ABSTRACT: BACKGROUND:This study was aimed to investigate the intestinal microbiota in racing pigeons with regard to Enterococcus species distribution, virulence factors and antibiotic susceptibility. Three methods (API, Multiplex sodA-PCR, 16S rRNA sequencing) were compared for Enterococcus species identification. Cloacal samples from 179 apparently healthy pigeons of 13 different flocks were tested. RESULTS:Multiplex sodA-PCR and 16S rRNA gene sequencing showed almost perfect agreement in Enterococcus species identification. Isolates were identified as Enterococcus columbae (34.5%), Enterococcus hirae (20.7%), Enterococcus faecalis (11.7%), Enterococcus faecium (11.7%), Enterococcus gallinarum (9%), Enterococcus mundtii (4.8%), Enterococcus casseliflavus (3.4%), Enterococcus cecorum (2.1%), Enterococcus durans (2.1%). More Enterococcus species were found after the race season than before. The study showed differences between Enterococcus species in relation to 68.8% (22/32) biochemical parameters. Six out of seven virulence genes were detected: gelE (43.5%), asa1 (42.1%), efaA (30.3%), ace (30.3%), cylA (27.6%), and esp (9%). None of the isolates harboured hyl gene. Overall 15.2% of Enterococcus isolates produced gelatinase, but 66.7% gelE genes were silent. Enterococcus faecalis showed the most often efaA, ace and gelatinase activity than other enterococcal species. Nearly all isolates (93.1%) were resistant to at least one antibiotic. The most frequent resistance was to enrofloxacin (80%), doxycycline with teicoplanin (73.1%), erythromycin (49.7%). The study revealed significant differences between some enterococcal species in the antibiotic susceptibility to different antibiotics. Enterococcus columbae and E. cecorum showed significantly more frequent resistance to chloramphenicol than other enterococci. The presence of VRE (19.3%), HLGR (2.8%) and no LRE were found. Overall 30.3% of isolates were positive for vancomycin resistance genes, where vanC1 (E. gallinarum), vanC2-C3 (E. hirae, E. casseliflavus), vanB (E. columbae) predominated. CONCLUSIONS:We conclude, that intestinal microbiota in racing pigeons is composed by 9 different Enterococcus species. Given that racing pigeons are kept in close contact with humans and backyard animals, combined with their long-distance flight abilities, they can serve as potential source of virulent and antibiotic resistant Enterococcus spp. in the environment.
