Antonie van Leeuwenhoek (2015) 107:1281–1289 DOI 10.1007/s10482-015-0422-6
ORIGINAL PAPER
Prevalence, diversity and characterization of enterococci from three coraciiform birds Petra Splichalova • Pavel Svec • Anuradha Ghosh • Ludek Zurek • Veronika Oravcova • Tomas Radimersky • Mirko Bohus • Ivan Literak
Received: 24 November 2014 / Accepted: 2 March 2015 / Published online: 14 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Coraciiform birds hoopoe (Upupa epops), common kingfisher (Alcedo atthis) and European roller (Coracius garrulus) were examined for enterococci in their cloacae and uropygial glands. The enterococcal isolates were identified at the species level using several genomic and proteomic methods, screened for antibiotic susceptibility and genotyped by pulsed-field gel electrophoresis (PFGE). Clonality of isolates from the common kingfisher was also assessed by multi-locus sequence typing (MLST). Using selective media, putative enterococcal isolates (n = 117) were recovered from 74 % (32 out of a total of 43) of Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0422-6) contains supplementary material, which is available to authorized users. P. Splichalova (&) V. Oravcova T. Radimersky I. Literak Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1–3, 612 42 Brno, Czech Republic e-mail: [email protected] P. Svec Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, Bld. A25, 625 00 Brno, Czech Republic
the bird samples and 114 isolates were confirmed as enterococci. Overall, among the total of 6 different species detected, Enterococcus faecalis was dominant (59 %) in all three bird species. The second most frequently isolated species was Enterococcus casseliflavus (32 %). PFGE revealed great diversity of strains from different bird species and anatomic location. Closely related strains were found only from nestlings from the same nest. No genes conferring resistance to vancomycin (vanA, vanB, vanC1 and van C2/C3) or erythromycin (erm A, ermB and mefA/E) were detected. MLST analysis and eBURST clustering revealed that sequence types of E. faecalis from the common kingfisher were identical to those of isolates found previously in water, chickens, and humans. M. Bohus Department of Environmental Ecology, Faculty of Natural Science, Comenius University in Bratislava, Mlynska dolina, 842 15 Bratislava, Slovak Republic I. Literak CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1–3, 612 42 Brno, Czech Republic
A. Ghosh L. Zurek Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
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Keywords Enterococcus Coraciiform birds sodA Rep-PCR 16S rRNA gene MALDI-TOF
Introduction Enterococci are ubiquitous bacteria in various environmental habitats and are commensals of mammals, birds, some reptiles and invertebrates (Aarestrup 2006). Some species have emerged as important causes of nosocomial and community acquired infections (Bogaard et al. 2002; Radhouani et al. 2012). In animals, enterococci can cause bovine mastitis, septicemia, endocarditis and sudden death in chickens (Aarestrup 2006); and diarrhea in rats, horses, dogs, cats and pigs (Aarestrup et al. 2002). They have innate resistance to many antimicrobial agents and they can carry a variety of acquired antibiotic resistance genes, which can be transmitted to other pathogenic bacteria (Murray 1991; Spera and Farber 1994; McMurphy et al. 2010; Radhouani et al. 2012). Acquired resistance of enterococci has also been detected in wild animals (Poeta et al. 2007). Over the past decade, members of the genus Enterococcus have been characterized using various classification methods and the number of newly described species has increased rapidly (Svec and Devriese 2009). Hoopoes, kingfishers and rollers are classified in the order Coraciiformes, a group that also includes beeeaters and wood hoopoes (Hackett et al. 2008). So far, only a few studies have focused on enterococci in coraciiform birds; Enterococcus faecalis was isolated from the uropygial gland of the hoopoe (Upupa epops, Upupidae) (Martin-Platero et al. 2006; Soler et al. 2008; Ruiz-Rodriguez et al. 2009, 2012) and Enterococcus phoeniculicola was described from the uropygial gland of the green wood hoopoe (Phoeniculus purpureus, Phoeniculidae) (Law-Brown and Meyers 2003). The present study aimed to assess the prevalence, species diversity, antibiotic resistance and clonality of enterococci isolated from three coraciiform birds. Enterococcal isolates cultured from the cloacae and the uropygial glands of coraciiform birds from central Europe, hoopoe, European roller (Coracius garrulus, Coraciidae), and common kin (Alcedo atthis, Alcedinidae), were identified by rep-PCR fingerprinting, sequencing of sodA and 16S rRNA genes, and matrix-
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assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and characterized by determination of antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Selected strains of E. faecalis from common kingfishers were also typed by multi-locus sequence typing (MLST).
Materials and methods Bird samples Smears from cloacae and uropygial glands were obtained from hoopoes originating from Sturovo, Slovakia (47°460 N 18°410 E; 1 female and her 3 nestlings were examined on 7 June 2008) and from Bzenec, Czech Republic (48°580 N 17°150 E; 3 nestlings from 1 nest were examined on 15 June 2008). Smears from cloacae were taken also from Common Kingfishers from Hradek and Kacov, Vlasim, Czech Republic (49°440 N 14°550 E; 7 nestlings from each of 2 nests, 7 June 2009). Smears from cloacae from European rollers were recovered in Martovce, Slovakia (47°450 N 18°070 E; 3 nestlings from 1 nest, 8 July 2009), Szatymaz, Hungary (46°200 N 20°02´E; 5, 3 and 3 nestlings from 3 nests, 24 June 2009) and Ludas Lake, Palic, Serbia (46°060 N 19°500 E; 4 nestlings from each of 2 nests, 24 June 2009). Smears and secretions were obtained with sterile cotton swabs. Secretions from the uropygial glands were obtained by squeezing the papilla of the gland where the secretion accumulates. Individual samples were placed in Amies transport medium (Oxoid, Great Britain) and Anaerobe medium (Oxoid, Great Britain), transported to the laboratory and stored at 4 °C until further analyses. Isolation of enterococci Slanetz-Bartley agar (Oxoid, Great Britain) and blood agar (Oxoid, Great Britain) with colistin (10 mg l-1) and nalidixic acid (10 mg l-1) were used for selective culturing and isolation of enterococci. Four to 12 colonies, 1–2 mm in diameter with wet appearance, were selected from individual samples and the isolates were characterized by Gram staining and catalase test. Gram-positive and catalase negative short rods and/or coccal isolates were maintained at -70 °C in glycerol for further analysis.
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Genus and species identification The genomic DNA was isolated by an alkaline extraction procedure (Svec et al. 2008) and used as a template for PCR using Enterococcus genus-specific primers to amplify the conserved sequence of the 16S rRNA gene (Deasy et al. 2000). For species identification, four different methods were used: (1) Rep-PCR fingerprinting with the (GTG)5 primer method (Svec et al. 2005, 2008); (2) sequencing of a 438 bp internal fragment of the sodA gene (Frolkova et al. 2012); (3) sequencing of the 16S rRNA gene (Deasy et al. 2000); (4) MALDI-TOF MS, performed using a Microflex MALDI-TOF mass spectrophotometer Microflex LRF20 (Bruker Daltonics, Germany). For the latter, briefly, a single colony from blood agar was mixed with matrix (a-cyano-4-hydroxycinnamic acid and trifluoroacetic acid) and the suspension was spotted onto a MALDI plate and ionized by nitrogen laser (wave-length 337 nm, frequency 20 Hz). The results were evaluated using flexControl—microflex v 3.3 (Bruker Daltonics, Germany) and the Maldi Biotyper v 3.0 (Bruker Daltonics, Germany) identification database. Species identity was confirmed by the congruence of results of at least three methods used. Genotyping by pulsed-field gel electrophoresis PFGE was performed according to Ghosh et al. (2011, 2012) with minor modifications. DNA was digested with 20 U of SmaI for 4 h at 37 °C. The PFGE run protocol included initial pulse time of 1 s and final time of 20 s at 200 V for 17.5 h. Comparison of the PFGE fingerprints was carried out in the BioNumerics v. 6.0 software (Applied Maths, Belgium) and the dendrograms were constructed using the band-based Dice correlation coefficient and the unweighted pair group mathematical average algorithm (UPGMA). Enterocococcus faecium ATCC 19434 was used as the reference strain. Restriction patterns were considered as closely related or indistinguishable with similarity C95 %. Multi locus sequence typing Six E. faecalis strains isolated from Common Kingfishers representing six different pulsotypes were further analyzed by MLST based on the analysis of seven housekeeping genes (gdh, gyd, pstS, gki, aro, xpt, yidL; Ruiz-Garbajosa et al. 2006). A single DNA
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band corresponding to the expected length of 395–583 bp was observed in all cases on 1.5 % agarose gel. PCR products were purified and sequenced as described previously (Frolkova et al. 2012). The sequences obtained were compared to the reference set of alleles using CodonCode Aligner ver. 2.0.4. (Imperial College London, Great Britain). MLST profiles were submitted to the MLST database (http://efaecalis.mlst.net) and assigned their MLST type (ST). eBURST clustering (http://eburst.mlst.net/) was performed using the E. faecalis MLST database. Phenotypic and genotypic resistance to antibiotics Antibiotic resistance was determined in all isolates using the disk diffusion method following the CLSI guidelines (CLSI 2008). Resistance was tested to ampicillin (10 lg per disc), tetracycline (30 lg), gentamicin (120 lg), vancomycin (30 lg) and erythromycin (15 lg). The presence of genes responsible for resistance to vancomycin i.e. vanA, vanB (DutkaMalen et al. 1995), vanC1, van C2/C3 (Janoskova and Kmet 2004), and to erythromycin i.e. ermA, ermB and mefA/E (Malhotra-Kumar et al. 2005) was tested using PCR. Primers and positive controls used for detecting the resistance genes were the same as published previously (Radimersky et al. 2010).
Results Identification and distribution of entercococci In total, 161 genomic DNA were isolated by alkaline extraction and 117 isolates from 32 out of 43 bird samples (74 %) were identified as enterococci. Out of 117, a total of 114 isolates were identified at the species level following the combinatorial approach. Rep-PCR identified 109 isolates (93 %), sodA sequence analysis identified 114 isolates (97 %) and sequencing of the 16S rRNA gene (404 bp) identified 99 isolates (85 %). MALDI-TOF MS resolved 102 isolates (87 %) to the species level. The distribution of Enterococcus spp. in individual birds is summarized in Table 1. Out of seven uropygial gland samples obtained from hoopoes, six samples (86 %) were positive for enterococci. Furthermore, 30 out of 43 (70 %) cloaca samples in all coraciid birds carried enterococci. Altogether, enterococci were
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Table 1 Distribution and species diversity of 114 enterococcal isolates from coraciiform birds Birds examined (n)
Hoopoe (7)
Common kingfisher (14)
European roller (22)
Sample origin/sp. ID.
Glandula uropygyi
Cloaca
Cloaca
Cloaca
E. faecalis E. casseliflavus
17
4
14
32
12
4
0
21
E. avium
2
1
0
0
E. hirae
0
1
0
1
E. gallinarum
0
0
0
3
E. mundtii
0
0
0
2
isolated from 71 % of hoopoes, 57 % of common kingfishers and 77 % of European rollers. Genotypic diversity From the 114 speciated isolates, 102 produced pulsotypes with SmaI. PFGE analysis revealed that E. faecalis strains (n = 67) presented 62 different pulsotypes, E. casseliflavus (n = 37) 30 pulsotypes, Enterococcus avium (n = 3) and Enterococcus gallinarum (n = 3) had three different pulsotypes each, and Enterococcus mundtii (n = 2) and Enterococcus hirae (n = 2) presented two different pulsotypes each. Overall, a low level of homology among strains of all species was found (Figs. 1, 2; Supplementary Figs. S1–S4). The only closely related strains originated from nestlings from the same nests. Isolates from the cloaca and the uropygial gland in hoopoes were genotypically distant (Figs. 1, 2). Six E. faecalis strains from Common Kingfishers representing individual pulsotypes were analyzed using MLST. In the locality of Hradek, sequence types ST34 and ST228 were found, and in the locality of Kacov ST228 and ST141 were found. Relatedness of these STs with E. faecalis STs available in the MLST database are shown in Fig. 3. ST34 is a SLV (single locus variant) of ST43, and DLV (double locus variant) of ST282, ST387, and ST128. ST141 is a SLV of ST178 as well as of ST132. ST228 is a SLV of ST247. The eBurst analysis shows that our STs are not related to each other (Fig. 3). Antibiotic resistance Two isolates of E. faecalis from a Kingfisher showed intermediate resistance to vancomycin and erythromycin. However, none of the resistance genes
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tested for (vanA, vanB, vanC1, vanC2/C3, ermA, ermB, and mefA/E) were detected. Both isolates were from the same nestling and shared the same PFGE profile.
Discussion In this study, strains of several Enterococcus spp. isolated from the cloacae and uropygial glands of wild coraciiform birds were studied. Genomic (rep-PCR, sodA gene and 16S rRNA gene sequencing) and proteomic (MALDI-TOF MS) methods were used for identification of enterococci to the species level. RepPCR and sodA gene sequencing were found to be most resolving and reliable. On the other hand, 404 bp of 16S rRNA gene sequence was too short to reliably distinguish several species. We were not able to distinguish E. hirae, E. mundtii, and E. durans. These species belong to the same group and have similar nucleotide sequences (Svec and Devriese 2009). The same problem was experienced with E. gallinarum and E. casseliflavus. These species have 99.8 % similarity of 16S rRNA gene and in the full gene length (1452 bp) have only three different nucleotides (Williams et al. 1991). For unknown reasons, goodquality mass spectra were not obtained for some strains of E. casseliflavus and E. faecalis using MALDI-TOF MS. All samples were prepared with the same procedure and only 12 samples (13 %) did not produce the good-quality spectra. The most frequent enterococcal species isolated from the cloaca of hoopoes and European rollers was E. faecalis followed by E. casseliflavus, E. gallinarum, E. avium, E. hirae, and E. mundtii. In samples from Common Kingfishers, E. faecalis was the only species isolated. The prevalence of E. faecalis among our
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Fig. 1 Clonality of E. faecalis from the uropygial gland and cloaca of coraciiform birds. Dendrogram based on SmaI restriction patterns resolved by pulsed-field gel electrophoresis
depicting the relationships of 62 representative E. faecalis strains. The scale indicates the level of pattern similarity
isolates was 51 % and this species represented 59 % of all identified enterococci. Enterococci have been previously isolated from the cloacae and faeces of wild birds. Marrow et al. (2009) isolated E. faecalis and E. gallinarum from raptors and owls with 80 % prevalence. E. faecium and E. durans were isolated from the Egyptian Vulture (Neophron percnopterus) with a prevalence of up to 64 % (Blanco et al. 2006). Radhouani et al. (2012) isolated E. faecium, E. faecalis, E. hirae and E. durans from the Common
Buzzard (Buteo buteo) with a prevalence of 74 %. Radimersky et al. (2010) isolated E. faecalis, E. faecium, E. durans, E. hirae, E. mundtii, E. gallinarum and E. casseliflavus from feral domestic pigeons (Columba livia) with a prevalence of 58 %. Enterococcal prevalence in our study was 70 % which is similar to that reported for wild birds previously. Enterococci have been also previously isolated from the uropygial gland of hoopoes. Martin-Platero et al. (2006) isolated symbiotic E. faecalis strain
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Fig. 2 Clonality of E. casseliflavus from the uropygial gland and cloaca of coraciiform birds. Dendrogram based on SmaI restriction patterns resolved by pulsed-field gel electrophoresis
depicting the relationships of 30 representative E. casseliflavus strains. The scale indicates the level of pattern similarity
Fig. 3 Clustering of three E. faecalis STs from the cloaca of common kingfisher. eBURST clustering of three E. faecalis multi-locus sequence types (STs) from this study (indicated by
solid line circles) in relation to one representative of 600 STs from MLST database. Each ST is represented as a node and lines connect single locus variants
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producing antimicrobial peptides active against a broad spectrum of pathogenic bacteria. In addition to E. faecalis, Soler et al. (2008) isolated E. faecium, E. mundtii, E. avium and E. gallinarum. Moreover, RuizRodriguez et al. (2012) cultured E. casseliflavus and E. saccharolyticus from hoopoes. These authors reported that symbiotic bacterial strains are likely vertically transmitted. We also studied one female and her 3 nestlings, but no enterococci were isolated from the mother’s gland. In our study, E. faecalis, E. casseliflavus, and E. avium were isolated from the uropygial gland of hoopoes. These results indicate that enterococcal species composition of cloaca and uropygial gland are similar. Enterococcus spp. are frequently detected in the digestive tract and faeces of birds and, unlike most hole-nesters, hoopoes do not keep their nest clean of faeces. Thus, the fecal contamination of nests of hoopoes would allow the colonization of the hoopoe’s gland by enterococci (Soler et al. 2008). However, PFGE pulsotypes of the isolates from the uropygial glands and cloacae were not identical or even related. Because of the small sample size (birds and isolates), we are not able to determine if strain composition of the uropygial gland and the cloaca is similar. Moreover, no bird-specific and/or locality-specific pulsotypes were obtained among the analyzed strains. Similar results were published by Sei-Yoon et al. (2010) who did not find any association between the origin of faecal samples of enterococci and PFGE profiles. In our study, indistinguishable pulsotypes were found in nestlings within the same nests. Since nestlings within the nest live in close contact with each other and are on the same diet, the composition of their intestinal microbiota is expected to be similar. Six E. faecalis strains representing individual pulsotypes were analysed by MLST and revealed 3 sequence types. Isolates belonging to ST34 have previously been isolated from people in Spain (Freitas et al. 2011), from chickens in Germany (Olsen et al. 2012) and from the waterway in Australia (Rathnayake et al. 2011a). Related isolates belonging to ST43, ST282, ST387, and ST128 have previously been isolated from people in Spain, France, and Poland (Rathnayake et al. 2011b) and from water in Australia (Rathnayake et al. 2011a). ST141 strains have previously been isolated from a human in Denmark (Fertner et al. 2011), from a human and chickens in Vietnam (Poulsen et al. 2012), and from chickens in Germany (Fertner et al. 2011). Related strains belonging to
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ST178 and ST132 have previously been isolated from people and chickens in Germany and Poland (Hammerum et al. 2010). ST228 strains have previously been isolated from people and chickens in Denmark (Fertner et al. 2011) and its related sequence type ST24 has previously been isolated from water in Australia (Rathnayake et al. 2011a). The eBurst analysis shows that our E. faecalis STs obtained from Common Kingfishers are not related to each other and they are not part of the hospital-associated clonal complexes CC2, CC9 and CC21 (Fertner et al. 2011; Rathnayake et al. 2011b). However, they are related to STs reported from water, people and poultry worldwide. Wild birds are a good model for studying the influx of antibiotic resistant enterococci to wildlife, since these birds are not exposed to antibiotics (Radhouani et al. 2012; Santos et al. 2013; Oravcova et al. 2013, 2014a, b). However, the spread of antibiotic resistance traits from people and/or the urban habitat to wildlife has been documented (Santos et al., 2013). Interestingly, enterococcal isolates from the present study lacked any of the tested genes conferring antimicrobial resistance. Based on our data, coraciiform birds are likely not in close contact with people and domestic animals that could serve as sources of antibiotic resistant bacteria. In conclusion, the results of this study indicate that enterococci are found as a part of the natural intestinal microbiota in wild coraciiform birds and the uropygial glands of hoopoes. In addition, there was a great genotypic diversity among E. faecalis and E. casseliflavus isolates from different coraciiform birds. None of the enterococcal isolates harboured any of the antimicrobial resistance genes tested. Acknowledgments We thank Alois Cizek for valuable advices, Gaspar Camlik, Pavel Cech, Vladimir Hosek, Orsolya Kiss, Zuzana Literakova, Karel Simecek, Otto Szekeres and Bela Tokody for the cooperation in the field and Jan Bardon for performing MALDITOF MS. This study was supported by the project CEB (CZ.1.07/ 2.3.00/20.0183) from the Ministry of Education, Youth and Sports of the Czech Republic and the project ‘‘CEITEC—Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.
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1289 Soler JJ, Martin-Vivaldi M, Ruiz-Rodriguez M, Valdivia E, Martin-Platero AM, Martinez-Bueno M, Peralta JM, Mendez M (2008) Symbiotic associations between hoopoes and antibiotic-producing bacteria that live in their uropygial gland. Funct Ecol 22:864–871 Spera RV, Farber BF (1994) Multidrug-resistant Enterococcus faecium. An untreatable nosocomial pathogen. Drugs 48:678–688 Svec P, Devriese LA (2009) Genus I. Enterococcus (ex Thiercelin and Jouhaud 1903) Schleifer and Kilpper-Ba¨lz 1984, 32VP. In: De Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds) Bergey’s manual of systematic bacteriology, The Firmicutes, vol 3. Springer, New York, pp 594–607 Svec P, Vancanneyt M, Seman M, Snauwaert C, Lefebvre K, Sedlacek I, Swings J (2005) Evaluation of (GTG)5-PCR for identification of Enterococcus spp. FEMS Microbiol Lett 247:59–63 Svec P, Novakova D, Zackova L, Kukletova M, Sedlacek I (2008) Evaluation of (GTG)5-PCR for rapid identification of Streptococcus mutans. Anton Leeuw Int J G 94:573–579 Williams AM, Rodrigues UM, Collins MD (1991) Intrageneric relationships of enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA. Res Microbiol 142:67–74
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ORIGINAL PAPER
Prevalence, diversity and characterization of enterococci from three coraciiform birds Petra Splichalova • Pavel Svec • Anuradha Ghosh • Ludek Zurek • Veronika Oravcova • Tomas Radimersky • Mirko Bohus • Ivan Literak
Received: 24 November 2014 / Accepted: 2 March 2015 / Published online: 14 March 2015 Ó Springer International Publishing Switzerland 2015
Abstract Coraciiform birds hoopoe (Upupa epops), common kingfisher (Alcedo atthis) and European roller (Coracius garrulus) were examined for enterococci in their cloacae and uropygial glands. The enterococcal isolates were identified at the species level using several genomic and proteomic methods, screened for antibiotic susceptibility and genotyped by pulsed-field gel electrophoresis (PFGE). Clonality of isolates from the common kingfisher was also assessed by multi-locus sequence typing (MLST). Using selective media, putative enterococcal isolates (n = 117) were recovered from 74 % (32 out of a total of 43) of Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0422-6) contains supplementary material, which is available to authorized users. P. Splichalova (&) V. Oravcova T. Radimersky I. Literak Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1–3, 612 42 Brno, Czech Republic e-mail: [email protected] P. Svec Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, Bld. A25, 625 00 Brno, Czech Republic
the bird samples and 114 isolates were confirmed as enterococci. Overall, among the total of 6 different species detected, Enterococcus faecalis was dominant (59 %) in all three bird species. The second most frequently isolated species was Enterococcus casseliflavus (32 %). PFGE revealed great diversity of strains from different bird species and anatomic location. Closely related strains were found only from nestlings from the same nest. No genes conferring resistance to vancomycin (vanA, vanB, vanC1 and van C2/C3) or erythromycin (erm A, ermB and mefA/E) were detected. MLST analysis and eBURST clustering revealed that sequence types of E. faecalis from the common kingfisher were identical to those of isolates found previously in water, chickens, and humans. M. Bohus Department of Environmental Ecology, Faculty of Natural Science, Comenius University in Bratislava, Mlynska dolina, 842 15 Bratislava, Slovak Republic I. Literak CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1–3, 612 42 Brno, Czech Republic
A. Ghosh L. Zurek Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
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Keywords Enterococcus Coraciiform birds sodA Rep-PCR 16S rRNA gene MALDI-TOF
Introduction Enterococci are ubiquitous bacteria in various environmental habitats and are commensals of mammals, birds, some reptiles and invertebrates (Aarestrup 2006). Some species have emerged as important causes of nosocomial and community acquired infections (Bogaard et al. 2002; Radhouani et al. 2012). In animals, enterococci can cause bovine mastitis, septicemia, endocarditis and sudden death in chickens (Aarestrup 2006); and diarrhea in rats, horses, dogs, cats and pigs (Aarestrup et al. 2002). They have innate resistance to many antimicrobial agents and they can carry a variety of acquired antibiotic resistance genes, which can be transmitted to other pathogenic bacteria (Murray 1991; Spera and Farber 1994; McMurphy et al. 2010; Radhouani et al. 2012). Acquired resistance of enterococci has also been detected in wild animals (Poeta et al. 2007). Over the past decade, members of the genus Enterococcus have been characterized using various classification methods and the number of newly described species has increased rapidly (Svec and Devriese 2009). Hoopoes, kingfishers and rollers are classified in the order Coraciiformes, a group that also includes beeeaters and wood hoopoes (Hackett et al. 2008). So far, only a few studies have focused on enterococci in coraciiform birds; Enterococcus faecalis was isolated from the uropygial gland of the hoopoe (Upupa epops, Upupidae) (Martin-Platero et al. 2006; Soler et al. 2008; Ruiz-Rodriguez et al. 2009, 2012) and Enterococcus phoeniculicola was described from the uropygial gland of the green wood hoopoe (Phoeniculus purpureus, Phoeniculidae) (Law-Brown and Meyers 2003). The present study aimed to assess the prevalence, species diversity, antibiotic resistance and clonality of enterococci isolated from three coraciiform birds. Enterococcal isolates cultured from the cloacae and the uropygial glands of coraciiform birds from central Europe, hoopoe, European roller (Coracius garrulus, Coraciidae), and common kin (Alcedo atthis, Alcedinidae), were identified by rep-PCR fingerprinting, sequencing of sodA and 16S rRNA genes, and matrix-
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assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and characterized by determination of antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Selected strains of E. faecalis from common kingfishers were also typed by multi-locus sequence typing (MLST).
Materials and methods Bird samples Smears from cloacae and uropygial glands were obtained from hoopoes originating from Sturovo, Slovakia (47°460 N 18°410 E; 1 female and her 3 nestlings were examined on 7 June 2008) and from Bzenec, Czech Republic (48°580 N 17°150 E; 3 nestlings from 1 nest were examined on 15 June 2008). Smears from cloacae were taken also from Common Kingfishers from Hradek and Kacov, Vlasim, Czech Republic (49°440 N 14°550 E; 7 nestlings from each of 2 nests, 7 June 2009). Smears from cloacae from European rollers were recovered in Martovce, Slovakia (47°450 N 18°070 E; 3 nestlings from 1 nest, 8 July 2009), Szatymaz, Hungary (46°200 N 20°02´E; 5, 3 and 3 nestlings from 3 nests, 24 June 2009) and Ludas Lake, Palic, Serbia (46°060 N 19°500 E; 4 nestlings from each of 2 nests, 24 June 2009). Smears and secretions were obtained with sterile cotton swabs. Secretions from the uropygial glands were obtained by squeezing the papilla of the gland where the secretion accumulates. Individual samples were placed in Amies transport medium (Oxoid, Great Britain) and Anaerobe medium (Oxoid, Great Britain), transported to the laboratory and stored at 4 °C until further analyses. Isolation of enterococci Slanetz-Bartley agar (Oxoid, Great Britain) and blood agar (Oxoid, Great Britain) with colistin (10 mg l-1) and nalidixic acid (10 mg l-1) were used for selective culturing and isolation of enterococci. Four to 12 colonies, 1–2 mm in diameter with wet appearance, were selected from individual samples and the isolates were characterized by Gram staining and catalase test. Gram-positive and catalase negative short rods and/or coccal isolates were maintained at -70 °C in glycerol for further analysis.
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Genus and species identification The genomic DNA was isolated by an alkaline extraction procedure (Svec et al. 2008) and used as a template for PCR using Enterococcus genus-specific primers to amplify the conserved sequence of the 16S rRNA gene (Deasy et al. 2000). For species identification, four different methods were used: (1) Rep-PCR fingerprinting with the (GTG)5 primer method (Svec et al. 2005, 2008); (2) sequencing of a 438 bp internal fragment of the sodA gene (Frolkova et al. 2012); (3) sequencing of the 16S rRNA gene (Deasy et al. 2000); (4) MALDI-TOF MS, performed using a Microflex MALDI-TOF mass spectrophotometer Microflex LRF20 (Bruker Daltonics, Germany). For the latter, briefly, a single colony from blood agar was mixed with matrix (a-cyano-4-hydroxycinnamic acid and trifluoroacetic acid) and the suspension was spotted onto a MALDI plate and ionized by nitrogen laser (wave-length 337 nm, frequency 20 Hz). The results were evaluated using flexControl—microflex v 3.3 (Bruker Daltonics, Germany) and the Maldi Biotyper v 3.0 (Bruker Daltonics, Germany) identification database. Species identity was confirmed by the congruence of results of at least three methods used. Genotyping by pulsed-field gel electrophoresis PFGE was performed according to Ghosh et al. (2011, 2012) with minor modifications. DNA was digested with 20 U of SmaI for 4 h at 37 °C. The PFGE run protocol included initial pulse time of 1 s and final time of 20 s at 200 V for 17.5 h. Comparison of the PFGE fingerprints was carried out in the BioNumerics v. 6.0 software (Applied Maths, Belgium) and the dendrograms were constructed using the band-based Dice correlation coefficient and the unweighted pair group mathematical average algorithm (UPGMA). Enterocococcus faecium ATCC 19434 was used as the reference strain. Restriction patterns were considered as closely related or indistinguishable with similarity C95 %. Multi locus sequence typing Six E. faecalis strains isolated from Common Kingfishers representing six different pulsotypes were further analyzed by MLST based on the analysis of seven housekeeping genes (gdh, gyd, pstS, gki, aro, xpt, yidL; Ruiz-Garbajosa et al. 2006). A single DNA
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band corresponding to the expected length of 395–583 bp was observed in all cases on 1.5 % agarose gel. PCR products were purified and sequenced as described previously (Frolkova et al. 2012). The sequences obtained were compared to the reference set of alleles using CodonCode Aligner ver. 2.0.4. (Imperial College London, Great Britain). MLST profiles were submitted to the MLST database (http://efaecalis.mlst.net) and assigned their MLST type (ST). eBURST clustering (http://eburst.mlst.net/) was performed using the E. faecalis MLST database. Phenotypic and genotypic resistance to antibiotics Antibiotic resistance was determined in all isolates using the disk diffusion method following the CLSI guidelines (CLSI 2008). Resistance was tested to ampicillin (10 lg per disc), tetracycline (30 lg), gentamicin (120 lg), vancomycin (30 lg) and erythromycin (15 lg). The presence of genes responsible for resistance to vancomycin i.e. vanA, vanB (DutkaMalen et al. 1995), vanC1, van C2/C3 (Janoskova and Kmet 2004), and to erythromycin i.e. ermA, ermB and mefA/E (Malhotra-Kumar et al. 2005) was tested using PCR. Primers and positive controls used for detecting the resistance genes were the same as published previously (Radimersky et al. 2010).
Results Identification and distribution of entercococci In total, 161 genomic DNA were isolated by alkaline extraction and 117 isolates from 32 out of 43 bird samples (74 %) were identified as enterococci. Out of 117, a total of 114 isolates were identified at the species level following the combinatorial approach. Rep-PCR identified 109 isolates (93 %), sodA sequence analysis identified 114 isolates (97 %) and sequencing of the 16S rRNA gene (404 bp) identified 99 isolates (85 %). MALDI-TOF MS resolved 102 isolates (87 %) to the species level. The distribution of Enterococcus spp. in individual birds is summarized in Table 1. Out of seven uropygial gland samples obtained from hoopoes, six samples (86 %) were positive for enterococci. Furthermore, 30 out of 43 (70 %) cloaca samples in all coraciid birds carried enterococci. Altogether, enterococci were
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Table 1 Distribution and species diversity of 114 enterococcal isolates from coraciiform birds Birds examined (n)
Hoopoe (7)
Common kingfisher (14)
European roller (22)
Sample origin/sp. ID.
Glandula uropygyi
Cloaca
Cloaca
Cloaca
E. faecalis E. casseliflavus
17
4
14
32
12
4
0
21
E. avium
2
1
0
0
E. hirae
0
1
0
1
E. gallinarum
0
0
0
3
E. mundtii
0
0
0
2
isolated from 71 % of hoopoes, 57 % of common kingfishers and 77 % of European rollers. Genotypic diversity From the 114 speciated isolates, 102 produced pulsotypes with SmaI. PFGE analysis revealed that E. faecalis strains (n = 67) presented 62 different pulsotypes, E. casseliflavus (n = 37) 30 pulsotypes, Enterococcus avium (n = 3) and Enterococcus gallinarum (n = 3) had three different pulsotypes each, and Enterococcus mundtii (n = 2) and Enterococcus hirae (n = 2) presented two different pulsotypes each. Overall, a low level of homology among strains of all species was found (Figs. 1, 2; Supplementary Figs. S1–S4). The only closely related strains originated from nestlings from the same nests. Isolates from the cloaca and the uropygial gland in hoopoes were genotypically distant (Figs. 1, 2). Six E. faecalis strains from Common Kingfishers representing individual pulsotypes were analyzed using MLST. In the locality of Hradek, sequence types ST34 and ST228 were found, and in the locality of Kacov ST228 and ST141 were found. Relatedness of these STs with E. faecalis STs available in the MLST database are shown in Fig. 3. ST34 is a SLV (single locus variant) of ST43, and DLV (double locus variant) of ST282, ST387, and ST128. ST141 is a SLV of ST178 as well as of ST132. ST228 is a SLV of ST247. The eBurst analysis shows that our STs are not related to each other (Fig. 3). Antibiotic resistance Two isolates of E. faecalis from a Kingfisher showed intermediate resistance to vancomycin and erythromycin. However, none of the resistance genes
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tested for (vanA, vanB, vanC1, vanC2/C3, ermA, ermB, and mefA/E) were detected. Both isolates were from the same nestling and shared the same PFGE profile.
Discussion In this study, strains of several Enterococcus spp. isolated from the cloacae and uropygial glands of wild coraciiform birds were studied. Genomic (rep-PCR, sodA gene and 16S rRNA gene sequencing) and proteomic (MALDI-TOF MS) methods were used for identification of enterococci to the species level. RepPCR and sodA gene sequencing were found to be most resolving and reliable. On the other hand, 404 bp of 16S rRNA gene sequence was too short to reliably distinguish several species. We were not able to distinguish E. hirae, E. mundtii, and E. durans. These species belong to the same group and have similar nucleotide sequences (Svec and Devriese 2009). The same problem was experienced with E. gallinarum and E. casseliflavus. These species have 99.8 % similarity of 16S rRNA gene and in the full gene length (1452 bp) have only three different nucleotides (Williams et al. 1991). For unknown reasons, goodquality mass spectra were not obtained for some strains of E. casseliflavus and E. faecalis using MALDI-TOF MS. All samples were prepared with the same procedure and only 12 samples (13 %) did not produce the good-quality spectra. The most frequent enterococcal species isolated from the cloaca of hoopoes and European rollers was E. faecalis followed by E. casseliflavus, E. gallinarum, E. avium, E. hirae, and E. mundtii. In samples from Common Kingfishers, E. faecalis was the only species isolated. The prevalence of E. faecalis among our
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Fig. 1 Clonality of E. faecalis from the uropygial gland and cloaca of coraciiform birds. Dendrogram based on SmaI restriction patterns resolved by pulsed-field gel electrophoresis
depicting the relationships of 62 representative E. faecalis strains. The scale indicates the level of pattern similarity
isolates was 51 % and this species represented 59 % of all identified enterococci. Enterococci have been previously isolated from the cloacae and faeces of wild birds. Marrow et al. (2009) isolated E. faecalis and E. gallinarum from raptors and owls with 80 % prevalence. E. faecium and E. durans were isolated from the Egyptian Vulture (Neophron percnopterus) with a prevalence of up to 64 % (Blanco et al. 2006). Radhouani et al. (2012) isolated E. faecium, E. faecalis, E. hirae and E. durans from the Common
Buzzard (Buteo buteo) with a prevalence of 74 %. Radimersky et al. (2010) isolated E. faecalis, E. faecium, E. durans, E. hirae, E. mundtii, E. gallinarum and E. casseliflavus from feral domestic pigeons (Columba livia) with a prevalence of 58 %. Enterococcal prevalence in our study was 70 % which is similar to that reported for wild birds previously. Enterococci have been also previously isolated from the uropygial gland of hoopoes. Martin-Platero et al. (2006) isolated symbiotic E. faecalis strain
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Fig. 2 Clonality of E. casseliflavus from the uropygial gland and cloaca of coraciiform birds. Dendrogram based on SmaI restriction patterns resolved by pulsed-field gel electrophoresis
depicting the relationships of 30 representative E. casseliflavus strains. The scale indicates the level of pattern similarity
Fig. 3 Clustering of three E. faecalis STs from the cloaca of common kingfisher. eBURST clustering of three E. faecalis multi-locus sequence types (STs) from this study (indicated by
solid line circles) in relation to one representative of 600 STs from MLST database. Each ST is represented as a node and lines connect single locus variants
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producing antimicrobial peptides active against a broad spectrum of pathogenic bacteria. In addition to E. faecalis, Soler et al. (2008) isolated E. faecium, E. mundtii, E. avium and E. gallinarum. Moreover, RuizRodriguez et al. (2012) cultured E. casseliflavus and E. saccharolyticus from hoopoes. These authors reported that symbiotic bacterial strains are likely vertically transmitted. We also studied one female and her 3 nestlings, but no enterococci were isolated from the mother’s gland. In our study, E. faecalis, E. casseliflavus, and E. avium were isolated from the uropygial gland of hoopoes. These results indicate that enterococcal species composition of cloaca and uropygial gland are similar. Enterococcus spp. are frequently detected in the digestive tract and faeces of birds and, unlike most hole-nesters, hoopoes do not keep their nest clean of faeces. Thus, the fecal contamination of nests of hoopoes would allow the colonization of the hoopoe’s gland by enterococci (Soler et al. 2008). However, PFGE pulsotypes of the isolates from the uropygial glands and cloacae were not identical or even related. Because of the small sample size (birds and isolates), we are not able to determine if strain composition of the uropygial gland and the cloaca is similar. Moreover, no bird-specific and/or locality-specific pulsotypes were obtained among the analyzed strains. Similar results were published by Sei-Yoon et al. (2010) who did not find any association between the origin of faecal samples of enterococci and PFGE profiles. In our study, indistinguishable pulsotypes were found in nestlings within the same nests. Since nestlings within the nest live in close contact with each other and are on the same diet, the composition of their intestinal microbiota is expected to be similar. Six E. faecalis strains representing individual pulsotypes were analysed by MLST and revealed 3 sequence types. Isolates belonging to ST34 have previously been isolated from people in Spain (Freitas et al. 2011), from chickens in Germany (Olsen et al. 2012) and from the waterway in Australia (Rathnayake et al. 2011a). Related isolates belonging to ST43, ST282, ST387, and ST128 have previously been isolated from people in Spain, France, and Poland (Rathnayake et al. 2011b) and from water in Australia (Rathnayake et al. 2011a). ST141 strains have previously been isolated from a human in Denmark (Fertner et al. 2011), from a human and chickens in Vietnam (Poulsen et al. 2012), and from chickens in Germany (Fertner et al. 2011). Related strains belonging to
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ST178 and ST132 have previously been isolated from people and chickens in Germany and Poland (Hammerum et al. 2010). ST228 strains have previously been isolated from people and chickens in Denmark (Fertner et al. 2011) and its related sequence type ST24 has previously been isolated from water in Australia (Rathnayake et al. 2011a). The eBurst analysis shows that our E. faecalis STs obtained from Common Kingfishers are not related to each other and they are not part of the hospital-associated clonal complexes CC2, CC9 and CC21 (Fertner et al. 2011; Rathnayake et al. 2011b). However, they are related to STs reported from water, people and poultry worldwide. Wild birds are a good model for studying the influx of antibiotic resistant enterococci to wildlife, since these birds are not exposed to antibiotics (Radhouani et al. 2012; Santos et al. 2013; Oravcova et al. 2013, 2014a, b). However, the spread of antibiotic resistance traits from people and/or the urban habitat to wildlife has been documented (Santos et al., 2013). Interestingly, enterococcal isolates from the present study lacked any of the tested genes conferring antimicrobial resistance. Based on our data, coraciiform birds are likely not in close contact with people and domestic animals that could serve as sources of antibiotic resistant bacteria. In conclusion, the results of this study indicate that enterococci are found as a part of the natural intestinal microbiota in wild coraciiform birds and the uropygial glands of hoopoes. In addition, there was a great genotypic diversity among E. faecalis and E. casseliflavus isolates from different coraciiform birds. None of the enterococcal isolates harboured any of the antimicrobial resistance genes tested. Acknowledgments We thank Alois Cizek for valuable advices, Gaspar Camlik, Pavel Cech, Vladimir Hosek, Orsolya Kiss, Zuzana Literakova, Karel Simecek, Otto Szekeres and Bela Tokody for the cooperation in the field and Jan Bardon for performing MALDITOF MS. This study was supported by the project CEB (CZ.1.07/ 2.3.00/20.0183) from the Ministry of Education, Youth and Sports of the Czech Republic and the project ‘‘CEITEC—Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.
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