swab, and liver, cloacal swabs) from six commercial poultry farms belonging to Manikgonj, Narsingdi, Narayangonj districts of Bangladesh. Study- I and-II included a complete molecular epidemiological study (E. coli and P. multocida), with phenotypic characterization through culture-based techniques and molecular identification. Particular poultry pathogens (PPs) K. pneumoniae were characterized in Study III. In Study IV, antimicrobial sensitivity was tested using commonly used farm scale disinfectants and heavy metals. A total of 262 poultry samples were examined to retrieve 613 bacterial isolates among which droppings samples yielded the most PPs (28.83%), whereas egg surface swabs (2.99%) were the lowest. The majority of poultry samples had a high frequency of infectious bacterial populations: Escherichia (n=392), Staphylococcus (n=82), Micrococcus (n=20), Pasteurella (n=78), and Klebsiella (n=41), respectively. After presumptive screening, 297 Escherichia were isolated, followed by 22 P. multocida, 18 K. pneumoniae, 32 S. lentus, 7 M. luteus, and 4 M. aloeverae. According to the Congo Red binding (CRB) and Biofilm formation (BF) tests, (46.94%) 184/392 and (81.71%) 67/82 of the poultry isolates were tested positive for pathogenic Escherichia, respectively (Study I). Furthermore, Claremont phylogroups divided all pathogenic Escherichia isolates into five predominate phylotypes, in which phylotype B23 (37.36 %) was the most predominate followed by A1 (33.91%), D2 (11.49%), B22 (9.20%), B1 (8.05%), and non-group (5.43%), while 16S rRNA-based phylogenetic analysis of all isolates showed five distinct clades-A to E, except for P. multocida, which was divided into two unique clades. However, antibiotic resistance genes linked to tetracycline (tetA and blaTEM), quinolone (qnrB), carbapenemase (blaNDM/blaNDM-5, blaSIM-1, blaIMP-+4, and blaOXA-48) were found in 100% of E. coli isolates. Also, ≥80% of the isolates harbored several pathogenic genes e.g. uidA, fimH, crl, and ial. E. coli Phylotypes had a significant connection with CRB (p=0.003), BF (p=0.04), and virulence factors associated genes (VFGs) (p=0.05), and drug resistance (p=0.01). In Studies II-IV, on an average 88.71% isolates were found to be biofilm producers, and among them K. pneumoniae revealed the highest (100%) biofilm-forming ability followed by P. multocida (81.82%), Micrococcus (75%), and Staphylococcus (57.14%) isolates. In addition, in Study III, we found that ten (55.56%) K. pneumoniae isolates possessed one ESBL gene (blaTEM), followed by two carbapenemase genes pools, pool-1 (blaSIM-1, blaIMP-+4, and blaOXA-48) and pool-2 (blaIMP-+4 and blaOXA-48). In Study (I-IV), more than 70% of the pathogens tested were extremely resistant to multiple antibiotics: doxycycline, tetracycline, nitrofurantoin, and chloramphenicol while being susceptible to three antibiotics (imipenem, gentamicin, cefoxitin). At different doses, the isolates were resistant to all tested heavy metals (Cr>Co>Ni>Cu>Zn) and five common farm-scale disinfectants (Virocid> Quatovet>Savlon>Virkon S>Lysol) (Study IV). Whole-genome sequencing (WGS) (Studies I and II) of six MDR-positive E. coli (Phylogroup D2 (DH53, RN29), B1 (RN 3(2)), P. multocida (PM4, PM7), and E. fergusonii (OTSVEF-60) isolates confirmed as P. multocida type ST122, E. fergusonii type ST-5637 and E. coli type B1: ST155, E. coli type D2: ST405 harboring a number of VFGs, and antimicrobial resistant genes (AMRGs). Moreover, AMRGs were found to coexist with metal resistance (mercury, copper, zinc, nickel, arsenic, cobalt, and cadmium) genes. qacE-sul2, dfrA12, and ant-(3′′)-I genes carrying a class I integron gene cassette were also found in the OTSVEF-60 genome (Study I). Additionally, the pan-genome analysis showed 756 distinct genes linked with diverse virulence, metabolic pathways, pathogenicity, and antibiotic resistance (Study I-II). So, this study will be valuable for developing strategies to combat PDs in Bangladesh recommending focusing on the prevalent source of PDs, pathogenic MDR E. coli phylotype B23 circulations, followed by P. multicoda.