Campylobacter ornithocola sp. nov., a new member of the Campylobacter lari group isolated from wild bird faecal samples

Abstract

During a study on the prevalence and diversity of campylobacteria in wild birds faecal samples in the city of Valdivia (South of Chile) 17 Gram-negative, curved rod isolates, were initially identified as Campylobacter lari by PCR-RFLP. Further identification by 16S rRNA sequence analysis revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by rpoB, atpA and cpn60 gene sequence analysis. The average nucleotide identity between the representative strain WBE38T and the type strain of the closest taxon Campylobacter lari subsp. concheus (LMG 11760) was 90.8%. The oxidase and urease activity of the novel isolates enabled them to be phenotypically differentiated from recognized Campylobacter species. Therefore, on the basis of phenotypic, genetic and genomic characterizations, this study clearly shows that these strains represent a novel species within the genus Campylobacter, for which the name Campylobacter ornithocola sp. nov. is proposed, with the type strain WBE38T (=CECT 9147T =LMG 29815T).

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Campylobacter ornithocola sp. nov., a novel member of the
Campylobacter lari group isolated from wild bird faecal samples
Alberto C
aceres,
1
Ivo Muñoz,
1
Gregorio Iraola,
2
Florencia Díaz-Viraqu
e
3
and Luis Collado
1,
*
Abstract
During a study on the prevalence and diversity of campylobacteria in wild birds faecal samples from the city of Valdivia
(southern Chile) 17 Gram-stain-negative, curved-rod-shaped isolates, were initially identified as Campylobacter lari by PCR–
RFLP. Further identification by 16S rRNA sequence analysis revealed that they formed a distinct group in the genus
Campylobacter. This unique position was confirmed by the results of analysis of rpoB,atpA and cpn60 gene sequences. The
average nucleotide identity between the representative strain WBE38
T
and the type strain of the most closely related taxon C.
lari subsp. concheus (LMG 11760) was 90.8 %. The oxidase and urease activity of the novel isolates enabled them to be
phenotypically differentiated from species of the genus Campylobacter with validly published names. Therefore, on the basis
of phenotypic, genetic and genomic characterizations, the results of this study clearly indicate that these strains represent a
novel species within the genus Campylobacter, for which the name Campylobacter ornithocola sp. nov. is proposed, with the
type strain WBE38
T
(=CECT 9147
T
=LMG 29815
T
).
The genus Campylobacter was proposed by Sebald and
Veron [1], to accommodate the species Vibrio fetus (now
Campylobacter fetus, the type species of the genus). Since
then the number of species of the genus Campylobacter has
greatly increased and, at the time of writing (September
2016), this genus encompassed 28 species and 9 subspecies
with validly published names [2–4]. Infection with members
of the genus Campylobacter is the leading cause of food-
borne bacterial gastroenteritis in the developed world,
where infection is principally the result of consumption of
contaminated meat and poultry. The most common symp-
toms of campylobacteriosis include severe diarrhoea, with a
proportion of patients developing chronic sequelae such as
Guillain–Barr
e syndrome, reactive arthritis and irritable
bowel syndrome [5]. Campylobacter jejuni and Campylobac-
ter coli are by far the most isolated and studied species, while
the epidemiology and the clinical role of the other less com-
mon species in human and animal diseases is far less under-
stood [5, 6].
In the present study, 17 novel isolates of members of the
genus Campylobacter recovered from wild bird faecal sam-
ples were subjected to a polyphasic approach including
molecular identification by PCR–RFLP, genotyping by
enterobacterial repetitive intergenic consensus PCR
(ERIC–PCR), phylogenetic analysis using the 16S rRNA,
rpoB,atpA and cpn60 genes and phenotypic characteriza-
tion, in order to determine their taxonomic positions.
Additionally, the whole genome of WBE38
T
was
sequenced, since it was selected as the type strain. On the
basis of our results, we propose and describe these strains
as representing a novel species within the genus
Campylobacter.
Over the period between January 2013 and October 2015, a
study was conducted to evaluate the prevalence and diver-
sity of campylobacteria in wild bird faecal samples from the
city of Valdivia, in the south of Chile, where a collection of
isolates of members of the genus Campylobacter was
obtained (L. Collado, paper in preparation). The samples
corresponded to bird’s faecal droppings excreted in public
urban parks. Although the specific type of bird correspond-
ing to each specimen remains unidentified, at the time of
sampling black-faced ibis (Theristicus melanopis), southern
lapwing (Vanellus chilensis) and chimango caracara (Mil-
vago chimango) were the main species observed at these
sites. Faecal samples were collected using sterile cotton-
tipped swabs that were immediately placed into 8 ml Bolton
broth (Oxoid). Incubation was performed at 37 C for 48 h
under micro-aerobic conditions (CampyGen, Oxoid). After
Author affiliations:
1
Institute of Biochemistry and Microbiology, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile;
2
Bioinformatics Unit,
Institut Pasteur Montevideo, Montevideo, Uruguay;
3
Molecular Biology Unit, Institut Pasteur Montevideo, Montevideo, Uruguay.
*Correspondence: Luis Collado, luiscollado@uach.cl
Keywords: wild bird; Campylobacter; Valdivia; Chile.
Abbreviation: ERIC, enterobacterial repetitive intergenic consensus.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, rpoB,atpA,cpn60 genes and the draft genome of the strain WBE38
T
are KX467974,
KX467989, KX467979, KX467985 and LXSU00000000, respectively.
Three supplementary figures are available with the online Supplementary Material.
TAXONOMIC DESCRIPTION
C
aceres et al., Int J Syst Evol Microbiol
DOI 10.1099/ijsem.0.001822
001822 ã2017 IUMS
1

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enrichment, an aliquot (400 µl) was transferred onto the
surface of a 0.45 µm membrane filter (Millipore) which was
placed on a Petri dish containing Blood Agar Base (Merck)
supplemented with 5 % defibrinated sheep blood (Quad
Five) and it was allowed to filter passively under
room temperature conditions for 30 min. After filtration,
the filters were removed with sterile forceps and discarded.
The inoculated plates were incubated under the aforemen-
tioned conditions [7]. The 17 novel isolates described here
were initially identified as Campylobacter lari by the PCR–
RFLP described by Marshall et al. [8] showing the C1 and
C1b restriction patterns with the endonucleases DdeI and
BsrI, respectively (data not shown). However, recently this
species has been divided into two subspecies (C. lari subsp.
lari and C. lari subsp. concheus) [9]. Additionally, several
C. lari-like novel species have been described, i.e.,
Campylobacter insulaenigrae [10], Campylobacter peloridis
[9], C. subantarcticus [11] and Campylobacter volucris [12],
that collectively have been referred to as the Campylobacter
lari group [13]. Since no available PCR method could differ-
entiate these highly related taxa, all isolates we identified as
C. lari by PCR–RFLP were further analyzed by a polyphasic
taxonomic approach.
The 17 isolates were first analysed by means of the ERIC–
PCR technique using the protocol of Houf et al. [14], in
order to avoid further analysis of strains with the same
genotype. The electrophoretic patterns were analysed using
the Jaccard index and a dendrogram was reconstructed by
using the unweighted pair group linkage analysis (UPGMA)
method with the Phoretix 1d Pro software (Totallab). As
shown in Fig. 1, all the isolates belonged to different ERIC–
PCR types and, consequently, were considered to represent
different strains. Nearly complete 16S rRNA gene sequences
(1350 bp) of five selected strains (WBE38
T
, WBE186,
WBE206, WBE215 and WBE241), were amplified as
described by Vandamme et al. [15]. Additionally, the rpoB,
atpA and cpn60 genes were also evaluated with the protocols
described by Korczak et al. [16], Miller et al. [17] and Hill
et al. [18], respectively. Due to the high number of strains,
only the analysis of rpoB gene sequences included all 17
strains. Both DNA strands of the PCR products were
directly sequenced with an ABI 3730 XL automatic DNA
WBE258
WBE212
WBE241
WBE215
WBE225
WBE249
WBE210
WBE206
WBE201
WBE193
WBE187
WBE38
WBE186
WBE188
WBE195
WBE196
WBE189
0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Fig. 1. Cluster analysis based on the patterns obtained by ERIC–PCR from the Campylobacter ornithocola sp. nov. strains.
C
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sequencer (ABI) by a commercial sequencing facility (Mac-
rogen, Seoul, Korea). Alignment of the sequences was per-
formed with the CLUSTAL W program [19]. Phylogenetic trees
were reconstructed with the MEGA 6 software [20], by using
the neighbour-joining method [21] with Kimura’s two-
parameter substitution model [22] and the stability of the
groupings, estimated by bootstrap analysis (500 replica-
tions). Similarity values between the 16S rRNA gene
sequence of WBE38
T
and those of the type strains of all
members of the genus Campylobacter with validly published
names were calculated with the EzTaxon-e server [23]
obtaining a similarity range of 99.5–90.7 %. The 16S rDNA
phylogenetic tree (Fig. 2) clearly indicated that the five
strains represented a single species within the genus Cam-
pylobacter, most closely related to C. subantarcticus (99.5 %
similarity) and C. lari subsp. concheus (99.4 % similarity).
The taxonomic position of the novel strains within the Cam-
pylobacter lari group and the demonstration that they
C. ornithocola WBE 215 (KX467977)
C. ornithocola WBE 241 (KX467978)
C. ornithocola WBE 206 (KX467976)
C. ornithocola WBE 186 (KX467975)
C. ornithocola WBE 38T (KX467974)
C. lari UPTC NCTC 11845 (CP007775)
C. lari subsp. concheus LMG 21009T (AM922330)
C. subantarcticus LMG 24377T (AM933371)
C. volucris LMG 24380T (FM883694)
Campylobacter sp. RM16704 (CP007769)
C. lari subsp. lari ATCC 35221T (AY621114)
C. peloridis LMG 23910T (AM922331)
C. hepaticus NCTC 13823T (KU886019)
C. insulaenigrae NCTC 12927T (AJ620504)
C. jejuni subsp. doylei LMG 8843T (DQ174144)
C. jejuni subsp. jejuni NCTC 11351T (AF372091)
C. coli LMG 6440T (AF372092)
C. cuniculorum LMG 24588T (DQ400345)
C. helveticus ATCC 51209T (U03022)
C. avium LMG 24591T (EU623473)
C. upsaliensis CCUG 14913T (DQ174157)
C. canadensis LMG 24001T (EF621894)
C. hyointestinalis subsp. lawsonii LMG 14432T (AF097685)
C. lanienae NCTC 13004T (AF043425)
C. iguaniorum LMG 28143T (KF425533)
C. hyointestinalis subsp. hyointestinalis NCTC 11608T (AF097689)
C. fetus subsp. testudinum LMG 27499T (CP006833)
C. fetus subsp. fetus ATCC 27374T (DQ174127)
C. fetus subsp. venerealis NCTC 10354T (DQ174131)
C. corcagiensis CIT045T (KF745861)
C. ureolyticus ATCC 33387T (L04321)
C. mucosalis ATCC 43264T (DQ174173)
C. concisus ATCC 33237T (L04322)
C. sputorum biovar paraureolyticus LMG 17590 (AF022768)
C. sputorum biovar sputorum LMG 7795 (X67775)
C. sputorum biovar fecalis LMG 6617 (AF550637)
C. curvus ATCC 35224T (DQ174165)
C. hominis NCTC 13146T (AJ251584)
C. gracilis ATCC 33236T (DQ174168)
C. rectus ATCC 33238T (L04317)
C. showae CCUG 30254T (DQ174155)
100
99
99
100
100
98
91
93
89
94
92
79
88
94
100
92
90
80
98
0.01
Fig. 2. Neighbour-joining tree based on 16S rRNA sequences showing the phylogentic position of Campylobacter ornithocola sp. nov.
within the genus Campylobacter. Bootstrap values>70 %, generated from 500 replicates, are shown at the nodes. Bar, 0.01 substitutions
per site.
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represent a novel lineage were confirmed by the rpoB,atpA
and cpn60 phylogenetic trees (Figs S1, S2 and S3 available in
the online Supplementary Material).
For the whole-genome sequencing of WBE38
T
, genomic
libraries were prepared with the Nextera XT DNA Sample
Preparation Kit (Illumina) and then sequenced using a
MiSeq Illumina platform, which produced 5 250 728 pair-
end reads (2150 cycles). After an initial quality check,
reads were assembled with SPAdes [24] and annotated with
Prokka [25], producing 160 contigs that were deposited in
the GenBank under the accession number LXSU00000000.
The version described in this paper is version
LXSU01000000. The average nucleotide identity (ANI) was
used as an alternative to DNA–DNA hybridization [26].
The ANIb (based on BLAST) values of WBE38
T
compared
with the sequenced type strains of all other species of the C.
lari group were calculated using JSpecies v1.2.1 [27] and
were below the 95 % species cut-off (Table 1) [26]. The
genomic DNA G+C content of WBE38
T
was calculated
using in-house R scripts and was 29.5 mol%, which is within
the range reported for the genus Campylobacter (29–47 mol
%) [28].
For the physiological and biochemical characterizations of all
the novel strains, phenotypic testing was performed as
described previously [29–32]. Growth of strains was deter-
mined on nutrient broth no. 2 (Oxoid) supplemented with
5 % defibrinated sheep blood (Quad Five) and 2% agar
(Merck). Micro-aerobic growth (using CampyGen, Oxoid)
was evaluated at 25 C, 37 and 42 C for 48 to 72 h. Aerobic
and anaerobic growth (using AnaeroGen, Oxoid) were evalu-
ated at 37 C for 72 h. Catalase activity was evaluated by add-
ing a 3 % H
2
O
2
solution and observing the reaction within
5 s. Oxidase activity was determined with Bactident Oxidase
strips (Merck). Indoxyl acetate hydrolysis was determined as
described by Mills and Gherna [33]. In addition to this, a set
of additional phenotypic tests (reduction of nitrates, esterase
activity, hydrolysis of hippurate, g-glutamyl transferase activ-
ity, reduction of triphenyltetrazolium chloride (TTC),
alkaline phosphatase activity, production of H
2
S, assimilation
of glucose and pyrrolidonyl arylamidase, L-arginine arylami-
dase and L-aspartate arylamidase activities) were evaluated by
using the API Campy identification system (bioM
erieux)
according to the manufacturer’s instructions. All tests were
performed at least twice with Campylobacter jejuni (DSM
4688
T
), Campylobacter coli (DSM 4689
T
), C. lari (DSM
11375
T
), C. subantarcticus (LMG 24377
T
), C. insulaenigrae
(LMG 22716
T
), C. volucris (LMG 24380
T
) and Escherichia
coli (ATCC 25922) used as controls. The novel isolates were
biochemically different from most of the species of the genus
Campylobacter with validly published names because oxidase
activity was not detected. This test is positive in all species
except Campylobacter gracilis and sporadic isolates of
Campylobacter concisus and Campylobacter showae [34].
However, the novel isolates could be differentiated from oxi-
dase-negative members of the genus Campylobacter by the
urease test. Table 2 shows the most important phenotypic
characteristics differentiating the novel strains from the other
species of the genus Campylobacter. Bacterial cell shape was
observed using a scanning electron microscope (LEO 420;
Zeiss) following the protocol described by Kawamura et al.
[35]. The organism exhibits a curved shape and cells became
spherical or coccoid after 72 h of incubation (Fig. 3).
In conclusion, the results from this taxonomic study clearly
demonstrate that the isolates recovered from wild birds fae-
cal samples comprise a novel species distinct from other
currently known species of the genus Campylobacter, based
on 16S rRNA, housekeeping genes, sequence comparison of
eight whole genomes, morphological, physiological and bio-
chemical properties. The name Campylobacter ornithocola
sp. nov. is proposed, with WBE38
T
(=CECT 9147
T
=LMG
29815
T
) as the type strain.
DESCRIPTION OF CAMPYLOBACTER
ORNITHOCOLA SP. NOV.
Campylobacter ornithocola [or.ni.tho¢co.la. Gr. n. ornis, -
ithos bird; L. suff. -cola (from L. n. incola) dweller; N.L. n.
ornithocola bird dweller].
Cells are Gram-negative curved rods, non-encapsulated,
non-spore-forming and are 0.3–0.5 µm wide and 1.2–3 µm
long. After incubation on Columbia agar (5 % sheep blood)
in a microaerobic atmosphere at 37 C for 48 h, colonies are
glossy, slightly convex, round with smooth margins. Coc-
coid cells were observed in old cultures. Swarming on solid
media was noted. Pigments are not produced. Grows on
blood agar at 37 C and at 42 C under micro-aerobic culture
conditions (does not require atmospheric hydrogen) and at
37 C in anaerobic conditions. No growth was observed at
37 C on aerobiosis and at 25 C under micro-aerobic condi-
tions. No haemolysis is seen on blood agar. Catalase and
urease activity is present but no oxidase. Esterase activity
and reduction of triphenyltetrazolium chloride (TTC)
are variable.
Table 1. Average nucleotide identity based on BLAST (ANIb) values (in
percentages) for C. ornithocola sp. nov. and the most closely related
members of the genus Campylobacter
Strains: 1, C. ornithocola sp. nov. WBE38
T
; 2, C. lari subsp. concheus
LMG 11760; 3, C. lari subsp. lari RM 2100; 4, C. subantarcticus LMG
24377
T
; 5, C. peloridis LMG 23910
T
; 6, C. volucris LMG 24379; 7, C. insu-
laenigrae NCTC 12927.
Strains 1 2 3 4 5 6 7
1–
290.8 –
389.5 92.8 –
488.4 89.7 88.8 –
586.0 86.2 86.1 85.3 –
682.0 82.3 83.1 81.8 82.4 –
780.9 81.0 81.2 80.2 81.1 82.9 –
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Table 2. Phenotypic characteristics that differentiate Campylobacter ornithocola sp. nov. from other species of the genus Campylobacter
Species: 1, C. ornithocola sp. nov (n=17); 2, C. avium; 3, C. canadensis; 4, C. coli; 5, C. concisus; 6, C. corcagiensis; 7, C. cuniculorum; 8, C. curvus; 9, C. fetus subsp. fetus; 10, C. fetus subsp. testudinum;
11, C. fetus subsp. venerealis; 12, C. geochelonis; 13, C. gracilis; 14, C. helveticus; 15, C. hepaticus; 16, C. hominis; 17, C. hyointestinalis subsp. hyointestinalis; 18, C. hyointestinalis subsp. lawsonii; 19. C.
iguaniorum; 20, C. insulaenigrae; 21, C. jejuni subsp. doylei; 22, C. jejuni subsp. jejuni; 23, C. lanienae; 24, C. lari subsp. concheus; 25, C. lari subsp. lari; 26, C. mucosalis; 27, C. peloridis; 28, C. rectus;
29, C. showae; 30, C. sputorum; 31, C. subantarcticus; 32, C. upsaliensis; 33, C. ureolyticus; 34, C. volucris. Data for reference taxa were taken from [3, 4, 9–12, 28, 36] . +, 100 % of strains positive; ,
100 % of strains negative; (+), 80–94 % of strains positive; V, 42–66 % of strains positive; (), 7–33 % of strains positive; ND, not determined.
Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Oxidase * + + + V+ + + + + + + + + + + + + + + + + + + + + + V+ + + + +
Catalase + V V ++ + + + (+) V V ++ + + + V+ + + + + () + V+V+
Urease + V  +                    ND    
†
+
†
Alkaline phosphatase     V+VND     ND   ()ND   +  (+) ND    ND   
Reduction of
Nitrate V+V+ () (+) + + + + (+) + (+) + V+ + + + + + + + ()ND + + (+) +‡+ + +
2,3,5,Triphenyltetra-
zolium
chloride (TTC)
()ND +  V V +    ND    ND ND V +ND + + ND    ND V  
Hydrolysis of
Hippurate +     ()   +  (+)      + +            
Indoxyl acetate ++V+V    V+ +      + + ND    +V  +V
H
2
S production (TSI)   V  +()        + + +     
†
+ND V+   
a-Haemolysis    () ()+ ()ND V   +  V V ND + + + ND +ND + + + + + V ND
Growth at/in/on
25 C (microaerobic)      ND   + + +             +       
37 C (microaerobic) + + + + + + + V+ + + + + + + + + + + + + + + + + + V+ + + + +
42 C (microaerobic) + + + + (+) + (+) V(+) V  V+ + () + + +   + + + + + + ()V+ + + V+
37 C (anaerobic) + ++ + + () + V+ +   ++ +    +ND +ND + + + + V+
†
37 C (aerobic)          V                       
Glycine 1% + V(+) (+) + + + + () + + V+ + V+ + () + (+) + V+ + V+ (+) + + 
H
2
requirement V  +  +    +  +V V ND    ND +ND + +     ND
DNA G+C content
(mol%)
29.5 35 ND 31 37–
41
31.9 32.4 45–
46
33–
35
ND 33–
34
33.6 44–
46
34 27.9 32.5 35–
36
31–
33
36 ND 31 30–
31
36 30 29–
30
36–
38
29 45–
46
44–
46
29–
33
30 32–
36
28–
30
29
* Oxidase negative test allows differentiation of the novel species from urease-positive thermophilic Campylobacter (UPTC) strains.
†This test was determined for the corresponding type strain, in this study.
‡We obtained a different result from that published by Debruyne et al. [12], when we evaluated the nitrate test with the API Campy (nitrate reduction negative for LMG 24377
T
).
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Pathogenicity is unknown; strains have been recovered
from faecal samples of wild birds. The type strain is
WBE38
T
(=CECT 9147
T
=LMG 29815
T
) which was isolated
from a faecal sample in Valdivia, Chile.
Funding information
This work was supported by the Comisión Nacional de Investigación
Científica y Tecnológica (CONICYT–Chile) under the project FONDECYT
N11130402.
Acknowledgements
We thank Bernhard Schink (University of Konstanz, Konstanz, Ger-
many) for his help with the specific etymology and nomenclature. We
also thank Ricardo Silva (Universidad Austral de Chile) for his help
with SEM.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Ethical statement
No experiments with humans or animals were carried out.
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