Genomic Comparisons and Zoonotic Potential of Campylobacter Between Birds, Primates, and Livestock

Abstract

Importance:
This study examined the link between public health and genomic variation of Campylobacter in relation to disease in humans, primates, and livestock. Use of large-scale whole genome sequencing enabled population level assessment to find new genes that are linked to livestock disease. With 184 Campylobacter genomes we assessed virulence traits, antibiotic resistance susceptibility, and potential for zoonotic transfer to observe there is a 'generalist' genotype that may move between host species.

Full text

Genomic Comparison of Campylobacter spp. and Their Potential for
Zoonotic Transmission between Birds, Primates, and Livestock
Allison M. Weis,
a
Dylan B. Storey,
a
*Conor C. Taff,
b
*Andrea K. Townsend,
b
*Bihua C. Huang,
a
Nguyet T. Kong,
a
Kristin A. Clothier,
c
Abigail Spinner,
d
Barbara A. Byrne,
e
Bart C. Weimer
a
School of Veterinary Medicine, Department of Population Health and Reproduction, 100K Pathogen Genome Project, University of California, Davis, California, USA
a
;
Department of Wildlife, Fish, & Conservation Biology, University of California, Davis, California, USA
b
; California Animal and Food Safety & Health Laboratory, University of
California, Davis, California, USA
c
; California National Primate Research Center, University of California, Davis, California, USA
d
; School of Veterinary Medicine, Department
of Pathology, Microbiology, and Immunology, University of California, Davis, California, USA
e
ABSTRACT
Campylobacter is the leading cause of human gastroenteritis worldwide. Wild birds, including American crows, are abundant in
urban, suburban, and agricultural settings and are likely zoonotic vectors of Campylobacter. Their proximity to humans and
livestock increases the potential spreading of Campylobacter via crows between the environment, livestock, and humans. How-
ever, no studies have definitively demonstrated that crows are a vector for pathogenic Campylobacter. We used genomics to eval-
uate the zoonotic and pathogenic potential of Campylobacter from crows to other animals with 184 isolates obtained from
crows, chickens, cows, sheep, goats, humans, and nonhuman primates. Whole-genome analysis uncovered two distinct clades of
Campylobacter jejuni genotypes; the first contained genotypes found only in crows, while a second genotype contained “generalist”
genomes that were isolated from multiple host species, including isolates implicated in human disease, primate gastroenteritis, and
livestock abortion. Two major ␤-lactamase genes were observed frequently in these genomes (oxa-184, 55%, and oxa-61, 29%), where
oxa-184 was associated only with crows and oxa-61 was associated with generalists. Mutations in gyrA, indicative of fluoroquinolone
resistance, were observed in 14% of the isolates. Tetracycline resistance (tetO) was present in 22% of the isolates, yet it occurred in 91%
of the abortion isolates. Virulence genes were distributed throughout the genomes; however, cdtC alleles recapitulated the crow-only
and generalist clades. A specific cdtC allele was associated with abortion in livestock and was concomitant with tetO. These findings
indicate that crows harboring a generalist C. jejuni genotype may act as a vector for the zoonotic transmission of Campylobacter.
IMPORTANCE
This study examined the link between public health and the genomic variation of Campylobacter in relation to disease in humans,
primates, and livestock. Use of large-scale whole-genome sequencing enabled population-level assessment to find new genes that are
linked to livestock disease. With 184 Campylobacter genomes, we assessed virulence traits, antibiotic resistance susceptibility, and the
potential for zoonotic transfer to observe that there is a “generalist” genotype that may move between host species.
Campylobacter is a motile Gram-negative spiral bacterium that
causes gastroenteritis in humans and other animals (1,2). In
livestock, Campylobacter may cause abortion in addition to gas-
troenteritis (3). It is one of the most common foodborne zoonotic
pathogens worldwide and is often transmitted via the fecal-oral
route through the consumption of contaminated food or water (1,
2). In the United States, campylobacteriosis is estimated to affect
more than 1.3 million people each year, with symptoms including
fever, abdominal cramping, and bloody diarrhea (1,4,5). Inter-
nationally, campylobacteriosis is a significant public health bur-
den; the incidence in developed nations is estimated to be 4.4 to
9.3 per 1,000 people yearly and is a substantial cause of morbidity
in developing nations (6). In rare and severe cases, infection can
lead to chronic autoimmune disorders, such as Guillain-Barré
syndrome (GBS) and Miller Fisher syndrome (7,8). Outbreaks in
the United States are largely attributed to contaminated poultry
and water and are commonly associated with unpasteurized milk
(1,4,5,9). Despite many efforts to contain and abate Campylo-
bacter jejuni outbreaks, national and international reduction goals
remain unmet and the number of new cases continues to increase
yearly (6,10–12).
Recent research has focused on uncovering the initial sources
of human infection. Birds are considered to be a primary host of
Campylobacter, which is a commensal organism in a broad range
of wild bird populations, including black-headed gulls (Chroico-
cephalus ridibundus), Sandhill cranes (Grus canadensis), European
starlings (Sturnus vulgaris), and American crows (Corvus brachy-
rhynchos)(
13–15). C. jejuni isolates from these birds have been
implicated in human disease (11,16,17). Other studies indicate
that some Campylobacter isolates found in wild birds may not be
pathogenic to humans (14,18–23). These conflicting reports in-
dicate that studies using higher resolution molecular methods
Received 10 June 2016 Accepted 30 September 2016
Accepted manuscript posted online 7 October 2016
Citation Weis AM, Storey DB, Taff CC, Townsend AK, Huang BC, Kong NT, Clothier
KA, Spinner A, Byrne BA, Weimer BC. 2016. Genomic comparison of Campylobacter
spp. and their potential for zoonotic transmission between birds, primates, and
livestock. Appl Environ Microbiol 82:7165–7175. doi:10.1128/AEM.01746-16.
Editor: C. A. Elkins, FDA Center for Food Safety and Applied Nutrition
Address correspondence to Bart C. Weimer, bcweimer@ucdavis.edu.
*Present address: Dylan B. Storey, West Sacramento, California, USA; Conor C. Taff,
Lab of Ornithology, Cornell University, Ithaca, New York, USA; Andrea K.
Townsend, Department of Biology, Hamilton College, Clinton, New York, USA.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.01746-16.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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December 2016 Volume 82 Number 24 aem.asm.org 7165Applied and Environmental Microbiology

may provide insights to more precisely assess Campylobacter iso-
lates from wild birds so as to gauge their importance to food safety
and public health.
To date, most studies have used 16S rRNA, fla typing, or mul-
tilocus sequence typing (MLST) to characterize isolates and exam-
ine zoonotic transmission. Unfortunately, the resolutions of these
methods have limited the conclusions that can be drawn. While
16S sequencing has been used historically to assess microbial di-
versity, it underestimates the genome variation within a species
(24,25). The MLST method, which uses five to seven genes to
build a classification system, is more robust than 16S sequencing
and has been used in many studies (26–28). To date, however, the
application of taxonomically conserved housekeeping genes,
while phylogenetically useful, does not include biologically infor-
mative genes, such as virulence factors (29), thereby limiting con-
clusions about potential pathogenicity. Recent studies have dem-
onstrated the importance of whole-genome sequencing (WGS) in
understanding C. jejuni pathogenicity and transmission (18,21,
23,30); however, to date, studies of Campylobacter in wild birds
have not taken advantage of WGS approaches to define the geno-
types that are associated with zoonotic transmission and viru-
lence.
The American crow is a North American passerine that forages
in a variety of settings, including dumps, feedlots, pastures, and
urban areas. As such, American crows have the potential to trans-
fer pathogens from human waste or infected animal manure to
human food, waterways, and livestock, potentially acting as both a
reservoir and a transport host (22). Previous studies indicate that
Campylobacter prevalence is high in crow species around the
world. Corvids from Japan, New Zealand, Malaysia, and Tanzania
harbor Campylobacter with prevalences ranging from 34% to 43%
(31–35). Likewise, 67% of free-ranging American crows in the
Sacramento Valley, California, tested positive for C. jejuni (22).
Crow social behavior may contribute to high infection rates in
crows, and foraging patterns may amplify the risk of zoonotic
transmission in specific areas, such as feedlots, where many spe-
cies intermingle (36). The sequencing of 16S rRNA revealed that
many of those isolates were similar to C. jejuni strains that were
isolated from human clinical samples (22).
We used WGS to compare the genomes of 100 Campylobacter
species isolates (97 C. jejuni isolates) that were collected from
crows between 2012 and 2014 in the Sacramento Valley, Califor-
nia, with 60 Campylobacter species isolates from nonhuman pri-
mates, chickens, sheep, cows, and goats from the same geographic
region. We examined evidence for genetic traits that are indicative
of pathogenicity and zoonotic potential from published Campy-
lobacter species whole-genome sequences for a total of 184 ge-
nomes.
MATERIALS AND METHODS
Isolates sequenced. In addition to the 100 Campylobacter species isolates
from crows (22), isolates were collected from rhesus macaques (Macaca
mulatta) between 2014 and 2015 from the California National Primate
Center (CNPRC), an open-air facility housing ⬃5,000 macaques (47 iso-
lates); from cases of abortion in bovine, ovine, and caprine fetuses (9
isolates); and from chickens collected by the UC Davis Veterinary Medi-
cine Teaching Hospital (VMTH) between 2014 and 2015. The genomes of
isolates collected during this study were compared to genomes in NCBI
GenBank, which were isolated mainly from humans, chickens, and an
ovine abortion case.
Crow and primate sampling. Sample collection of Campylobacter was
done as described previously (22). Briefly, fecal or cloacal swab samples
were collected from American crows (hereafter referred to as “crows”) in
Yolo County, California, between May 2012 and June 2014 and isolated
using Amies clear gel collection and transport swabs (Remel BactiSwab;
Thermo Fisher Scientific, Waltham, MA). Individual transport swabs
were stored on ice (4 to 7°C) for 2 to 6 h prior to culture. Bacteria were
isolated and identified at the VMTH as described previously (22) and in
the paragraph below. All crow sampling was done using protocols ap-
proved by the Institutional Animal Care and Use Committee of the Uni-
versity of California, Davis (IACUC 16897). Primate sampling was done
using fecal swabbing at the CNPRC during routine primate health care on
primates for diarrhea/gastroenteritis in accordance with the approved
protocols with the same methods and reagents as those used at the VMTH
(described above and below), and Campylobacter isolates were given to
this study from the CNPRC after bacterial isolation.
Campylobacter testing. Campylobacter culture was performed from
fecal samples inoculated onto 5% sheep blood agar (SBA) containing
cefoperazone, vancomycin, and amphotericin B and from aborted fetus
samples on 5% SBA containing amphotericin B and novobiocin (Campy
CVA; Hardy Diagnostics, Santa Maria, CA, USA). Plates were incubated at
37°C in microaerobic conditions (CampyGen; Oxoid Limited, Hamp-
shire, United Kingdom) for 4 to 6 days, as described by Weis et al. (22).
Bacterial colonies were Gram stained and subcultured onto 5% SBA
(Hardy Diagnostics) for further characterization. Microaerobic isolates
with a characteristic appearance on the culture medium and Gram stain
(small, curved Gram-negative rods) that were catalase positive were iden-
tified as Campylobacter spp. and as C. jejuni if they hydrolyzed hippurate
(Dalynn Biologicals Inc., Calgary, Canada). Additionally, each isolate was
evaluated for susceptibility to nalidixic acid and cephalothin by using
30-␮g antibiotic disks for further species confirmation (BD Biosciences,
New Jersey, USA) (22).
Genomic analysis. These genomes were part of the 100K Pathogen
Genome Project using previously published methods (37–45).
DNA extraction, library preparation, and next-generation sequenc-
ing. High-molecular-weight genomic DNA (gDNA) was isolated from
bacterial colonies grown on 5% SBA plates (UC Davis Vet Med Biological
Services) at 37°C in microaerophilic conditions (as described above).
DNA was extracted using whole-genome isolation kits (Qiagen, Valencia,
CA, USA) with previously published modifications (46). The bacterial
cells were processed using the protocols of the 100K Pathogen Genome
Project (UC Davis, Weimer laboratory) as previously described (46–48).
Briefly, bacteria were lysed with an enzyme preparation, vortexed, and
processed according to the manufacturer’s recommendations to obtain
purified gDNA (Qiagen). Genomic DNA purity and integrity were as-
sessed on the Agilent 2200 TapeStation with the genomic DNA Screen-
Tape (Agilent Technologies, Santa Clara, CA, USA) as previously de-
scribed (38,39,42). Genomic DNA ratios that were greater than 1.8 for
A
260/280
and A
260/230
were used for library construction.
Isolated gDNA was sheared using the Covaris E220 with the 96 micro-
Tube plate (Covaris, Inc., Woburn, MA, USA) (37). The fragmented DNA
size was determined with the Agilent Bioanalyzer 2100 high-sensitivity
DNA kit (Agilent Technologies) to confirm the normal size distribution
around a 300-bp peak. Libraries were constructed using the KAPA HTP
library preparation kit (KK8234, KR0426 [v3.13]; Kapa Biosystems, Wil-
mington, MA, USA) with dual surface plasmon resonance imaging (SPRI)
size selection (40). Libraries were constructed using the Agilent Bravo
option B (Agilent Technologies). Libraries were indexed using Bioo Sci-
entific NEXTflex-96 DNA barcodes v13.05 (Bioo Scientific Corp., Austin,
Texas, USA) and Integrated DNA Technologies Weimer 384 TS-LT DNA
barcodes. Library quantification was done using the KAPA library quan-
tification kit (KK4824; Kapa Biosystems) to ensure the final library con-
centration prior to normalization and pooling for sequencing (40).
Sequencing was performed with BGI@UCDavis (BGI@UCDavis, Sacra-
mento, CA, USA) using the Illumina HiSeq 2000 platform with PE100
Weis et al.
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(Illumina Inc., San Diego, CA, USA) or the Illumina HiSeq 3000 platform
with PE150 at the UC Davis Genome Center (Davis, CA, USA) (43,45).
Sequence assembly, annotation, and whole-genome analyses. As-
sembly of paired-end reads was done with ABySS 1.5.2 using the following
parameters: Kmer length ⫽64 (49). Annotations were done with the
Prokka pipeline using the following parameters: –force –addgenes – com-
pliant – genus Campylobacter –usegenus –rfam (50). Genomic distances
were determined using the genome-to-genome distance calculator
(GGDC), an in silico DNA-DNA hybridization (isDDH) technique, using
the webserver at http://ggdc.dsmz.de/distcalc2.php as published previ-
ously (51,52) and implemented locally as PanCake (53). The DNA-DNA
hybridization (DDH) model “formula 2” was used as is recommended for
draft genomes. Distance matrices were translated into the Newick tree
format with Trex webserver software using the neighbor-joining method
(54,55), and distance matrixes were clustered and visualized using the R
statistical programming language (56). Single-gene (16S rRNA genes and
cdtC) analyses were performed by extracting the sequences from each
genome and aligning them using MUSCLE through Geneious (v6.1.8) to
align sequences and generate phylogenetic trees (57,58). Trees were ed-
ited using Dendroscope 3.0 (59). SplitsTree4 was used to compute trees
and splits using the equal angle method with the “use weights” and “run
convex hull” parameters (60).
Genome alignments were done using Mauve under progressiveMauve
(61,62). Contigs were reordered using the “reorder contigs” option in
Mauve under the default parameters using C. jejuni subsp. jejuni NCTC
11168 as the reference genome, and then reordered genomes were aligned
to each other using progressiveMauve. This publication made use of the
Campylobacter MLST database (http://pubmlst.org/campylobacter) for in
silico MLST (29).
Genomic assessment of virulence factors and antibiotic resistance
genes. Genomes were compared against a database (63,64) consisting of
virulence factor genes from six published Campylobacter genomes (Cam-
pylobacter fetus 82-40, C. jejuni 81-176, C. jejuni 81116, C. jejuni RM
11168, C. jejuni RM1221, and C. jejuni subsp. doylei 269.97) and plasmid
C. jejuni 81-176 pVir. All published Campylobacter virulence factor genes
were examined for each isolate to determine genotype variation; subse-
quently, five specific features associated with virulence and infection were
selected for comparison and occurrence. These targets included (i) cdtA,
cdtB, and cdtC, which code for cytolethal distending toxin (CDT), the
main toxin in Campylobacter (65–67); (ii) putative type IV secretion sys-
tem (T4SS) virB genes, associated with invasion (68); (iii) secreted inva-
sion proteins CiaB and FlaC (69–71); and (iv) adherence genes jlpA,porA,
pebA, and cadF (63,72,73). Virulence factor proteins were defined using
the Pathogenic Bacterial Virulence Factor database (63,64) using USE-
ARCH (74) and by hand using previously published genes (75–78)in
Geneious using Prokka annotations.
Antibiotic resistance genes were analyzed in every genome using the
Resistance Gene Identifier software and the Comprehensive Antibiotic
Resistance database (CARD) (79). Using these tools, Campylobacter ge-
nomes were assessed for the presence of known antibiotic resistance genes
(75–78). Specifically, we examined each genome for five antibiotic resis-
tance genes or operons that are relevant to Campylobacter antibiotic re-
sistance: (i) the multidrug-resistant efflux complex CmeABC and its reg-
ulatory gene cmeR (76,77); (ii) the MacAB efflux locus, which confers
resistance to macrolides (79); (iii) the tetO locus, a ribosomal protection
protein that confers resistance to tetracycline and its derivatives (75); (iv)
the oxa-184 and oxa-61 genes, which are members of the class D ␤-lacta-
mase family and confer resistance to ␤-lactam antibiotics (78); and (v)
specific point mutations in the gyrA gene known to mediate resistance to
fluoroquinolones in Campylobacter.
Accession number(s). All raw genome sequences generated in this
study are available in the NCBI SRA as part of the 100K Pathogen Genome
Project under BioProject accession number PRJNA186441. Accession
numbers are listed in Table S1 in the supplemental material.
RESULTS
Assembly and annotation. Each of the 160 sequenced Campylo-
bacter genomes were assembled and annotated using ABySS and
Prokka with the same settings and conditions (49,50). The ge-
nomes of all three species ranged from 1,491,293 to 2,006,566 bp
(the smallest being Campylobacter lari), with an average of
1,772,774 bp assembled in an average of 55 contigs per genome.
They contained an average of 1,799 coding DNA sequences
(CDS), 40 tRNAs per genome, and one transfer-messenger RNA
(tmRNA). For a full list of the genome structural details, see
Table S1 in the supplemental material as well as the SRA acces-
sion numbers.
Each Campylobacter genome was examined for 16S rRNA
genes sequence variation and MLST pattern, including the se-
quence type and clonal complex. However, the discriminative res-
olution in many cases was poor and inconclusive, particularly be-
tween some Campylobacter coli and C. jejuni 16S rRNA gene
sequences (see Fig. S1 in the supplemental material). Although
indiscriminant in some sequences, this analysis did confirm the
observations of Weis et al. (22), who observed a “crow-only” host
clade.
MLST was considered the gold standard for genotyping an
isolate in the pregenomics era; however, this method relies on the
perfect match of an isolate from within the already existing data-
base (29). If the sequence is not contained in the database, no
assignment can be made and other methods are needed to deter-
mine the relationship. In this study, 100 C. jejuni and C. coli ge-
nomes matched known MLST patterns. Unfortunately, 60 ge-
nomes had no match in the database of commonly used
housekeeping genes (see Table S2 in the supplemental material).
Consequently, further comparisons were done to determine spe-
cific similarities using the entire genome sequence and genome
distances.
Whole-genome analysis. Genome distance calculations re-
solved individual species into distinct groups of C. jejuni,C. coli,
and C. lari (Fig. 1; see also Fig. S2 in the supplemental material). A
phylogenetic tree constructed from the genome distances placed
83% (72/87) of the C. jejuni isolates from crow origin into a sep-
arate clade from those isolates obtained from primates or humans,
while 17% (15/87) of the isolates from crows clustered closely with
human, primate, and sheep C. jejuni (Fig. 1). This finding dem-
onstrated that multiple genotypes exist within each bacterial spe-
cies, and in the case of C. jejuni, it also resolved host origin, which
may be evidence for host species adaptation. These observations
confirm that the genome sequence has sufficient analytical reso-
lution to link host range with genotype as observed previously in
environmental samples using Vibrio spp. (80).
To further examine that link between genotype and host
source, we used genome distance measurements to define two
major groups within C. jejuni: (i) a crow-only cluster and (ii) a
distinct cluster that contained isolates from many host species that
we defined to be “generalists,” a concept similar to what other
groups have previously defined for this organism (81,82). The
generalist group contains phylogenetically similar organisms ob-
tained from seven different host species (Fig. 1). Several crow iso-
lates were interspersed among the generalist C. jejuni clade. Nota-
bly, the genome distance of two crow isolates that clustered closely
with human isolate C. jejuni ICDCCJ07001, an isolate known to
cause GBS, suggests that crows may carry organisms that are re-
Campylobacter in Multiple Host Species
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sponsible for this disease in humans. Similar relationships were
observed for isolates that caused human gastroenteritis (i.e., C.
jejuni M1) and isolates associated with abortion in livestock. Gen-
eralist genotypes that were linked closely to the three disease pre-
sentations were distinctly different from crow-only genotypes,
warranting further examination of genomes to determine the spe-
cific genes and genotypes that were associated with the disease
phenotype across the host range.
After careful investigation of the DDH analyses between the
crow-only and generalist groups, it is possible that the isolates in
the crow-only clade are potentially a novel third subspecies of C.
jejuni. For instance, comparing two separate isolates from crows,
BCW_3810 from the crow-only clade and BCW_6872 from the gen-
eralist clade, the DDH estimate is 74% (71% to 76.8%) where ⬎70%
is the same species. This indicates that the crow-only and the gen-
eralist isolates are both C. jejuni; however, the DDH estimate to
belong to the same subspecies is 79%, leaving the crow-only iso-
late as a separate subspecies from the generalist C. jejuni subsp.
jejuni isolate. Therefore, using this whole-genome analysis
method, it is possible that these isolates are a separate subspecies.
Genome structure. Genome synteny was examined in the 160
genomes sequenced in this study, with an additional 24 genomes
(184 total comparisons) from the NCBI SRA that spanned host
sources and disease phenotypes. This analysis found that the col-
lection of genomes did not contain any major structural differ-
ences (e.g., inversions or translocations). As expected, structural
alignments between isolates that were placed closely together us-
ing the genome distance analysis also aligned the closest structur-
ally (see Fig. S4 in the supplemental material). Between the crow-
only and the generalist clades, the genomes were more diverse
between clades than within each clade, indicating that the major
differences between each clade were at the level of allelic variation.
Whole-genome antibiotic resistance genotypes. All Campy-
lobacter genomes examined, regardless of clade, species, or host
origin, contained the cmeABC efflux complex (data not shown),
while 79.4% (127/160) contained the cmeR regulatory gene (Fig. 2)
and all contained the macAB efflux loci (data not shown). The tetO
locus, however, was found primarily in C. jejuni isolates from
crows and ruminant abortion cases. In crow-derived C. jejuni,
23.7% (23/97) of genomes contain tetO, 65.2% (15/23) from the
FIG 1 Genome distance calculations from draft genomes show two main groups within C. jejuni: a crow-only clade and a generalist clade (A). The generalist
clade subset (B) shows C. jejuni isolates from a mixture of crow, primate, sheep, cow, goat, and human (GenBank) hosts. Tips are labeled with accession number
(GenBank), strain name, source (when available), MLST (when available). Scale bar indicates genomic distance.
Weis et al.
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FIG 2 Genomic distance and antibiotic resistance genes. Dendrogram built from genomic distance calculations shows host in colored dots (key). The top arm
of the dendrogram shows the C. jejuni isolates, and the bottom arm of the dendrogram shows the primate C. coli isolates. Tetracycline resistance gene tetO can
be found predominantly in crow and sheep (abortive) isolates, and oxa-184 is found more often in crow populations whereas oxa-61 is found more often in the
sheep and primate populations. Point mutations in gyrA are represented by purple, indicating Thr-Ile 86 mutation, and blue, indicating Thr-Val 86 (found only
in C. lari). Gray indicates the presence of gyrA but no fluoroquinolone resistance-related SNP.
December 2016 Volume 82 Number 24 aem.asm.org 7169Applied and Environmental Microbiology

crow-only clade and 34.7% (8/23) from the generalist clade. Two
C. jejuni isolates from primates in the generalist clade contained
tetO. Almost all (91%, 10/11) abortion isolates contained tetO
(Fig. 2). These results signify that tetO is widespread in abortion
isolates (91% of the population was positive), is potentially linked
to the disease phenotype, and may be useful in differentiating host
specificity.
Out of the total genomes sequenced in this study, 84% of iso-
lates contained class D ␤-lactamase genes. Of those, 55% (88/160)
of the genomes contained the oxa-184 gene and 29.4% (47/160)
contained the oxa-61 gene. Almost all C. jejuni isolates from crows
contained oxa-184 (except for 5 generalists), whereas the C. jejuni
isolates in the generalist clade typically contained oxa-61. The C.
coli isolates from primates contained the oxa-61 loci (Fig. 2; see
also Fig. S3 in the supplemental material). The two prominent
␤-lactamase genes (oxa-61 and oxa-184) were differentially pres-
ent within the genomes and were indicative of host source.
Genomes were further examined for the presence of specific
point mutations in gyrA known to mediate resistance to fluoro-
quinolones. Of the Campylobacter isolates from nonhuman pri-
mates, 32% contained a mutation that would result in a Thr-Ile 86
mutation in the GyrA protein, whereas only 4% of Campylobacter
isolates from crows contained this mutation. Importantly, all
Campylobacter isolates from crows with the gyrA mutation were
from the generalist clade; none were identified in the crow-only
clade (Fig. 2; see also Fig. S3 in the supplemental material). No
other known substitutions in gyrA were found in the C. jejuni
genomes; however, all three C. lari isolates from crows contained
a Thr-Val 86 mutation as well. These results demonstrated that
while Campylobacter isolates from crows (i.e., wildlife) contain
fewer antibiotic resistance genes than Campylobacter isolates from
agriculture-associated sources and captive animals, the presence
of fluoroquinolone resistance genes in a small number of C. jejuni
isolates from crows (all generalists) may be indicative of zoonotic
transmission.
Whole-genome virulence loci analysis. Virulence genes were
specifically examined in conjunction with each Campylobacter
isolate’s host species (Table 1). Nearly all genomes (95%) con-
tained one or more cytolethal distending toxin gene (cdtA,cdtB,
and cdtC). All genomes contained genes associated with invasion
(ciaB and flaC) and adherence genes (jlpA,porA,pebA, and cadF).
Genes involved in the type IV secretion system (T4SS) were
identified much less frequently in these genomes than the other
virulence genes that were studied. virB4 was present in 31% of
genomes, while the remaining vir genes were found in only 8% to
21% of the evaluated genomes. Most T4SS genes were found in
tandem with other T4SS genes in one genome (Table 1). Nearly all
genomes were potentially pathogenic because of the presence of
known virulence loci in all clades, suggesting that all 160 genomes
are potentially pathogenic in humans.
Cytolethal distending toxin. Since all loci were present, ge-
nomes were further investigated for specific point mutations
within each virulence gene as a possible differentiation metric for
virulence by host species. To evaluate the hypothesis that adher-
ence molecules in Campylobacter may provide the critical differ-
ence between zoonotic and nonzoonotic species and can inform
us of the origin and functional differences between the generalist
and crow-only groupings, the membrane-binding protein CdtC
that facilitates adhesion to the host cell and promotes cell entry of
CdtB (65–67) was specifically identified and examined as a candi-
date for this hypothesis. Alignments of cdtC from each genome
revealed single nucleotide polymorphism (SNP) variants that
would lead to protein coding changes segregated by Campylobac-
ter species. Within C. jejuni,cdtC allelic variation exactly recapit-
ulated clades defined by whole-genome distance calculation into
crow-only or generalist C. jejuni (Fig. 3). Differentiation of this
gene into host-specific clades indicated that significant pheno-
typic differences can be explained by small changes in a single
gene.
Analysis of CdtC protein alignment found specific amino acid
changes separating the protein sequences into four main groups,
i.e., C. coli,C. lari, and two groups with C. jejuni (generalist and
crow only) (Fig. 3; see also Fig. S5 in the supplemental material).
The CdtC alignment, when assessed for SNPs, revealed that the
crow-only CdtC alleles were nearly identical. The generalist clade
contained seven alleles of cdtC that coded for amino acid changes
(Fig. 3A). All C. coli and C. lari cdtC alleles were identical within
each bacterial species and were more similar to each other than to
C. jejuni. This finding indicates that within C. jejuni,cdtC may be
under evolutionary pressure in relation to host species coloniza-
tion and transmission. This suggests that the allelic variation of
cdtC may be a fundamental factor in disease after transmission
and may impact zoonotic and disease phenotypes. We further
hypothesized that a combination of adherence specialization and
antibiotic selection may provide clues to zoonotic potential and
disease potential.
Abortion isolates. To test this hypothesis, we examined ge-
nomes from isolates that caused abortion in multiple livestock
species. Previous studies demonstrate that abortive C. jejuni iso-
lates are associated with the occurrence of tetO (83). We observed
that 91% (10/11 isolates) of abortion cases contained tetO in the
TABLE 1 Occurrence of common virulence factors in 160
Campylobacter isolates sequenced in this study
Toxin type/function and
gene Frequency (%)
CDT toxin
cdtA 96
cdtB 95
cdtC 97
Invasion
ciaB 100
flaC 100
Adherence
cadF 100
jlpA 100
porA 100
pebA 100
Type IV secretion system
virB1 12
virB2 17
virB3 13
virB4 31
virB6 21
virB8 21
virB9 8
virB10 20
virB11 12
Weis et al.
7170 aem.asm.org December 2016 Volume 82 Number 24Applied and Environmental Microbiology

genome of the C. jejuni isolate. Only one abortion-associated C.
jejuni isolate (BCW_6919) did not contain tetO (Fig. 2). When
CdtC was assessed using the abortion isolates, the resultant phy-
logenetic tree was very similar to the genomic distance tree, and
the CdtC variant was identical in all abortion isolates and genomic
near-neighbors (Fig. 3B). Genomic distance calculations of the
abortion isolates reveal several instances where C. jejuni isolates
from crows, chickens, humans, and primates cluster with those
that caused abortion (Fig. 4). Taken together, these data indicate
that CdtC and TetO may play a more important role in disease
than previously thought. This may have broad implications for
pathogenicity, zoonotic transmission, and disease phenotype as
highlighted by the findings for livestock abortion.
DISCUSSION
This study describes the first large-scale (184 isolates) genomic
comparison of Campylobacter isolates collected from wild birds,
livestock, nonhuman primates, and humans. Whole-genome se-
quencing evaluation identified distinct clades within Campylobac-
ter spp., particularly C. jejuni, and provided a much more robust
vehicle for isolate comparison than 16S rRNA genes or MLST
approaches, as additional molecular resolution was needed to un-
cover new discoveries for allelic variation and disease. While evi-
dence of major structural alterations in genomes was not detected,
gene-level differences were identified that may contribute to host
adaptation and pathogenic potential. Since this tool clearly de-
fined bacterial species, we hypothesized that this approach may be
predictive of host source, especially when coupled with the meta-
data and genomic markers that link transmission capability, such
as those in the generalist clade.
Nearly all (95%) of the isolates from crows, primates, and live-
stock contained loci associated with invasion and virulence. Al-
though the exact mechanistic details of how C. jejuni invades hu-
man host cells and causes disease are still unclear, several virulence
pathways have been implicated in C. jejuni pathogenesis. CDT is a
holotoxin composed of three proteins, CdtA, CdtB, and CdtC,
and has been shown to disrupt the host cell cycle, leading to cell
arrest and even host cell death (65–67,84). The flagellar apparatus
of C. jejuni consists of many proteins, including filament proteins
FlaA and FlaB, and has been shown to act in motility of the bac-
teria and as a putative secretion system, secreting Cia (Campylo-
bacter invasion antigen) proteins and FlaC into the host epithelial
cells (69–71,84,85). Type IV secretion system proteins typically
encoded on plasmids (pVir) have been shown previously to con-
tribute to virulence although their function is still unknown (68).
The C. jejuni isolates in this study contained many of these viru-
lence loci and can be characterized as potential pathogens by their
genomes. Whether those virulence genes are expressed still needs
to be determined. Phylogenetic analysis based on one protein se-
quence in particular (cdtC) recapitulated the full genome phylo-
genetic tree, which may indicate a role in each isolate’s host pre-
dilection and zoonotic potential.
Our results show that several genomes from crows (17%; 15/
87) displayed high similarity to sequences of isolates implicated in
FIG 3 Alignment of CdtC sequences replicates the whole-genome phylogenetic tree and correctly splits species and clades. The cdtC nucleotide sequence was
extracted from every genome, aligned, and built into a phylogenetic tree (A). Replicating the full genome tree, cdtC drives the separation between C. lari,C. coli,
and between the C. jejuni generalists and the C. jejuni crow-only host grouping. Within the generalists, CdtC sequences from the abortion isolates are identical
to those from select chickens, crows, and primates (B). Numbers represent bootstrapping, and the scale bar is at the bottom.
Campylobacter in Multiple Host Species
December 2016 Volume 82 Number 24 aem.asm.org 7171Applied and Environmental Microbiology

human disease, suggesting that these isolates are potential patho-
gens of public health importance and zoonotic transfer. The C.
jejuni isolates from crows fell into two major clades: 83% (72/87)
made up a crow-specific clade of C. jejuni, whereas 17% (15/87)
was associated with other hosts and made up part of the generalist
clade.
The Campylobacter isolates collected from macaques that were
evaluated in this study were split between C. coli and C. jejuni. The
C. coli isolates from primates all clustered together by genomic
distance calculations and were very similar to one another (all 30
C. coli isolates were in the same C. coli clade). Most (81%; 13/16) of
the primate C. jejuni isolates and all of the livestock isolates were
members of the generalist clade. Campylobacter is widely accepted
to be a genomically diverse group. However, we observed that the
crow-only clade was very genomically similar, whereas the gener-
alist clade contained four major clades that contain 60 different
genotypes that are associated with multiple host sources. It is likely
that these are the genomes that represent high diversity and likely
high transmissibility.
Zhao et al. (86) recently found that the Campylobacter geno-
type, using WGS, is predictive of antibiotic resistance phenotypes
using 114 isolates of Campylobacter (C. jejuni and C. coli) and 18
antibiotic resistance genes. They found between 95% and 100%
correlation to phenotype. The findings of Zhao et al. provide ad-
ditional evidence to verify that the antibiotic resistance genes and
SNPs observed in this study may be predictive of phenotype from
crow isolates. With this hypothesis, we examined all of the isolates
from crows to find that they had either genes or SNPs associated
with antibiotic resistance genes, and 85% of isolates contained
class D ␤-lactamase genes. Specifically, 80% contained oxa-184
whereas 5% contained oxa-61. All five of the C. jejuni isolates from
crows that contained oxa-61 were part of the generalist clade,
whereas the crow-only clade only contained oxa-184. Over 23% of
C. jejuni isolates from crows contained tetO, 4.5% of C. jejuni
isolates from crows contained a gyrA SNP, and one isolate had
both tetO and the gyrA SNP. From the generalist clade of C. jejuni
isolates from crows, 53.3% (8/15) were positive for tetO and all
four crow C. jejuni isolates with the gyrA SNP were from the gen-
eralist clade (26.6%; 4/15). Humans infected with C. jejuni are
frequently treated with fluoroquinolones (e.g., ciprofloxacin) (87,
88). Because of this, food animals (post 2005) are no longer
treated with fluoroquinolones in the United States (89). While the
FIG 4 Genomic distance dendrogram of C. jejuni isolates implicated in causing abortion compared with GenBank C. jejuni from humans and with crow and
primate isolates from this study. This dendrogram reveals greater than assumed genomic distance between those isolates with the same MLST and those thought
to be clonal and shows two novel isolates from sheep and goats that associate more closely with a crow isolate and a chicken isolate from the Sacramento Valley.
Tips are labeled as strain identifier, isolate source, and MLST (when available). Abortive C. jejuni isolates are indicated by stars.
Weis et al.
7172 aem.asm.org December 2016 Volume 82 Number 24Applied and Environmental Microbiology

point mutations conferring resistance to fluoroquinolones were
absent in chicken and livestock isolates, mutations were fre-
quently identified in primate isolates (36.7% [11/30] of C. coli
isolates and 25% [4/16] of C. jejuni isolates). Similarities between
the generalists (32% of C. jejuni isolates) from crow, sympatric
macaque, and sheep isolates suggest that transmission between
avian, primate, and ungulate hosts can occur. The prevalence of
antibiotic resistance genes in isolates from a wide host range may
pose a public health risk from this agent.
These results add to recent studies exploring the importance of
wild birds in disseminating Campylobacter throughout a large
geographic area (13–15). Here, we have developed a method using
genomics, antibiotic resistance markers, and specific virulence
factors to detect isolates of pathogenic and zoonotic potential.
Isolates in the generalist category contain clinically important an-
tibiotic resistance genes and, through potential dissemination of
Campylobacter isolates via crows, represent a risk to human and
animal health and should be regarded as a public health threat.
Further, we show that using the whole genome is more accurate
and informative for phylogenetics, virulence, and antibiotic resis-
tance assessment than all other previous forms of analysis.
In conclusion, this study demonstrated the power of popula-
tion-scale WGS with Campylobacter isolates from wild bird pop-
ulations, captive nonhuman primates, isolates from human infec-
tions, and livestock abortion isolates. This approach provided a
robust and powerful method to determine genome-scale patho-
genicity and the potential for zoonotic exchange in ecological set-
tings to link disease and host range, where previously used molec-
ular tools lacked the necessary resolution. Here, we showed that
whole-genome distance calculations classified each species accu-
rately. Further, C. jejuni isolates from crows segregated into two
main clades, that of a crow-only clade not associated with disease
and a generalist clade, which contained genomes from multiple
hosts many of which were associated with disease (gastroenteritis,
GBS, or abortion). Antibiotic resistance genes such as ␤-lactama-
ses also segregated along the genome distance clades. This was also
observed for cdtC, again, recapitulating the genome distance
groupings. Remarkably, the isolates from livestock that caused
abortion all contained the identical cdtC allele, implicating that
this specific cdtC allele was unique to this livestock disease. Inter-
estingly, tetO was found with all but one of the abortion cases,
suggesting that cdtC and tetO may be coevolving to produce a
generalist genome that is zoonotic and abortion linked. Use of
WGS and population genomics provided a high-resolution view
of Campylobacter genomes that was linked to host range, diseases,
and possible zoonotic transmission genotypes.
ACKNOWLEDGMENTS
We would like to acknowledge Karen Pacheco and JoAnn Yee at the
VMTH for their help with isolation and identification of crow Campylo-
bacter isolates and Abigail Spinner at the CNPRC for primate isolates. We
thank the 100K Pathogen Genome Project team. We also thank Walter
Boyce and Woutrina Smith for collaborations and discussions.
Studies were funded in part by the University of California, Davis to
Andrea K. Townsend, by the Agriculture and Food Research Initiative
under competitive grant no. 2014-67012-21622 from the USDA National
Institute of Food and Agriculture to Conor C. Taff, and by the 100K
Pathogen Genome Project under FDA grant no. 5U01FD003572-04.
FUNDING INFORMATION
This work, including the efforts of Andrea K. Townsend, was funded by
UDSA, Agriculture and Food Research Initiative Competitive Grant
(2014-67012-21622). This work, including the efforts of Bart C. Weimer,
was funded by FDA/CFSAN (5U01FD003572-04).
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