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. 2016 Mar 25;54(4):1082–1088. doi: 10.1128/JCM.03202-15

Comparative Detection and Quantification of Arcobacter butzleri in Stools from Diarrheic and Nondiarrheic People in Southwestern Alberta, Canada

Andrew L Webb a,b, Valerie F Boras c, Peter Kruczkiewicz d, L Brent Selinger b, Eduardo N Taboada d,✉, G Douglas Inglis a,✉
Editor: P H Gilligan
PMCID: PMC4809925  PMID: 26865686

Abstract

Arcobacter butzleri has been linked to enteric disease in humans, but its pathogenicity and epidemiology remain poorly understood. The lack of suitable detection methods is a major limitation. Using comparative genome analysis, we developed PCR primers for direct detection and quantification of A. butzleri DNA in microbiologically complex matrices. These primers, along with existing molecular and culture-based methods, were used to detect A. butzleri and enteric pathogens in stools of diarrheic and nondiarrheic people (n = 1,596) living in southwestern Alberta, Canada, from May to November 2008. In addition, quantitative PCR was used to compare A. butzleri densities in diarrheic and nondiarrheic stools. Arcobacter butzleri was detected more often by PCR (59.6%) than by isolation methods (0.8%). Comparison by PCR-based detection found no difference in the prevalence of A. butzleri between diarrheic (56.7%) and nondiarrheic (45.5%) individuals. Rates of detection in diarrheic stools peaked in June (71.1%) and October (68.7%), but there was no statistically significant correlation between the presence of A. butzleri and patient age, sex, or place of habitation. Densities of A. butzleri DNA in diarrheic stools (1.6 ± 0.59 log10 copies mg−1) were higher (P = 0.007) than in nondiarrheic stools (1.3 ± 0.63 log10 copies mg−1). Of the 892 diarrheic samples that were positive for A. butzleri, 74.1% were not positive for other bacterial and/or viral pathogens. The current study supports previous work suggesting that A. butzleri pathogenicity is strain specific and/or dependent on other factors, such as the level of host resistance.

INTRODUCTION

Nearly 1.7 billion cases of diarrheal disease are reported globally each year (1), although this is an underestimation of true rates of enteritis, as many afflicted individuals do not have access to or choose not to pursue medical assistance (2). For those seeking diagnosis, the majority of cases of acute enteritis are not linked to an identified etiological agent (3, 4). Ascertaining the etiology of enteric disease is essential for the development of effective therapeutics and preventative mitigation strategies. Direct contact with animals and ingestion of untreated water and/or undercooked animal products are recognized risk factors for acute enteritis (3), which suggests that a significant number of cases of enteritis are caused by unidentified biotic pathogens of human or zoonotic origin. Critical components of the epidemiology of arcobacteriosis and the population structure of Arcobacter butzleri have yet to be resolved, in large part because effective culture and/or molecular-based detection methods for this bacterium have yet to be developed.

Arcobacter butzleri is ubiquitous in the environment (e.g., river water contaminated with human and/or nonhuman animal feces) (5–7). That the bacterium is detected in such a variety of sources suggests that pathways for transmission among animals and environmental sources exist, but accurate source tracking of A. butzleri is hampered by a lack of standard detection and isolation methods. Most methods for the isolation of A. butzleri from microbiologically complex matrices rely on selective enrichments and/or antibiotics to inhibit the growth of nontarget microorganisms (8, 9). In addition, the incubation temperature and atmosphere utilized for isolation have been inconsistent; temperatures vary from 25°C (10) to 37°C (11), and atmospheres range from aerobic (9, 12) to microaerobic (5 to 6% O2, 6 to 10% CO2, 0 to 7% H2, and 79 to 85% N2) and anaerobic (10, 13–15). Accumulated evidence indicates that no single medium, temperature, or atmosphere will isolate all strains of A. butzleri. For example, Merga et al. (16) compared five media and plating techniques and found that the most effective strategy detected A. butzleri in only 70.7% of positive samples.

A number of researchers have utilized primers to detect A. butzleri in nonselective enrichment (17, 18). However, no primers have been specifically designed to detect and quantify A. butzleri DNA extracted directly from complex matrices without an intermediate enrichment step. Primer development for the detection of microorganisms can be divided into two broad steps: (i) the in silico design of primers targeting taxon-specific gene sequences ascertained from comparative analysis of genome data and (ii) the in vitro validation of primer sensitivity (i.e., the minimum detectable amount of target DNA), specificity (i.e., the lack of detection of nontarget taxa), and inclusivity (i.e., the detection of all subtypes within a target taxon). During primer design, potential gene targets must be identified and compared to a sequence database to identify marker sites that have conserved nucleotide length, composition, and presence within the target species while being absent from nontarget species. As genomic databases cannot contain the entirety of genetic diversity of bacteria, and data are particularly lacking for the genetically diverse A. butzleri, developed primers must also be carefully evaluated to ensure sensitivity, specificity, and inclusivity. This is especially true for development of primers to detect DNA in complex matrices, such as feces.

We hypothesized that A. butzleri is a significant enteric pathogen that is underdiagnosed because of the limitations of culture-based detection. Thus, A. butzleri DNA will be more prevalent in stools from diarrheic than from nondiarrheic individuals (i.e., cohorts in the same space and time). Furthermore, A. butzleri loads will be higher in diarrheic stools, and the bacterium will be present in diarrheic stools in the absence of other recognized bacterial and viral pathogens. To test these hypotheses, the following objectives were established: (i) to use comparative whole-genome sequence analysis to select unique, highly conserved, nonvariable loci to develop direct detection and quantification primers for A. butzleri; (ii) to evaluate the sensitivity, specificity, and inclusivity of the developed primers; (iii) to contrast isolation and PCR detection frequency of A. butzleri in stools of diarrheic and nondiarrheic people (n ≈ 1,600) living in southwestern Alberta, Canada, as a model health region; (iv) to use quantitative PCR (qPCR) to contrast A. butzleri DNA loads in stools from diarrheic and nondiarrheic people; and (v) to determine the frequency at which A. butzleri occurs with other recognized bacterial and viral pathogens.

MATERIALS AND METHODS

Primer design and in silico evaluation.

The online tool Rapid Annotation Using Subsystem Technology (RAST) (19) was used to identify open reading frames (ORFs) for genomic sequences from 12 A. butzleri strains available in the NCBI database (BioProject accession numbers PRJNA233527, PRJNA58557, PRJNA158699, PRJNA61483, and PRJNA200766), including eight sequenced by our research group (7), along with whole-genome sequences from 10 additional A. butzleri strains (PRJNA309088) provided by Catherine Carrillo (Canadian Food Inspection Agency). The Basic Local Alignment Search Tool (BLAST) (20) and a program developed in-house (Concatenator) were used to compare ORFs between A. butzleri strains; those that were redundant or missing from any strains or that varied in terms of their length or sequence were removed from consideration. The RAST (19) and BLAST (20) tools were also used to compare the A. butzleri genomic sequences to those of four Arcobacter skirrowii (PRJNA307998) and six Arcobacter cryaerophilus (PRJNA307600) strains that were sequenced as part of the current project; any A. butzleri ORFs that were detected in A. skirrowii or A. cryaerophilus were removed from consideration. The program Geneious (version 5.3.6; Biomatters Ltd., Auckland, New Zealand) was used to concatenate and align the remaining sequences and to identify sites for PCR primer design. Primers for endpoint and qPCR were designed for optimal use with HotStarTaq Plus DNA polymerase (Qiagen Inc., Toronto, ON, Canada) and QuantiTect SYBR green (Qiagen Inc.).

Primer evaluation. (i) Primer specificity.

Selected PCR primers were tested for specificity against genomic DNA from 22 type strains within the order Campylobacterales, including Arcobacter spp. (i.e., A. butzleri, A. cryaerophilus, and A. skirrowii), Campylobacter spp. (i.e., Campylobacter coli, C. concisus, C. curvus, Campylobacter fetus subsp. fetus, C. hominis, C. hyointestinalis subsp. hyointestinalis, C. insulaenigrae, C. jejuni, C. jejuni subsp. doylei, C. lanienae, C. lari, C. mucosalis, C. showae, C. sputorum subsp. sputorum, and C. upsaliensis), and Helicobacter spp. (i.e., H. canadensis, H. pullorum, and H. pylori). Amplification reaction mixtures consisted of 2.0 μl of 10× PCR buffer containing 15 mM MgCl2 (Qiagen Inc.), 2.0 μl of UltraPure bovine serum albumin (BSA; 1.0 mg ml−1; Ambion, Life Technologies Inc., Burlington, ON, Canada), 0.4 μl of deoxynucleoside triphosphate (dNTP) mix (10 mM; Bio Basic Canada Inc., Markham, ON, Canada), 0.1 μl of HotStarTaq Plus DNA polymerase (5.0 U μl−1; Qiagen Inc.), 1.0 μl of ddAbutzF (10 μM; Integrated DNA Technologies, Coralville, IA), 1.0 μl of ddAbutzR (10 μM; Integrated DNA Technologies), 2.0 μl of DNA template, and 11.5 μl of nuclease-free water (Qiagen Inc.). The PCR consisted of activation at 95°C for 5.0 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 90 s, and elongation at 72°C for 60 s, followed by a final elongation at 72°C for 5 min and storage at 4°C. Amplicons were visualized on a QIAxcel capillary electrophoresis machine (Qiagen Inc.) using the AM320 separation and resolution method, with 15- to 3,000-bp alignment markers and 100- to 2,500-bp size markers.

(ii) Primer inclusivity.

Primers were evaluated for their ability to amplify DNA from 130 A. butzleri isolates representing 92 different subtypes. The PCR reagents and conditions used for primer evaluation were the same as described for primer specificity. The identities of isolates were confirmed by sequencing the near-complete 16S rRNA gene (21). Isolate subtypes were identified using a comparative genomic fingerprinting method (CGF40) specific to A. butzleri (7).

(iii) Primer sensitivity.

To determine the limit of detection of developed primers, DNA extracted from porcine feces seeded with A. butzleri was tested; pigs were selected as a monogastric model for human beings. Multiple fresh samples of feces were collected from three pigs obtained from the University of Alberta Swine Unit (Edmonton, AB, Canada) and were stored at −20°C. No antibiotics were administered to the pigs. To produce cells for incorporation into feces, A. butzleri ATCC 49616 was cultured in triplicate on Columbia agar (DF0944-17-0; Difco) containing 10% sheep blood (CBA) in a microaerobic atmosphere (i.e., 5% O2, 3% H2, 10% CO2, and 82% N2) at 37°C for 48 h. Biomass from the three cultures was removed from the surface of the medium and combined in Columbia broth (CB; Difco). The absorbance (A600) was adjusted to 0.5 and corresponded to approximately 2.0 × 109 cells ml−1. The suspension was diluted with CB in a 10-fold dilution series. Feces were thawed, and 1.0 ml from each dilution of A. butzleri cells was thoroughly mixed into 10 g of the feces. The control treatment consisted of 10 g of feces mixed with 1.0 ml of sterile CB. Three subsamples (0.2 ± 0.02 g) were removed from the seeded feces and stored at −20°C for later DNA extraction. To enumerate A. butzleri cells by culture, 1.0 g of the seeded feces was suspended in 9.0 ml of CB and diluted in a 10-fold dilution series, and 100 μl of each dilution was spread on CBA in duplicate, cultures were incubated in a microaerobic atmosphere (i.e., 5% O2, 3% H2, 10% CO2, and 82% N2) at 37°C, and colonies were enumerated at the dilution yielding 30 to 300 CFU after 48 and 96 h. The experiment was conducted two times on separate occasions.

DNA was extracted from the frozen feces subsamples using a QIAamp DNA stool minikit (Qiagen Inc.) according to the manufacturer's specifications for pathogen detection. As an internal amplification control (IAC), 2 μl of DNA (1 × 106 copies μl−1) from a synthesized gene designed using the genome of Pyrococcus yayanosii (22) was added to the feces subsamples prior to extraction; this bacterium is an obligate piezophilic hyperthermophilic archaeon isolated from deep-sea hydrothermal sites (23). The IAC targets a 268-bp sequence in a putative carbohydrate kinase (PfkB family; GenBank accession number AEH23732.1) using the primers IAC-f (3′-GGTATGCTAGCCCCGCTTAGGGT-5′) and IAC-r (3′-TGCTCCAGAAAAGATGTCCAGCGG-5′ and was synthesized by Integrated DNA Technologies. The presence and quantities of the IAC were confirmed by real-time PCR amplification on a Stratagene Mx3005P quantitative PCR (qPCR) system (Agilent Technologies, Santa Clara, CA) using the following reagents: 10 μl of 2× Quantitect SYBR green (Qiagen Inc.), 2.0 μl of UltraPure BSA (1.0 mg ml−1; Ambion), 1.0 μl of primer IAC-f (10 μM; Integrated DNA Technologies), 1.0 μl of primer IAC-r (10 μM; Integrated DNA Technologies), 2.0 μl of DNA template, and 4.0 μl of nuclease-free water (Qiagen Inc.). Samples were quantified in duplicate reactions. The amplification conditions were one cycle at 95°C for 15 min, followed by 40 cycles of 15 s at 94°C, 30 s at 64°C, and 30 s at 72°C for data acquisition. Direct endpoint detection of A. butzleri DNA was carried out as described above for primer specificity. Quantitative PCR of A. butzleri was carried out on a Stratagene Mx3005P qPCR system (Agilent Technologies) using the following reagents: 10 μl of 2× Quantitect SYBR green master mix (Qiagen Inc.), 2.0 μl of UltraPure BSA (1.0 mg ml−1; Ambion), 1.0 μl of ddAbutzF (10 μM; Integrated DNA Technologies), 1.0 μl of ddAbutzR (10 μM; Integrated DNA Technologies), 2.0 μl of DNA template, and 4.0 μl of nuclease-free water (Qiagen Inc.). Samples were quantified in duplicate reactions. The amplification conditions were 1 cycle at 95°C for 15 min, followed by 40 cycles of 30 s at 94°C, 90 s at 65°C, and 60 s at 72°C for data acquisition. At the end of amplification, melt curve analysis was conducted. The quantitative PCR data were analyzed using MxPro (version 4.10; Agilent Technologies Inc.).

Detection and quantification of A. butzleri in diarrheic and nondiarrheic stools. (i) Ethics approval.

Scientific and ethics approval to isolate, detect, and quantify A. butzleri from diarrheic and nondiarrheic people (i.e., healthy volunteers) was obtained from the Regional Ethics Committee of the former Chinook Health Region and from the University of Lethbridge Human Subject Research Committee.

(ii) Acquisition of stool samples.

A total of 1,506 stool samples were obtained from diarrheic individuals submitting samples to the Chinook Regional Hospital in Lethbridge, AB, Canada, between 1 May and 25 November 2008. Stool samples from diarrheic people were suspended in Cary-Blair medium (24) for transportation to the Chinook Regional Hospital. In addition, stool samples were obtained from 90 nondiarrheic volunteers from 27 October to 12 November 2008. Samples were kept at 4°C for no longer than 24 h. Information provided with the samples included stool collection date, along with the age, sex, and place of habitation (i.e., postal code) of the submitting individual. Using the same method as described for seeded porcine feces, subsamples (0.2 ± 0.02 g) were taken from stools and stored at −20°C for later DNA extraction.

(iii) Isolation of A. butzleri.

Media for isolation of A. butzleri were CBA, Karmali agar (CM0935; Oxoid) with Karmali supplement (KS; SR0167, Oxoid), Karmali agar (CM0935; Oxoid) with Bolton supplement (KB; SR0183E, Oxoid), Arcobacter selection and isolation agar (ASIA) (25), and Johnson-Murano agar (JMA) (26). The isolation method varied by medium: membrane filtration (13) was used for CBA; direct plating of 100 μl of the processed sample was used for KS, KB, and ASIA; and Bolton broth (CM0983, Oxoid) with Bolton supplement (SR0183E, Oxoid) (BBS) was used for enrichment culture with subsequent isolation on KS, KB, ASIA, and JMA. The CBA cultures were incubated at 37°C for up to 10 days, and all other agar media were incubated at both 30°C and 37°C for 72 h. All cultures were maintained in a high-hydrogen atmosphere (i.e., 5% O2, 30% H2, 10% CO2, and 55% N2). For enrichment cultures, 25 μl of each sample was added to 2.0 ml of BBS and incubated at both 30°C and 37°C. At 24 and 48 h, 10 μl of the enrichment was streaked on KS, KB, ASIA, and JMA.

Two colonies per morphology per medium per sample were collected, streaked for purity on CBA, and examined microscopically for cell size, shape, and motility. Genomic DNA was extracted from isolates using the DNeasy blood and tissue kit (Qiagen Inc.) according to manufacturer's specifications and an automated system (model 740; Autogen, Holliston, MA). Arcobacter butzleri DNA was identified by taxon-specific PCR using the same reagents and conditions as specified for primer specificity and by sequencing of the near-complete 16S rRNA gene (21). All recovered A. butzleri isolates were subtyped using CGF40 (7).

(iv) Extraction of total DNA from feces and direct detection of A. butzleri DNA.

The IAC was added to all stool subsamples, and genomic DNA was extracted using the QIAamp DNA stool minikit (Qiagen Inc.). Quantitative PCR for the IAC and endpoint PCR for A. butzleri were conducted as described for seeded porcine feces. Amplifications were scored as positive or negative, and only samples that were positive for the IAC in the absence of A. butzleri amplification were considered to be true negatives.

(v) Specificity of primers in stools by sequencing of direct PCR amplicons.

To confirm the specificity of amplification, 90 arbitrarily selected amplicons were sequenced. In order to generate enough product for sequence analysis, the A. butzleri PCR volume was doubled to 40 μl, containing 4.0 μl of 10× PCR buffer with 15 mM MgCl2 (Qiagen Inc.), 4.0 μl of UltraPure BSA (1.0 mg ml−1; Ambion), 0.8 μl of dNTP mix (10 mM; Bio Basic), 0.2 μl of HotStarTaq Plus (5.0 U μl−1; Qiagen Inc.), 2.0 μl of ddAbutzF (10 μM; Integrated DNA Technologies), 2.0 μl of ddAbutzR (10 μM; Integrated DNA Technologies), 4.0 μl of DNA template, and 23 μl of nuclease-free water (Qiagen Inc.). The PCR mix was activated at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 60 s, and elongation at 72°C for 30 s, a final elongation at 72°C for 5 min, and storage at 4°C. Products were purified with a MinElute 96 UF purification kit (Qiagen Inc.) and rehydrated to 20.0 μl. Sequencing was conducted by Eurofins MWG Operon, and sequences were aligned in Geneious (version 5.3.6; Biomatters) and identified using the BLAST program in the NCBI database.

(vi) Quantification of A. butzleri DNA extracted from stools.

DNA from human diarrheic (n = 69) and nondiarrheic (n = 50) stools collected during the same period (i.e., 27 October to 11 November 2008) that tested positive for A. butzleri by direct detection PCR was quantified by qPCR using the same conditions as for seeded porcine feces.

(vii) Comparison of A. butzleri prevalence to known pathogens.

The current study was part of a larger study examining the prevalence of bacterial and viral pathogens in stools from diarrheic and nondiarrheic people living in southwestern Alberta. All samples were processed by staff at the Chinook Regional Hospital for Aeromonas spp. (i.e., A. caviae, A. hydrophila, A. salmonicida, A. sobria, and A. veronii) (27), Edwardsiella spp. (Edwardsiella hoshinae and E. tarda) (28), Campylobacter spp. (C. coli, C. fetus, C. lari, and C. jejuni) (29), Escherichia coli O157:H7 (30), Plesiomonas shigelloides (28), Salmonella enterica (30), Shigella spp. (S. boydii, S. dysenteriae, S. flexneri, and S. sonnei) (30), Staphylococcus aureus (31), Vibrio spp. (Vibrio alginolyticus, V. cholerae, V. fluvialis, V. metschnikovii, V. mimicus, V. parahemolyticus, and V. vulnificus) (32), and Yersinia spp. (Yersinia enterocolitica, Y. pestis, Y. pseudotuberculosis, and Y. ruckeri) (33). In addition, RNA viruses (noroviruses GI, GII, GIII, and GIV, sapovirus, rotavirus, and astrovirus) were detected using TaqMan PCR (34; D. Leblanc, G. D. Inglis, V. F. Boras, J. Brassard, and A. Houde, submitted for publication).

(viii) Data analysis.

All statistical analyses were carried out using SigmaPlot (version 12.0; Systat Software, San Jose, CA). The chi-square test of independence was used to calculate significant differences in prevalence of A. butzleri between diarrheic and nondiarrheic people by culture-based isolation and by PCR-based detection, as well as for calculating significant differences in prevalence of A. butzleri in diarrheic people by age, sex, and location. The chi-square test of independence was also used to ascertain possible difference in rates of coinfection of A. butzleri with known pathogens in diarrheic humans. In order to determine if significant differences existed in the rate of coinfection of A. butzleri with more than two tested pathogens, the rate of coinfection for each pathogen was compared to the mean rate of coinfection with all other pathogens. The Mann-Whitney rank sum test was used to calculate significant difference between abundances of A. butzleri in stools from diarrheic and nondiarrheic people.

Nucleotide sequence accession numbers.

Sequences have been deposited in NCBI under BioProject accession numbers PRJNA307998 and PRJNA307600.

RESULTS

Primer design and in silico evaluation.

Comparative whole-genome sequence analysis of Arcobacter species revealed 1,906 conserved ORFs. Of the 66 ORFs that were not present in A. skirrowii or A. cryaerophilus, 48 did not contain sufficient length or sequence variation, and 42 were also longer than 300 bp. These 42 ORFs were concatenated for further analysis. The gene sequence for PCR amplification was required to be no more than 200 bp long, with a primer length between 19 and 23 nucleotides, a GC content of 35% to 65%, a melting temperature of 60°C to 68°C, and self-annealing or cross-annealing stretches less than 4 bp in length. The designed primers (ddAbutzF, 5′-AGTGATGGTGGAGTTGCTAGTC-3′, and ddAbutzR, 5′-GTTGCAGGAGCTTTTTCACTCC-3′) targeted a sequence that was identified as part of a putative gene encoding the gamma subunit of quinohemoprotein amine dehydrogenase (NCBI reference sequence WP_004510536.1). In silico analysis of 22 A. butzleri strains (BioProject accession numbers PRJNA233527, PRJNA58557, PRJNA158699, PRJNA61483, PRJNA200766, and PRJNA309088) identified a single copy of the target sequence per genome. The predicted PCR product was 137 bp and was unique to A. butzleri as determined by BLAST analysis (35). In addition, the primer target sequences were identical to all available A. butzleri genomes, and the closest nontarget match possessed 79% query coverage.

Primer evaluation. (i) Primer specificity.

Of the 22 taxa within Campylobacterales that were evaluated, only A. butzleri produced a detectable PCR amplification product when tested with the ddAbutz primers.

(ii) Primer inclusivity.

All 130 isolates (100%) were amplified by endpoint PCR using the ddAbutz primers.

(iii) Primer sensitivity.

The ddAbutz primers amplified A. butzleri DNA at concentrations as low as 0.6 log10 copies mg−1 by endpoint PCR and qPCR. This equated to a minimum detection limit of 1.1 copies per reaction.

Detection and quantification of A. butzleri in diarrheic and nondiarrheic stools. (i) Isolation of A. butzleri.

The overall rate of detection of A. butzleri by culture-based isolation using a variety of media and plating methods was low (0.8%), and there was no difference (P = 0.81) in detection between diarrheic and nondiarrheic individuals (see Table S1 in the supplemental material). For culture-positive samples, 8 of 13 were positive by a single method, and membrane filtration on CBA was the most inclusive (46%). No A. butzleri isolates were obtained by direct plating of processed stools onto KS. No medium and plating technique was specific to A. butzleri; each selected for at least one nontarget bacterium (see Table S2). There were too few A. butzleri-positive stools to compare the effectiveness of direct plating with that of enrichment techniques.

(ii) Total DNA extraction and detection of A. butzleri DNA.

Of the 1,596 human stool samples tested, an IAC and/or A. butzleri amplicon was not observed in extracted DNA from 26 samples (1.6%). Of the remaining 1,570 stools, 1,482 samples were obtained from diarrheic people and 88 were obtained from nondiarrheic people. The overall prevalence of A. butzleri was 60%, and there was no difference (P = 0.13) in prevalence of A. butzleri DNA between diarrheic (57%) and nondiarrheic (46%) stools. The rate of detection of A. butzleri in diarrheic individuals varied throughout the sample period, with peaks at the beginning and the end of the summer (Fig. 1). No correlation was observed between A. butzleri prevalence in diarrheic stools with sex (P = 0.37), age (P ≥ 0.26), or place of habitation (P = 0.15) (Table 1).

FIG 1.

FIG 1

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Rate of infection of A. butzleri in stools from diarrheic humans as determined by targeting the single-copy quinohemoprotein amine dehydrogenase gene with novel ddAbutz primers using direct endpoint PCR. The total numbers of human stools processed (by month) were 209 (May), 232 (June), 199 (July), 228 (August), 225 (September), 198 (October), and 191 (November).

TABLE 1.

Direct PCR detection of A. butzleri in diarrheic stools

Subject characteristics No. of samples Rate of infection (%) P value
Sex
    Male 599 61.8 0.37
    Female 873 59.5
Age (yrs)
    0–4 215 62.3 0.53
    5–18 112 55.4 0.26
    19–64 747 61.3 0.46
    65+ 398 59.0 0.52
Habitationa
    Rural 560 57.7 0.15
    Urban 887 61.4
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a

Rural or urban location of habitation was ascertained from postal codes submitted by diarrheic individuals.

(iii) Specificity of PCR primers in diarrheic stools by PCR amplification.

All 90 (100%) of the amplicons from human stools that were sequenced were identified as A. butzleri by BLAST analysis. Trimmed sequences were 93 bp to 95 bp in length. Because the sequences were identical, a single consensus sequence was compared to the NCBI database.

(iv) Quantification of A. butzleri.

Overall cell density in human stool samples was 1.4 ± 0.62 log10 cells mg−1, but quantities of DNA were higher (P = 0.007) in stools of diarrheic (1.6 ± 0.59 log10 copies mg−1) than nondiarrheic (1.3 ± 0.63 log10 copies mg−1) people.

(v) Comparison of prevalence of A. butzleri prevalence to that of known pathogens.

Of the 1,482 diarrheic samples examined, 390 (26%) were positive for recognized bacterial and/or viral pathogens. Of the samples positive for A. butzleri, 661 (74%) were not positive for other bacterial and/or viral pathogens. None of the recognized pathogens were more likely to be codetected with A. butzleri (P ≥ 0.26) (Table 2).

TABLE 2.

Detection of A. butzleri and recognized enteric pathogens in diarrheic stools

Pathogen No. of positive samples No. of co-infections with A. butzleri Rate of coinfection (%) P value
Aeromonas spp.a 9 6 66.7
C. coli 16 9 56.3 0.94
C. difficilea 7 5 71.4
C. jejuni 183 103 56.3 0.68
E. coli O157:H7 17 11 64.7 0.54
Salmonella spp. 25 15 60.0 0.79
Astrovirus 20 10 50.0 0.49
Norovirus GI 16 7 43.8 0.26
Norovirus GII 110 66 60.0 0.53
Norovirus GIIIa 0 0
Norovirus GIVa 1 1 100
Rotavirus 14 6 42.9 0.26
Sapovirus 26 16 61.5 0.66
Total 444 255 57.4
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a

Pathogen was not detected in enough samples to be statistically viable.

DISCUSSION

Efficiency of A. butzleri detection methods.

In the current study, we compared the detection of A. butzleri by isolation to detection by PCR. We found that the rate of detection of A. butzleri in human stools by isolation was low (0.8%) compared to the rate of PCR-based detection (60%). Others have found that PCR was more effective than culturing for detection of A. butzleri in human stools (12), seawater (36), and wastewater and chicken carcasses (37). Fera et al. (12) suggested that the decreased rate of detection observed in selective and enrichment media may be the result of competition by nontarget members of the source microbiota, along with difficulty replicating source conditions for growth in a controlled system. In addition, the use of enrichment culture has been shown to reduce the diversity of other enteric pathogens (38, 39), and antimicrobial agents in A. butzleri selective media may also reduce diversity (40). This is problematic because antimicrobial agents are often required to inhibit growth of nontarget organisms that could exclude A. butzleri. We frequently isolated presumptive A. butzleri based on colony morphology that turned out to be Alistipes spp., Bacteroides spp., Catabacter spp., Citrobacter spp., Helicobacter spp., and particularly Campylobacter spp., which were very commonly recovered. Previous studies have noted a similar lack of specificity for culture isolation of A. butzleri from fecal samples (10, 16).

Prevalence of A. butzleri in human stools.

We observed that the overall prevalence of A. butzleri in human stools was 60%, which is much higher than the rates of 25% or less reported by others (9, 12, 41, 42). We attribute the high rate of A. butzleri detection in the current study to our use of primers designed and validated for maximum efficiency in complex matrices. While previous studies evaluated primer sensitivity and/or specificity, they typically did not examine inclusivity. In contrast, we emphasized inclusivity as a critical component of our primer design and evaluation. PCR inclusivity is the ability of primers to amplify all subtypes of the target taxon, and it is reduced as a result of poor binding efficiency at the primer binding site. It is therefore important to select a target site that lacks sequence variation within the targeted bacterium so that it is not susceptible to competitive binding by nontarget organisms. The PCR primers used in previous studies target universal gene sequences such as 16S rRNA (42), 23S rRNA (9), hsp60 (17), and gyrA (41). The strategy that we employed identified nonuniversal gene sequences that were conserved within A. butzleri, thereby circumventing the potential pitfalls of PCR amplification of universal genes. To validate primer inclusivity, we examined 130 A. butzleri isolates representing 92 different CGF subtypes, and the primers successfully amplified the gamma subunit of the quinohemoprotein amine dehydrogenase gene (NCBI reference sequence WP_004510536.1) for all 130 isolates. In comparison, previous studies have evaluated inclusivity of their primers against a relatively small number (one to seven) of A. butzleri isolates (17, 43–45).

Comparative detection of A. butzleri in diarrheic and nondiarrheic stools.

Arcobacter butzleri is the fourth most commonly isolated Campylobacter-like organism from diarrheic humans (10), but few studies have compared the prevalence of A. butzleri in diarrheic and nondiarrheic humans. We hypothesized that if A. butzleri is an emerging pathogen, it would be significantly more prevalent in stools from diarrheic than nondiarrheic people. Even though we detected a much higher prevalence of A. butzleri in stools than previous studies, we found no significant difference between diarrheic and nondiarrheic groups. Collado et al. (9) also found no difference in prevalence between stools from diarrheic and nondiarrheic people in Chile, although there were too few A. butzleri-positive stools for statistical comparison. In South Africa, Samie et al. (46) used PCR to compare prevalences of A. butzleri in stools from diarrheic and nondiarrheic individuals and found no significant difference. These findings contrast with those regarding recognized enteric pathogens, which are more prevalent in diarrheic than in nondiarrheic individuals (47).

Comparative quantification of A. butzleri in diarrheic and nondiarrheic stools.

In situations where the pathogenicity of enteric bacteria is uncertain (48, 49), quantification of microorganism density can provide evidence in support of pathogenicity (i.e., an increase in density of a microorganism in diseased individuals). For example, Phillips et al. (50) observed that viral loads of the recognized pathogen norovirus GII were much greater in diarrheic than in nondiarrheic individuals, and Brassard et al. (51) observed that viral loads of the emerging pathogen Torque teno virus were much greater in diarrheic than in nondiarrheic people. To our knowledge, the current study is the first to compare densities of A. butzleri in diarrheic and nondiarrheic people. Although we observed that A. butzleri DNA loads were low in both diarrheic and nondiarrheic individuals, the density of A. butzleri DNA in stools from diarrheic people was slightly higher than in stools from nondiarrheic individuals. It is uncertain whether the difference in DNA loads between the two groups is biologically relevant (i.e., that pathogenic subtypes exist and contribute to the differential density) or is confounded by the diseased status of the diarrheic group. This warrants further investigation.

Epidemiology of diarrheic individuals infected with A. butzleri.

The prevalence of A. butzleri in diarrheic human stools increased with the onset of summer, and it remained relatively high throughout the sample period, but there was no correlation between rate of detection of A. butzleri and patient age or sex. Previous studies also found no correlation between A. butzleri infection and patient age or sex (41, 46). In comparison, host infection by pathogenic campylobacters is influenced by both age and sex (46, 52, 53), as is infection by other emerging pathogens, such as Helicobacter pylori (46) and Torque teno virus (51). We found no correlation between A. butzleri infection and place of habitation (i.e., whether patients lived in an urban or rural area). However, we were unable to ascertain the degree to which people living in urban versus rural locations interacted with livestock (e.g., through occupational exposure). Thus, it is not possible to determine the degree to which occupation influenced intestinal colonization by A. butzleri in the current study.

Coisolation of A. butzleri with recognized pathogens.

We examined whether A. butzleri was present in diarrheic persons in the absence of recognized pathogens. We found that 74% of A. butzleri-positive samples were not positive for recognized pathogens. The most commonly detected bacterial pathogen was C. jejuni, but the rate of coinfection with A. butzleri was not significantly greater than with other pathogens. Although it is difficult to directly compare our results with previous studies (because the pathogens detected varied, as did the methods of detection), others reported that significant numbers of samples, ranging from 16% (46) to 60% (41), were positive for A. butzleri and not for recognized pathogens. Considering that most cases of enteritis are not attributed to a single pathogenic species (3, 4) and that the majority of cases of enteritis are not linked to an etiological agent (2), the isolation of A. butzleri in the absence of other pathogens does not necessarily indicate that A. butzleri causes disease. Furthermore, our observation that A. butzleri is equally and highly prevalent in diarrheic and nondiarrheic individuals supports the conclusion that A. butzleri does not possess species-wide pathogenicity.

Conclusions.

We examined the prevalence and abundance of A. butzleri in stools from diarrheic and nondiarrheic people living in southwestern Alberta. We tested the hypothesis that, as it is an emerging enteric pathogen, the prevalence and abundance of A. butzleri will be greater in diarrheic than in nondiarrheic people. Culture-based isolation and novel direct detection PCR primers were used to detect A. butzleri in 1,596 human stools. We found that the vast majority of A. butzleri infections were not detected by culture-based isolation, that there was no difference in prevalence of A. butzleri between diarrheic and nondiarrheic cohorts, and that A. butzleri DNA loads were only slightly greater in diarrheic stools. Thus, we conclude that either A. butzleri is not a pathogen or the strain of A. butzleri and/or the status of the host regulates pathogenicity (e.g., A. butzleri is an opportunistic pathogen in a similar manner to that of H. pylori [54]). The application of high-throughput subtyping methods such as CGF40 (7) is necessary to ascertain whether specific strains of A. butzleri are associated with disease in humans, with confirmation using models of pathogenicity or virulence.

Supplementary Material

Supplemental material
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ACKNOWLEDGMENTS

We thank the following people for their assistance: Judy Baxter, Deborah Sweeny, and the staff of the Chinook Regional Hospital Department of Laboratory Medicine (Alberta Health Services) for contributing stools of diarrheic individuals and for isolating and identifying recognized bacterial pathogens; Kathaleen House (Agriculture and Agri-Food Canada, Lethbridge, Canada) for processing stool samples and isolating arcobacters, campylobacters, and helicobacters; Rachel Vivian, Kathaleen House, Gwen Leusink, Mitch Stevenson, and Greg Frick (Agriculture and Agri-Food Canada, Lethbridge, Canada) for identifying Campylobacter and Helicobacter spp.; Rachel Vivian and Jenny Gusse (Agriculture and Agri-Food Canada, Lethbridge, Canada) for extracting DNA from stool samples; Danielle Leblanc and Elyse Poitras (Agriculture and Agri-Food Canada, St. Hyacinthe, Canada) for detecting RNA viruses by TaqMan PCR; human volunteers for contributing nondiarrheic stool samples; Matthew Thomas for designing the IAC; and Catherine Carrillo (Canadian Food Inspection Agency, Ottawa) for providing the genome sequences of 10 A. butzleri strains.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.03202-15.

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