Investigation into the occurrence of Gram-negative bacteria and associated antibiotic resistance in grey-headed flying foxes (Pteropus poliocephalus)

Reports of bacteria exhibiting antimicrobial resistance (AMR) in wildlife have increased globally, with many identified as zoonotic strains. However, reports of AMR in fruit bats (Chiroptera: Pteropodidae) are scarce and few studies have characterised bacterial isolates. Fruit bats are known reservoirs of zoonotic viruses, but their carriage of non-viral zoonotic pathogens, including Gram-negative bacteria, is rarely examined. Previously, neither screening for AMR nor the isolation of Gram-negative bacterial pathogens had been undertaken in Australian fruit bats. This project examined the occurrence and characteristics of target Gram-negative bacteria, namely Escherichia coli and Klebsiella pneumoniae, in grey-headed flying foxes (GHFF; Pteropus poliocephalus), an endemic Australian fruit bat. Escherichia coli and K. pneumoniae were cultured from faecal samples collected from wild and captive GHFF. Isolates were characterised using PCR and WGS methods to determine strain types, identify anthropogenic lineages, and detect virulence factors and acquired AMR genes. Additionally, faecal DNA and isolates were screened for class 1 integrons, an AMR determinant associated with diverse Gram-negative bacteria and anthropogenic microbial pollution. Class 1 integrons harbouring diverse AMR genes were detected in faecal DNA from wild (5.3%) and captive (41.2%) GHFF. The identification of novel class 1 integrons suggests GHFF have a role in the emergence of new AMR determinants. The high occurrence of class 1 integrons observed in captive GHFF demonstrates the association between increased acquisition of AMR and close proximity to humans. Antimicrobial resistant E. coli were detected in wild (3.5%) and captive (6.5%) GHFF. Collectively, E. coli isolates exhibited resistance to 10 antimicrobial classes including carbapenems, cephalosporins, fluoroquinolones, trimethoprim/sulfamethoxazole and tetracyclines. All GHFF antimicrobial resistant E. coli isolates belonged to human/domestic animal associated lineages and 69.2% carried virulence traits associated with extra-intestinal pathogenic E. coli (ExPEC). Diverse K. pneumoniae strains and closely related members of the K. pneumoniae species complex (KpSC) were detected in wild (9.0%) and captive (80.0%) GHFF. Two novel strains of K. africana (previously only detected in humans) were identified in GHFF (n=8). Overall, low prevalence of human associated K. pneumoniae (2.2%) and AMR (1.1%), and no hypervirulent strains were detected. Typical enteropathogenic E. coli (tEPEC), which are usually only associated with human carriage, were highly prevalent (15.3%, range 0%-87%) across five wild GHFF colonies. Novel sequence types and bfpA allele variants were identified, suggesting GHFF may have evolved host-specific tEPEC as part of their microbiome. This project demonstrated the transmission of anthropogenic E. coli, KpSC and AMR determinants into GHFF. Overall, the occurrence of AMR determinants in faecal bacteria was low in wild GHFF, but included resistance to critically important antimicrobials, however, it was higher in captive GHFF. These findings indicate GHFF play a role in the dissemination of AMR associated with Gram-negative bacteria. The detection of MDR ExPEC and widespread occurrence of tEPEC are both associated with potential zoonotic risks. These findings have potential implications for human and GHFF health and highlights the need for surveillance of GHFF for AMR and pathogenic bacteria, and to elucidate transmission pathways between GHFF, humans, domestic animals and the environment.