Despite the literature describing gut microbiota in pigeons, there is a little information regarding the enterococcal microbiota and distribution of Enterococcus species in domestic pigeons [1, 18]. Most of available works have concerned feral pigeons [2,3,4, 19, 20]. Evaluation of the cloacal content of racing pigeons in this study revealed a high prevalence of Enterococcus isolation. Our results contradicted the previous findings suggesting that Enterococcus species were naturally associated with humans and generally were rarely found in pigeons [2]. A total of 65–67% enterococcal species were correctly identified using API & Multiplex PCR as well as API & 16S sequencing. Jackson at al [18]. obtained higher agreement for poultry or swine isolates by API and PCR. In our study, Multiplex PCR and sequencing were the most adequate methods used for identification of pigeon Enterococcus species and the accuracy of these methods was approx. 97%. The highest accuracy (compared to the gold standard) was observed for Multiplex PCR, a bit lower for 16S sequencing and much lower for API.
According to the literature, the 16S rRNA gene sequence-based results used for bacteria identification should be analysed by two or more databases due to the difficulties in interpretation [21]. According to the recommendations the obtained results were compared with two freely available, quality-controlled, web-based public databases: GenBank and EzTaxon. Although both databases attributed equally all Enterococcus isolates to the same species, there was moderate to poor agreement between databases in the classification of isolates to a given species and did not seem to differ between enterococcal species. Our results support the findings, that the analysis by GenBank combined with EzTaxon is more discriminative than analysis by GenBank alone [21].
Similarly to other studies [1, 18] we showed that E. columbae predominated in intestinal microbiota of racing pigeons. On the other hand, there were pigeon lofts lacking E. columbae species. Enterococcus columbae was not found in city pigeons in Egypt [4]. Interestingly, among domestic birds only pigeons have their own specific Enterococcus species. Enterococcus columbae has not been recognized as a component of normal gut microbiota in other species. This phenomenon has not been clarified yet, however the certain features of the pigeon microbiota may have an impact because of their rudimentarily developed caeca. The main findings support the fact that pigeon enterococcal gut microbiota is composed of host-specific bacteria [1, 22]. The frequent occurrence of E. hirae in this study contrasts with the findings of other authors [1, 4] who did not find E. hirae in pigeon intestines. Besides of pigeons, E. hirae is one of the most commonly encountered enterococcal species in poultry [23,24,25]. According to the literature, E. faecalis followed by E. faecium are the most prevalent enterococcal species colonizing the gastrointestinal tract in humans, poultry and wild birds [8, 12, 23]. We found that E. faecalis and E. faecium were the third most common Enterococcus species identified in racing pigeons. In contrast to domestic pigeons, it seems that E. faecalis and E. faecium largely dominated the enterococcal intestinal microbiota in feral pigeons [3, 19]. Screening of the intestinal enterococcal microbiota revealed that E. gallinarum, originally described in chickens, was rarely found in racing pigeons [23, 24]. Enterococcus cecorum was less commonly found in pigeon lofts, and occurred irregularly but usually after race season. Interestingly, among the known Enterococcus species, only E. cecorum was described as the cause of enterococcal infections in racing pigeons [6, 7]. Recently, E. cecorum commonly considered as a part of physiologic intestinal microbiota of animals, has grown into one of the leading bacterial cause of lameness in broiler chickens [7,8,9, 24]. Similarly to Zigo et al. [22] E. mundtii was a rarely identified in racing pigeons. However, other authors observed E. mundtii more than twice as prevalent in feral pigeons [3]. This study revealed lower prevalence of E. durans racing pigeons than in poultry and feral pigeons [1, 4, 22,23,24,25]. It seems that Enterococcus raffinosus is absent in racing pigeons, but can occur in feral pigeons [4]. In contrast to poultry, Enterococcus avium and Enterococcus aquimarinus were not found in pigeons [2, 3, 22,23,24]. Based on the obtained results and literature data we deduced that pigeon microbiota is composed by similar enterococcal species (except E. columbae) that have been demonstrated in poultry [23, 25].
In our study the enterococcal intestinal microbiota in racing pigeons was the most abundant with Enterococcus species (up to 7) after the race season and E. columbae, E. faecium, E. hirae were the most prevalent. Before racing season the microbiota was composed by 4 Enterococcus species, with E. columbae and E. hirae as dominant. Similarly to our study Zigo et al. [22] identified more Enterococcus species in intestinal microbiota after race season. In contrast to our results, they recorded 3 dominant Enterococcus species before as well as after race season in pigeons. In our study, 2–5 Enterococcus species were found during the race season and E. columbae, E. faecium, E. faecalis, E. gallinarum predominated. Our results were consistent with the mentioned authors who showed that intestinal microbiota during the race season was dominated by E. faecalis, E. faecium, E. columbae [22]. We concluded that the race season may affect the presence of commensal bacteria in pigeons and may have impact on the differences in enterococcal gut microbiome composition between pigeon lofts. Moreover, some geographical differences in the occurrence of enterococci were observed.
To our best knowledge, this is the first study which provides differences in biochemical characteristics between intestinal Enterococcus species originated from racing pigeons. So far most studies concerning pigeons enterococci have provided biochemical properties only for E. columbae. Similarly to the literature [1, 5], few E. columbae isolates revealed activity of arginine dihydrolase (ADH), pyroglutamic acid arylamidase (PYRA), and ability to metabolize glycogen (GLYG). In contrast to Baele at al. [1], many E. columbae isolates were positive for alkaline phosphatase (PAL), and trehalose (TRE). We found that Enterococcus columbae was very similar to E. cecorum in terms of biochemical properties with exception of 7/32 characteristics (βNAG, HIP, PUL, LARA, MβDG, TAG, βMAN).
So far, among studies concerning pigeons enterococcal virulence factors were examined only in wood pigeons (Columba palumbus) [19]. In this study we confirmed the presence of 6 different virulence genes in Enterococcus isolated from intestinal microbiota of racing pigeons. A higher prevalence of gelE, asa1 and ace was in line with other studies concerning enterococci in farm animals [9, 16] but not with the results for enterococci isolated from food-stuffs [26]. The lower detection rate of cylA and esp in racing pigeons was in near agreement with poultry isolates [9, 16, 27]. Incidences of esp detection in enterococci isolated from cattle and pigs were higher than in our study [16, 27]. We confirmed that pigeon enterococci did not contain hyaluronidase gene. The absence of hyl was noted also in enterococci obtained from wild game meat and farm animals [16, 17, 27]. In contrast to pigeon, hyl was often reported in human, wild bird, food and water isolates [13, 15, 24, 28]. Jung et al. [9] found hyl among single pathogenic E. cecorum from poultry.
The study revealed that small number of racing pigeon enterococci produced gelatinase, but 66.7% gelE genes were silent. Among virulence genes found in E. columbae, gelE was the predominant one. High occurrence of efaA, ace and gelatinase activity was observed in E. faecalis. Our findings differed from those of Martín et al. [19] who did not confirm silent gelE genes in enterococci from wood pigeons. Moreover, they did not find any of the examined virulence genes in E. columbae. Our results corroborate similar findings by authors who described the highest gelatinase activity in E. faecalis [14, 19]. Among enterococci from feral pigeons, mainly E. faecium hydrolyzed gelatinase [4].
Enterococci harbouring virulence factors are more likely to cause infections than those without them. However, E. cecorum and E. columbae retrieved from infection case in racing pigeon were negative for virulence factors [7]. None of virulence factors was found significantly more often in the pathogenic poultry isolates compared to the commensal ones [9]. More data are needed on the distribution of enterococcal virulence factors for the purpose of establishing their role in the pathogenesis of Enterococcus-associated diseases in pigeons.
The prevalence of antimicrobial resistant enterococci in racing pigeons investigated in this study was very high. A total of 93.1% isolates showed resistance to at least one antimicrobial, and 29% were resistant to 4 antibiotics. On the contrary, Radimersky et al. [3] reported a lower number of isolates resistant to at least one antibiotic in feral pigeons. The most frequent resistance to tetracycline was observed in strains obtained from feral or racing pigeons and from wild birds [3, 12, 18, 28]. We concluded that frequent resistance to enrofloxacin and doxycycline may be associated with a common usage of these antibiotics in domestic pigeons. According to the actual legislation, only several veterinary medicinal products used in pigeons have been authorised for marketing within the territory of Poland, including amoxicillin, enrofloxacin, norfloxacin, doxycycline. On the other hand resistance to antibiotics which are not approved for use in pigeons (e.g. aminoglycosides, macrolides, phenicols) or even in animals (glycopeptide antibiotics) may suggest acquisition of resistance from other sources. The resistance to vancomycin was more frequent than observed in enterococci from feral pigeons in Brazil, Czech Republic [2, 3], and 5-times less frequent than in feral pigeons in Egypt [4]. Our study revealed significant differences between some enterococcal species in susceptibility to different antibiotics (doxycycline, nitrofurantoin, chloramphenicol, erythromycin, teicoplanin). Similarly to other authors [18] we showed more frequent resistance of E. columbae to enrofloxacin. We concluded that the most VRE belonged to E. hirae and E. columbae, while the most HLGR isolates belonged to E. faecium. On the contrary, Butaye et al. [18] did not find VRE in pigeons, whereas HLGR were represented mainly by E. faecalis. For the first time enterococci from racing pigeon were screened against linezolid. In contrast to feral pigeons [4], no LRE were found in racing pigeons.
This study, to the best of our knowledge provides, for the first time, detailed vancomycin resistance genotypes of a variety of enterococci isolated from racing pigeons. In contrast to the meat of wild game animals (including pigeons), wild birds, feral pigeons [3, 4, 17, 28], racing pigeons did not harbour vanA-enterococci and only sporadically vanB. Similarly to our results, other authors did not find vanA-mediated glycopeptide resistance in enterococci isolated from fecal samples in pigeons [29]. However vanB predominated in feral pigeons [4]. Out of the vancomycin resistance genes tested, vanC1 and vanC2-C3 predominated in racing pigeons. Vancomycin resistance caused by the vanC genes predominated in Enterococcus isolates in chickens [4]. Our data confirm significantly higher prevalence of vanC2-C3 among E. casseliflavus and E. hirae. However in chickens the vanC gene predominated among E. faecium [4]. We concluded that vancomycin resistance of E. columbae was caused by the vanC1, vanB or vanC1-C2 genes.