folia biologica et geologica 58/1, 105–114, ljubljana 2017
CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE OF 
FREE-LIVING BIRDS CAPTURED IN SLOVENIA
KULTIVABILNA BAKTERIJSKA MIKROBIOTA IZ SAPIŠČ 
PROSTOŽIVEČIH PTIC, UJETIH V SLOVENIJI
Jure ŠKRABAN1,a, Tjaša MATJAŠIČ1,a, Franc JANŽEKOVIČ1, Gottfried WILHARM2 & Janja 
TRČEK1,3*
http://dx.doi.org/10.3986/fbg0024
abStRact
cultivable bacterial microbiota from choanae of free-liv-
ing birds captured in Slovenia
We have analysed the structure of cultivable choanal 
microbiota from free-living birds in relation to bird diet, its 
richness and the relative number of opportunistic bacteria 
acquired from the environment. For this purpose, we have 
taken choanal swabs from 25 free-living birds representing 
13 different bird species captured in Slovenia. From the 
grown cultures, 98 bacterial colonies were isolated and their 
16S rRNA genes sequenced. Most of the bacteria belonged to 
the phylum Actinobacteria (52 %), Proteobacteria (31 %), 
Firmicutes (15 %) and Bacteroidetes (4 %). Thirty-two per-
cent of sampled birds were colonized by known human op-
portunists and 44 % of birds by at least one known plant 
pathogen. Hierarchical clustering of the analyzed microbio-
ta grouped the birds according to their predominant diet. 
The richness of choanal microbiota from birds feeding 
mainly on insects was poorer compared to the birds feeding 
on diverse animal and plant material. The study has shown 
that the free-living birds carry an important reservoir of op-
portunistic human and plant pathogenic bacteria in their 
upper respiratory tract. To get a deeper insight into its com-
position, a bigger pool of birds will have to be analyzed in 
the future.
Keywords: birds, microbiota, choanae, pathogenic bac-
teria, diet
iZVleČeK
Kultivabilna bakterijska mikrobiota iz sapišč prosto-
živečih ptic, ujetih v Sloveniji
Sestavo kultivabilne bakterijske mikrobiote v sapiščih 
prostoživečih ptic smo analizirali z vidika vpliva prehrane, 
bogatosti mikrobiote in prisotnosti oportunističnih bakterij. 
Petindvajsetim prostoživečim pticam, ki so bile ujete v Slo-
veniji in so pripadale 13 vrstam, smo odvzeli bris sapišča. Po 
nacepitvi brisov na mikrobiološka gojišča in gojenju, smo 
izolirali 98 bakterijskih kolonij in jim določili nukleotidno 
zaporedje gena za 16S rRNK.  Večina izoliranih bakterij je 
pripadala deblu Actinobacteria (52 %), Proteobacteria (31 
%), Firmicutes (15 %) in Bacteroidetes (4 %). Pri približno 
eni tretjini ptic (32 %) smo iz sapišča izolirali vsaj eno 
oportunistično bakterijsko vrsto, ki lahko povzroča okužbe 
pri ljudeh. Pri slabi polovici ptic (44 %) pa smo v sapišču 
našli vsaj eno bakterijsko vrsto, ki lahko okuži rastline. Z 
metodo hierarhičnega združevanja smo pokazali, da imajo 
ptice s podobno prehrano, podobno bakterijsko mikrobioto 
sapišč. Ptice, ki se prehranjujejo pretežno z žuželkami so 
imele manj bogato mikrobioto kot ptice, ki se prehranjujejo 
z bolj raznoliko živalsko in rastlinsko hrano. Raziskava je 
tudi pokazala, da so zgornja dihala prostoživečih ptic 
pomemben rezervoar oportunističnih bakterij, ki lahko 
okužijo ljudi in rastline. Da bi dobili globji vpogled v sestavo 
mikrobiote zgornjih dihal, bi v prihodnosti morali povečati 
število analiziranih ptic. 
Ključne besede: ptice, mikrobiota, sapišče, patogene 
bakterije, prehrana 
 1 Department of Biology, Faculty of Natural Sciences and Mathematics, University of Maribor, Maribor, Slovenia
 2 Robert Koch Institute, Wernigerode Branch, Germany
 3 Faculty of Chemistry and Chemical Engineering, University of Maribor, Maribor, Slovenia
 * Corresponding author: telephone: +386 2 2293749, e-mail: janja.trcek@um.si
 a both authors contributed equally to the work
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
106 folia biologica et geologica 58/1 – 2017
Free-living birds are recognized vectors for spreading 
pathogenic bacteria across long distances with well-
known transmission of various respiratory infections 
to humans (Murthy et al. 2008, Pan et al. 2012, Tsio-
dras et al. 2008). Despite of this, our knowledge on the 
avian respiratory tract microbiota is very limited. 
While some data exist for the lower respiratory tract, 
almost nothing is known about the bacteria living in 
the upper respiratory regions. 
Data on microbiota of the lower respiratory tract 
in domestic birds have shown that it harbors poten-
tially pathogenic bacteria. Majority of cultured bacte-
ria found in the lungs and trachea of birds belonged to 
phyla Proteobacteria, Firmicutes, Tenericutes, Actino-
bacteria, Bacteroidetes and Chlamydia/Verrucomicro-
bia (Murthy et al. 2008, Charlton et al. 1993, Byrum 
& Slemons 1995). Additionally, culture independent 
analyses detected groups of fastidious or poorly repre-
sented taxons belonging to Fusobacteria, Acidobacte-
ria, Chloroflexi, Cyanobacteria and Deinococcus 
-Thermus in the lower respiratory tract of poultry. 
Among them were also potential pathogens (Myroides 
spp., Collinsella aerofaciens, Bacteroides fragilis, Ente-
rococcus cecorum, Kurthia zopfii, Kushneria sp. and 
Bordetella sp.) (Shabbir et al. 2015). Even though the 
pathogen Riemerella sp. has been isolated from the 
upper respiratory tract of some species of domestic and 
free-living birds (Vancanneyt et al. 1999), a deeper 
insight into the structure of the upper respiratory mi-
crobiota in birds is lacking. Thus far, a very limited 
number of research attempted to analyze the bacterial 
composition of the upper respiratory tract in free-liv-
ing birds, besides, they used selective media for cultur-
ing specific groups of pathogenic bacteria, and thus 
substantially limiting the overall view on microbial 
diversity of the upper respiratory tract (Lamberski et 
al. 2003, Stenkat et al. 2014). In such a way, Lamber-
ski et al. (2003) analyzed two species of hawks which 
harbored pathogens like Salmonella sp. and Pasteurella 
sp.. Stenkat et al. (2014) focused more on water birds 
and also found potential avian and human pathogens 
(Klebsiella pneumoniae, Escherichia coli and Pseudo-
monas aeruginosa among others).
More knowledge about the microbiota of the upper 
respiratory tract of birds is necessary for better under-
standing the influence, positive or negative, of this mi-
crobiome on animal health and the risks of spreading 
potential infections between free-living birds, in the 
environment and subsequently to humans (Walden-
strom et al. 2003, Abulreesh et al. 2007).
1 INTRODUCTION
2 MATERIALS AND METHODS
bird sampling, culturing of bacteria and identi-
fication
All of the 25 healthy adult birds included in this study 
were captured in fine mist nets for bird ringing during 
fall, between September 18 and December 10, 2013 in 
Maribor and its surroundings (Slovenia). The birds 
were caught in the frame of bird ringing scheme coor-
dinated by EURING.
Choanal swabs (pre moistened with sterile saline) 
were immediately taken from each bird and put in a 
transport medium (Amies agar gel medium transport 
swabs – no charcoal, Copan) until further processing. 
All samples were sent to the laboratory within 2 to 3 
hours after sampling and inoculated on nutrient agar 
(NA, Sigma). Inoculated plates were incubated 4 - 7 
days at 30°C. After incubation, each colony morpho-
type per bird was isolated and stored at -80°C until 
further processing. Colony morphotypes were differ-
entiated based on form, margin and pigmentation of 
the colonies.
Total DNA was isolated and cleaned using a com-
mercial kit (NucleoSpin Tissue, Macherey-Nagel). Full 
lengths of 16S rRNA genes were amplified with PCR. 
The final concentrations of the PCR reaction mix con-
tained 0.2 mM dNTP (Thermo Scientific), 1x PCR buf-
fer with KCl (Thermo Scientific), 2.5 mM MgCl2 
(Thermo Scientific), 1.0 µM of forward primer (5’-AAA 
TTG AAG AGT TTG ATC ATG GC-3’), 1.0 µM of re-
verse primer (5’-AAG GAG GTG ATC CAG CCG CA-
3’) and 0.025 units/µL of Taq polymerase (Thermo Sci-
entific). Amplicons were obtained using the following 
PCR protocol: initial denaturation at 95°C for 5 min 
followed by 30 cycles of denaturation at 95°C for 30 s, 
annealing at 56°C for 30 s and elongation at 72°C for 
1.5 min, followed by a final extension at 72°C for 10 
min. The PCR product was purified with a commercial 
kit (GeneJET PCR Purification Kit, Thermo Scientific) 
and sequenced (Eurofins Genomics). The obtained se-
quences were compared to EMBL/GenBank/DDBJ da-
tabases and identified using BLAST. The closest hits to 
type strains, with 98.7 or higher % similarity, were 
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
107folia biologica et geologica 58/1 – 2017
identified at species level. In case of two or more differ-
ent hits with similarity score above 98.7 %, the isolate 
was identified at the genus level.
Hierarchical clustering
Ward method with Euclidian distances was used for 
the clustering of the choanal microbiota of the investi-
gated birds, for which the data on the presence or ab-
sence of bacterial species were included in the analysis.
Statistical analysis
To test the differences in the presence of microbial 
groups between different species of birds, we used 
Fisher’s exact test, where P < 0.05 was considered sig-
nificant. Where the differences in the frequencies of 
microbial groups were significant, odds ratio was cal-
culated (P < 0.05).
The differences in species richness between the 
groups of birds were tested with Student - T test, P < 
0.05 was considered significant.
3 RESULTS AND DISCUSSION
In this study we performed identification of bacteria 
isolated on a complex nutrient agar medium from 
birds’ choanae with the aim to assess microbial diver-
sity from this specific niche and find a possible corre-
lation with birds’ diet. For this, we have sampled 25 
table 1: Richness of choanal microbiota.
Preglednica 1: bogatost mikrobiote sapišč.
Bird Species Number of birds Average number of different isolates 
per bird species
European robin (Erithacus rubecula) 4 2.8 ± 2.0a
Garden warbler (Sylvia borin) 1 4
Common reed bunting (Emberiza schoeniclus) 1 3
Willow warbler (Phylloscopus trochilus) 1 1
Dunnock (Prunella modularis) 4 2.5 ± 1.1a
Common redstart (Phoenicurus phoenicurus) 1 5
Common chiffchaff (Phylloscopus collybita) 1 3
yellowhammer (Emberiza citrinella) 1 4
Eurasian blackcap (Sylvia atricapilla) 2 2.5 ± 1.5a
Song thrush (Turdus philomelos) 1 12
Pigeon (Columba livia) 4 3.3 ± 2.3a
Common chaffinch (Fringilla coelebs) 1 10
Eurasian tree sparrow (Passer montanus) 3 5.7 ± 1.9a
a, standard deviation
birds, from which 98 bacterial  colonies were isolated 
and sequenced. The number of different bacterial spe-
cies per bird ranged from 12 (Song thrush (Turdus phi-
lomelos) to 1 (Willow warbler (Phylloscopus trochilus)) 
(Table 1).
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
108 folia biologica et geologica 58/1 – 2017
Majority of isolates belonged to phyla Actinobacte-
ria (52 %) and Proteobacteria (31 %). The phyla Fir-
micutes and Bacteroidetes were represented by only 15 
% and 4 %, respectively. The isolates belonged to 22 
families. The majority constituted families Microbacte-
riaceae (36 %), Pseudomonadaceae (11 %), Enterobacte-
riaceae (10 %), Micrococcaceae (7 %), Flavobacteriace-
ae, Xanthomonadaceae and Staphylococcaceae (all 4 
%). Other families were found only sporadically. Of 13 
different bird species, which have been sampled, 85 % 
were colonized with members of Microbacteriaceae. 
Pseudomonadaceae, Nocardiaceae and Enterobacteria-
ceae were found in 38 %, 31 % and 31 % of bird species, 
respectively. Xanthomonadaceae, Moraxellaceae, 
Staphylococcaceae were found in 23 % and Micrococ-
caceae in 15 % of analyzed bird species. Other isolates 
were found only per single bird. Although two previous 
studies analyzed choanal swabs from birds, there are 
some substantial differences in experimental approach-
es in comparison to our study and also in the birds 
analyzed. Lamberski et al. (2003) analyzed choanal 
swabs from captive and free-living red-tailed and Coo-
per’s hawks, but the samples were grown on blood and 
MacConkey media. In this way they found the micro-
biota to be composed of Bacillus sp., Corynebacterium 
sp., Escherichia sp., Salmonella sp., Pasteurella sp., 
Streptococcus sp. and coagulase positive and negative 
staphylococci. Since we have performed the isolation 
on a complex nutrient medium in order to detect a 
wider range of environmental bacteria our results only 
partially overlapped. We have also isolated the genus 
Bacillus sp. and coagulase negative staphylococci, but 
otherwise the choanal microbiota of our birds greatly 
differed. This can be explained also by the fact that we 
have sampled different species of birds, with different 
diets (Cooper’s hawk feeds exclusively on small and 
mid-sized birds and red-tailed hawk is opportunistic 
carnivorous feeder) and in different geographical loca-
tions (Slovenia vs. Unites States). The other group, 
Stenkat et al. (2014) used blood, MacConkey and Bril-
liant green agar to investigate pharyngeal bacterial mi-
crobiota in water rails, spotted crakes, barn swallows, 
mute swans, reed warblers and black cormorants, and 
found numerous ubiquitous bacteria belonging pre-
dominantly to Enterobacteriaceae, Pseudomonadaceae, 
Aeromonadaceae, Bacillaceae, Staphylococcaceae and 
Streptococcaceae which are frequently present in the 
environment and on food. We have also found mem-
bers of the forementioned bacterial families, except the 
family Aeromonadaceae, which is more associated with 
water habitats and the family Streptococcaceae, which 
was absent in our study, possibly due to different 
growth media (nutrient agar as opposed to blood agar).
Out of 98 isolates from choanal swabs, 13 (13.3 %) 
have been known to cause opportunistic infections in 
humans. Species previously described as being associ-
ated with human infections were Acinetobacter calcoa-
ceticus (Nonaka et al. 2014), Cellulosimicrobium fun-
kei (Petkar et al. 2011), Curtobacterium citreum (Ri-
vera et al. 2012), Curtobacterium flaccumfaciens 
(Francis et al. 2011), Exiguobacterium sibiricum (Tena 
et al. 2014), Hafnia alvei (Gunthard & Pennekamp 
1996), Microbacterium oleivorans (Kim & Lee 2012), 
Microbacterium resistens (Panackal 2013), Pantoea 
agglomerans (Rezzonico et al. 2010), Pseudomonas ae-
ruginosa (yamazaki et al. 2012), Serratia grimesii 
(Kumar et al. 2013), Staphylococcus epidermidis 
(Vuong & Otto 2002) and Staphylococcus gallinarum 
(Tibra et al. 2010). Eight out of 25 (32 %) sampled birds 
carried one or more human opportunistic bacteria in 
their choanae. Five out of 25 (20 %) birds were colo-
nized by one, two birds were simultaneously colonized 
by two opportunists and the song thrush (Turdus phi-
lomelos) by three different putative pathogens. Pigeons 
also seemed to be frequent carriers of potential patho-
gens. Three out of four sampled pigeons were colo-
nized by Staphylococcus gallinarum (in our study found 
only in pigeons) and the fourth bird was colonized by 
Acinetobacter calcoaceticus. Two out of three sampled 
eurasian tree sparrows which, as pigeons, also live in 
close proximity to humans, also carried opportunists 
(Curtobacterium citreum, Curtobacterium flaccumfaci-
ens and Exiguobacterium sibiricum) in choanal micro-
biota.
In addition to human opportunistic bacteria, 7 po-
tential plant pathogens were also isolated from choa-
nae of 11 (44 %) sampled birds. These were Agrobacte-
rium larrymoorei (Bouzar & Jones 2001), Clavibacter 
michiganensis (Xu et al. 2010), Curtobacterium fla-
ccumfaciens (Francis et al. 2011), Plantoea agglome-
rans (Rezzonico et al. 2010), Pseudomonas aeruginosa 
(yamazaki et al. 2012), Pseudomonas flavescens (Fett, 
Cescutti & Wijey 1996) and Rhodococcus fascians 
(Crespi et al. 1992). Ten (40 %) birds were colonized by 
one plant pathogen and only one tree sparrow by two 
(Agrobacterium larrymoorei and Curtobacterium fla-
ccumfaciens). The most frequently isolated plant patho-
gen was Rhodococcus fascians, which was isolated from 
four different birds belonging to four different species 
(Eurasian tree sparrow, Common chaffinch, yellow-
hammer and Dunnock) with different feeding habits 
(seeds/insects, seeds/insects, insects and insects), re-
spectively. This suggests that it is commonly present in 
bird population.
Previous investigations have shown that the com-
position of intestinal microbiota in birds depends on 
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
109folia biologica et geologica 58/1 – 2017
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110 folia biologica et geologica 58/1 – 2017
table 2: identified bacterial isolates from choanae of free-living birds. the birds were grouped based on choanal 
microbiota composition with hierarchical clustering on group 1 (predominantly insectivorous birds) and group 2 
(birds with mixed diet of invertebrates and seeds).
Preglednica 2: identificirani bakterijski izolati iz sapišč prostoživečih ptic. Ptice smo s hierarhičnim zduževanjem 
združili v dve skupini, skupina 1 (pretežno žužkojede ptice) in skupina 2 (ptice z mešano prehrano sestavljeno iz 
nevretenčarjev in semen).
Bacterial isolates found in group 1 Bacterial isolates found in group 2 Bacterial isolates found in group 1 and 2
Aeromicrobium ponti/
A. tamlense Agrobacterium larrymoorei Acinetobacter calcoaceticus
Aeromicrobium sp. nov.a Agromyces terreus Chryseobacterium indoltheticum
Agrococcus versicolor Agromyces sp. nov.b Frigoribacterium faeni
Agromyces allii Arthrobacter aurescens Microbacterium hydrocarbonoxydans/M. phyllosphaerae
Bacillus aryabhattai Arthrobacter nitroguajacolicus/A. aurescens Microbacterium phyllosphaerae
Citrobacter gillenii Arthrobacter oxydans Micrococcus sp.
Clavibacter michiganensis Brochothrix campestris
Paenibacillus xylanexedens/ 
P. amylolyticus/
P. tundra
Curtobacterium plantarum Cellulosimicrobium funkei Pseudomonas flavescens
Enterococcus plantarum Chryseobacterium daecheongense Rathayibacter festucae
Hafnia alvei Chryseobacterium sp. nov.c Rhodococcus fascians
Microbacterium hominis Curtobacterium citreum Stenotrophomonas rhizophila
Microbacterium oleivorans Curtobacterium flaccumfaciens
Microbacterium oxydans Exiguobacterium sibiricum
Microbacterium sp. Leucobacter exalbidus
Microbacterium xylanilyticum Microbacterium hydrocarbonoxydans
Micrococcus yunnanensis Microbacterium resistens
Ochrobactrum thiophenivorans Microbacterium testaceum
Pantoea agglomerans Oerskovia sp.
Pantoea anthophila Okibacterium fritillariae
Plantibacter flavus Pantoea agglomerans
Pseudomonas aeruginosa Pseudoclavibacter helvolus
Pseudomonas moraviensis Pseudomonas cedrina
Pseudomonas orientalis Pseudomonas extremorientalis
Pseudomonas psychrotolerans Pseudoxanthomonas koreensis
Sanguibacter keddieii Sphingobacterium faecium
Serratia grimesii Staphylococcus gallinarum
Staphylococcus epidermidis Staphylococcus sp.
Stenotrophomonas chelatiphaga  
Variovorax paradoxus
a,b,c, potentially new species isolated from garden warbler (Sylvia borin)a, song thrush (Turdus philomelos)b and Eurasian 
tree sparrow (Passer montanus)c
various factors, among them being the host species and 
feeding patterns. The differences extend to functional 
properties such as the greater capacity for amino acid 
metabolism and energy harvest in carnivores com-
pared to herbivores (Waite & Taylor 2014). There-
fore, since the gastrointestinal and respiratory tracts 
are connected, it is reasonable to assume that these fac-
tors also influence the composition of the respiratory 
microbiota. To assess the differences in the choanal 
microbiota of the sampled birds, we have performed 
hierarchical clustering which grouped the birds into 
two groups (Fig. 1). Choanal microbiota of the birds 
with similar diet grouped together. Birds which feed 
predominantly on insects or have more monotonous 
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
111folia biologica et geologica 58/1 – 2017
diet clustered in one group, those that have more mixed 
diet of animals (invertebrates) and seeds throughout 
the year formed a separate group (Fig. 1). The number 
of bacterial species was used to assess the difference in 
choanal microbiota richness between the two groups. 
The first group which contains the birds predominant-
ly feeding on insects, or which have a more monoto-
nous diet in general, had a significantly lower average 
number of species (2.9 ± 1.6) in comparison to birds 
enjoying a more mixed diet of animals and plants 
throughout the season (7.8 ± 3.4) (P = 0.0002) (Table 1). 
The choanal microbiota differed between the two 
groups not only in terms of species richness, but also in 
terms of bacterial composition. Majority of isolates 
were found in only one of the two groups of birds (29 
– the first group, 27 – the second group) and only 11 
bacterial species colonized choanae of birds belonging 
to both groups (Table 2).
Stenkat et al. (2014) have previously found cor-
relations between certain bacterial families and feed-
ing habits, although they targeted specific pathogenic 
groups of bacteria. Enterobacteriaceae and Aeromon-
adaceae were correlated to piscivores, Staphylococca-
ceae and Streptococcaceae to aerial insectivores, and 
Pseudomonadaceae and Bacillaceae to herbivores. Our 
findings corroborate this, as hierarchical clustering 
grouped the choanal microbiota of the sampled birds 
into two groups based on the bird diet.
When comparing the presence or absence of indi-
vidual bacterial species, pigeons showed to be far more 
likely colonized with Staphylococcus gallinarum than 
other sampled birds (Fisher’s exact test (P = 0.013); 
odds ratio (pigeons/other birds) = 31.5, P = 0.012). Fur-
thermore, the presence of the genus Staphylococcus sp. 
was indicative of the birds with a more diverse diet 
throughout the season; these birds also clustered in 
one of the two groups (Fig. 1, Fisher’s exact test 
(P = 0.040); odds ratio (second group/first group) 
= 12.0; P = 0.044).
Apart from finding numerous human and plant 
opportunists, we have also isolated one novel species 
from garden warbler (Sylvia borin) (Aeromycrobium 
choanae sp. nov.) (Ber et al. 2017), and two potentially 
novel species from song thrush (Turdus philomelos) 
(Agromyces sp., 16S rRNA gene sequence similarity 
< 97 %) and Eurasian tree sparrow (Passer montanus) 
(Chryseobacterium sp., 16S rRNA sequence similarity 
< 98.7 %). Their description is part of ongoing research. 
4 CONCLUSIONS
Our study has shown that the choanal microbiota of 
free-living birds with a diet composed predominantly 
of insects, or with a generally monotonous diet, was 
poorer in terms of species richness, compared to birds 
with a more diverse diet during the year. Previously, 
correlation between selected bacterial families and diet 
has been determined, however our analyses have 
shown that the differences in microbiota extend be-
yond selected bacterial families. Hierarchical cluster-
ing of bacteria showed a correlation between the birds 
feeding patterns and the upper respiratory microbiota 
composition. Our study has also shown that free-living 
birds carry a wide array of known human and plant 
pathogens in their upper respiratory tract, but also 
possible novel species. Given the impact microbiota 
has on the bird ś health and bird ś potential for spread-
ing pathogens in the environment, it will be necessary 
to extend the analysis of choanal microbiota and fac-
tors that shape its structure, on more free-living bird 
species.
5 POVZETEK
Da bi ocenili mikrobno diverziteto v sapiščih prostoži-
večih ptic, smo 25 pticam odvzeli brise sapišč, ki smo 
jih nacepili na hranilni agar. Po gojitvi smo izolirali 98 
bakterijskih kolonij in jih na podlagi nukleotidnega 
zaporedja za 16S rRNK identificirali. Število različnih 
bakterijskih izolatov pri posamezni ptici se je gibalo 
med 12 (cikovt, Turdus philomelos) in 1 (severni kova-
ček, Phylloscopus trochilus). Večina izolatov je pripada-
la deblom Actinobacteria (52 %), Proteobacteria (31 %), 
Firmicutes (15 %) in Bacteroidetes (4 %). Izolati so ve-
činoma pripadali družinam Microbacteriaceae (36 %), 
Pseudomonadaceae (11 %), Enterobacteriaceae (10 %), 
Micrococcaceae (7 %), in Flavobacteriaceae, Xantho-
monadaceae in Staphylococcaceae (vse 4 %). Največ 
ptic (11) je bilo koloniziranih z bakterijami, ki so pri-
padale družini Microbacteriaceae, nato Pseudomona-
daceae (pet ptic), Nocardiaceae (štiri ptice), Enterobac-
teriaceae (štiri ptice), Xanthomonadaceae (tri ptice), 
ŠKRABAN, MATJAŠIČ, JANŽEKOVIČ, WILHARM & TRČEK: CULTIVABLE BACTERIAL MICROBIOTA FROM CHOANAE 
112 folia biologica et geologica 58/1 – 2017
Moraxellaceae (tri ptice), Staphylococcaceae (tri ptice) 
in Micrococcaceae (dve ptici). Ostale družine bakterij 
smo detektirali le pri posamezni ptici.
Od skupno 98 bakterijskih izolatov, smo našli 13 
(13,3 %) takih, ki lahko povzročajo okužbe pri ljudeh: 
Acinetobacter calcoaceticus, Cellulosimicrobium funkei, 
Curtobacterium citreum, Curtobacterium flaccumfaci-
ens, Exiguobacterium sibiricum, Hafnia alvei, Micro-
bacterium oleivorans, Microbacterium resistens, Panto-
ea agglomerans, Pseudomonas aeruginosa, Serratia gri-
mesii, Staphylococcus epidermidis in Staphylococcus 
gallinarum. Pri osmih pticah (32 %) smo v sapišču našli 
vsaj eno oportunistično bakterijo. Petina ptic je bila 
koloniziranih z eno, dve ptici z dvema, cikovt pa hkra-
ti s tremi oportunističnimi vrstami bakterij. Tudi ptice 
urbanih okolij (golob in domači vrabec) so bile koloni-
zirane s človeškimi oportunisti. Golobi s Staphyloco-
ccus gallinarum in Acinetobacter calcoaceticus, vrabci 
pa s Curtobacterium citreum, Curtobacterium flaccum-
faciens in Exiguobacterium sibiricum.
Poleg oportunističnih bakterij, ki povzročajo 
okužbe pri ljudeh, smo pri 44 % vzorčenih ptic našli 
bakterije, ki so patogene za rastline: Agrobacterium 
larrymoorei, Clavibacter michiganensis, Curtobacteri-
um flaccumfaciens, Plantoea agglomerans, Pseudomo-
nas aeruginosa, Pseudomonas flavescens in Rhodoco-
ccus fascians. Največkrat smo detektirali bakterijo 
Rhodococcus fascians, ki je bila prisotna pri štirih raz-
ličnih vrstah ptic (domači vrabec, ščinkavec, rumeni 
strnad in siva pevka). Prvi dve vrsti se prehranjujeta z 
raznovrstno hrano sestavljeno iz semen in žuželk, za-
dnji dve pa pretežno z žuželkami, kar bi lahko pome-
nilo, da je bakterija med pticami splošno prisotna.
Na sestavo in delovanje črevesne mikrobiote pri pti-
cah vplivajo različni dejavniki, kot sta vrsta gostitelja in 
vrsta hrane (mesojedci/rastlinojedci). Ker so prebavila 
in dihala povezana, ti dejavniki verjetno vplivajo tudi 
na sestavo in delovanje mikrobiote v dihalih. Z metodo 
hierarhičnega združevanja smo ptice na podlagi sestave 
bakterijske mikrobiote sapišč združili v dve skupini. V 
prvi skupini so bile pretežno žužkojede ptice, v drugi pa 
ptice z bolj raznovrstno prehrano rastlinskega in žival-
skega izvora. Tudi bogatost mikrobiote, ki smo jo oceni-
li na podlagi števila prisotnih bakterijskih vrst, je med 
obema skupinama ptic bila različna. Pri žužkojedih pti-
cah smo zaznali manjše število vrst (2,9 ± 1,6) v primer-
javi s pticami, ki se hranijo z bolj raznovrstno hrano ži-
valskega in rastlinskega izvora (7,8 ± 3,4) (P = 0,0002). 
Obe skupini ptic sta imeli tudi različno sestavo mikrobi-
ote, saj smo večino bakterijskih vrst našli le pri eni ali 
drugi skupini (29 bakterijskih vrst pri žužkojedih pti-
cah, 27 bakterijskih vrst pri pticah z raznoliko prehra-
no) in le 11 bakterijskih vrst smo detektirali pri obeh 
skupinah ptic. Pri vrtni penici (Sylvia borin), cikovtu 
(Turdus philomelos) in vrabcu (Passer montanus) smo v 
sosledju našli tudi novo in dve domnevno novi vrsti 
bakterij; Aeromycrobium choanae sp. nov., Agromyces sp. 
in Chryseobacterium sp..
Z metodo hierarhičnega združevanja smo poka-
zali, da imajo ptice s podobno prehrano, podobno 
bakterijsko mikrobioto sapišč. Ptice, ki se prehranju-
jejo pretežno z žuželkami, so imele manj bogato mi-
krobioto kot ptice, ki se prehranjujejo z bolj raznoliko 
živalsko in rastlinsko hrano. Raziskava je tudi poka-
zala, da so zgornja dihala prostoživečih ptic pomem-
ben rezervoar oportunističnih bakterij, ki lahko oku-
žijo ljudi in rastline, in tudi novih vrst bakterij. Da bi 
dobili globji vpogled v sestavo mikrobiote zgornjih 
dihal, bi v prihodnosti morali povečati število analizi-
ranih ptic. 
ACKNOWLEDGEMENTS – ZAHVALA
This research was funded by the Slovenian Research Agency through programs IP-0552 and P2-0006. For help 
with bird sampling, we thank bird ringing researcher Iztok Vreš (Slovenian Museum of Natural History).
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