Diagnostic Microbiology and Infectious Disease 75 (2013) 434–436

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Diagnostic Microbiology and Infectious Disease

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Assessment of the prevalence and diversity of emergent campylobacteria in humanstool samples using a combination of traditional and molecular methods☆

Luis Collado ⁎, Magali Gutiérrez, Mario González, Heriberto FernándezInstitute of Clinical Microbiology, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile

☆ Thisworkhas been supported by a postdoctoral grant f⁎ Corresponding author. Tel.: +56-63-293848; fax: +

E-mail address: luiscollado@uach.cl (L. Collado).

0732-8893/$ – see front matter © 2013 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.diagmicrobio.2012.12.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 September 2012Received in revised form 5 December 2012Accepted 19 December 2012Available online 30 January 2013

Keywords:CampylobacterArcobacterFecal samplesMolecular detectionSouthern Chile

This study aims to assess the diversity of campylobacteria (Campylobacter and Arcobacter) in human fecalsamples from patients with diarrhea (n = 140) and asymptomatic controls (n = 116) in Chile, using acombination of traditional culture and molecular methods. The culture methods detected campylobacteria in10.7% of the patients with diarrhea and in 1.7% of the controls. In contrast, the molecular methods detectedcampylobacteria more often than the traditional culture, with a prevalence of 25.7% and 5.2%, respectively.The traditional methods only recovered the species Campylobacter jejuni, Campylobacter coli, and Arcobacterbutzleri, whereas the molecular methods additionally detected the emergent species Campylobacter concisusand Campylobacter ureolyticus.

romFondecyt (no. 3110016).56-63-215295.

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© 2013 Elsevier Inc. All rights reserved.

The genus Campylobacter currently includes 25 species and8 subspecies (Man, 2011). With the exception of Campylobacterjejuni and Campylobacter coli, the role of the other less commonspecies in human and animal diseases is far less understood(Lastovica and Allos, 2008). However, in recent years, increasingattention has been paid to those emergent campylobacters due totheir pathogenic potential (Man, 2011). On the other hand, thegenus Arcobacter, at present, includes 17 species, whose role inhuman diseases is not yet well established (Collado and Figueras,2011; Levican et al., in press). Nevertheless, Arcobacter butzleri,Arcobacter cryaerophilus, and Arcobacter skirrowii have been associ-ated with gastrointestinal diseases (Vandenberg et al., 2006).Although emergent campylobacters and arcobacters are not cur-rently considered microorganisms of major public health concern,data increasingly suggest that their significance in human andanimal infections is underestimated for several reasons including theisolation procedures that favor the recovery of the most commonCampylobacter species (Vandenberg et al., 2006). Therefore, ourmain objective was to assess the prevalence and diversity ofcampylobacteria (Campylobacter and Arcobacter) in human stoolsamples using a combination of traditional culture and moleculardetection and identification methods.

A total of 256 fecal samples collected in the city of Valdivia(southern Chile) between November 2010 and March 2012 wereanalyzed. These included 140 specimens collected from patients withdiarrhea and 116 samples from healthy volunteers. Informed consentdocuments were obtained from all the enrolled persons or fromtheir parents.

The presence of Campylobacter and Arcobacter in the samples wasanalyzed by genus-specific polymerase chain reaction (PCR) (Harmonand Wesley, 1997; Linton et al., 1996). In positive samples, speciesidentification was carried out by the multiplex PCR (m-PCR) methodsused for the characterization of the 6 most common clinical campylo-bacters (Yamazaki-Matsune et al., 2007) and the 5 most commonarcobacters (Douidah et al., 2010). Additionally, specific PCR methodswere applied for the emergent species Campylobacter ureolyticus andCampylobacter concisus (Bullman et al., 2012; Man et al., 2010).

For Campylobacter isolation, samples were inoculated in themodified charcoal cefoperazone deoxycholate agar (mCCDA; Oxoid,Basingstoke, Hampshire, England). In parallel, a sample suspensionwas transferred to the surface of a 0.45-μm membrane filter(Millipore) placed on a Petri dish containing blood agar (BA) andwas allowed to filter passively for 30 min. After filtration, the filterswere removed and the plates were incubated under micro-aerobicconditions (6% O2, 6% CO2, 3% H2, and 85% N2) at 37 ºC for up to 6days, examined every 2 days (Lastovica and Allos, 2008). Arcobac-ters were isolated by enrichment in CAT (cefaperazone, amphoter-icin B, and teicoplanin) broth followed by filtration over BA (asdescribed above). The plates were incubated at 30 °C for 48 to 72 hunder aerobic conditions (Collado and Figueras, 2011).

Table 1Prevalence of campylobacteria in the studied population.

Feature No. of tested samples/no. (%) of positive samples by culture/no. (%) of positive samples bymolecular detection

Diarrheic Healthy

Age (no. of tested samples)b5 years (76) 57/7 (12.3)/18a(31.6) 19/1 (5.3)/2b (10.5)≥5 years (180) 83/8 (9.6)/18 (21.7) 97/1 (1.0)/4 (4.1)5 to 17 years (47) 24/2 (8.3)/4a (16.6) 23/0 (0.0)/2b (8.7)18 to 35 years (78) 30/2 (6.7)/7 (23.3) 48/1 (2.1)/2 (4.2)36 to 50 years (25) 15/1 (6.7)/2 (13.3) 10/0 (0.0)/0 (0.0)N50 years (30) 14/3 (21.4)/5 (35.7) 16/0 (0.0)/0 (0.0)

Total (256) 140/15 (10.7)/36 (25.7)c 116/ 2 (1.7) /6 (5.2)Gender (no. of tested samples)Female (130) 72/10 (13.8)/17 (23.6) 58/0 (0.0)/2 (3.4)Male (126) 68/5 (7.4)/19 (27.9) 58/2 (3.4)/4 (6.9)

Total (256) 140/15 (10.7)/36 (25.7)c 116/2 (1.7)/6 (5.2)

a In 1 sample, both Campylobacter sp. and Arcobacter sp. were present.b In 1 sample, 2 Campylobacter species were present.c Statistically significant in comparison with isolation (P = 0.002).

435L. Collado et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 434–436

Identification of isolates was carried out by the API Campy(bioMérieux, Marcy-l´Etoile, France) and the PCR methods aforemen-tioned. The identity of Campylobacter and Arcobacter strains wasconfirmed with the PCR– restriction fragment length polymorphismmethods described by Marshall et al. (1999) and Figueras et al.(2008), respectively.

Statistical analysis was performed using the Z test for proportionswith the EPIDAT v. 3.1 software (Pan American Health Organization(PAHO)/Xunta de Galicia, Spain). A P value of b0.05 was considered asstatistically significant.

The traditional culture methods detected campylobacteria in10.7% of the persons with diarrhea and in 1.7% of the healthy group.In contrast, the molecular methods detected campylobacteria moreoften, with a prevalence of 25.7% and 5.2%, respectively. However,this difference was only significant in diarrheic samples (P =0.002). The prevalence by culture, in children under 5 years old(12.3%), agrees with previous data reported in Valdivia, whichranged between 9.2% and 14.1% (Fernández, 2011). Our samplingwas not, however, restricted to this age group. Interestingly, fromthe 15 samples shown to contain Campylobacter by the culturemethod, more than half (n = 8) were from persons older than 5years (Table 1).

The ability to detect different species also differed betweentraditional and molecular methods. As shown in Table 2, onlyC. jejuni, C. coli, and A. butzleri were recovered using the traditionalapproach, whereas by using molecular methods, C. concisus andC. ureolyticus were also detected. Most positive samples werefound to contain 1 species, but some samples were found to

Table 2Comparison of the results of Campylobacter and Arcobacter isolation with those of direct m

Species No. (%) of positive samples among diarrheic and he

Diarrheic (n = 140)

Isolation Molecular detection

Campylobacter jejuni 14 (10.0)a 15 (10.7)Campylobacter coli 0 (0.0) 0 (0.0)Campylobacter concisus 0 (0.0) 16 (11.4)Campylobacter ureolyticus 0 (0.0) 5 (3.6)Arcobacter butzleri 1 (0.7) 2 (1.4)Total 15 (10.7) 38 (27.1)

a No significant difference was found between isolation of Campylobacter sp. by mCCDAb Statistically significant in comparison to detection in healthy samples (P b 0.05).

contain more than 1 species of campylobacteria (Table 1). Bycombining the data from both detection methodologies, the mostprevalent species associated with gastroenteritis were found to beC. concisus (11.4%) and C. jejuni (10.7%) followed by C. ureolyticus(3.6%) and A. butzleri (1.4%) (Table 2). In healthy persons, themost prevalent species was C. concisus (3.4%), followed by C.ureolyticus (1.7%), while C. jejuni and C. coli showed the sameprevalence (0.9%). Among all detected species, only C. jejuni and C.concisus were significantly associated with diarrhea (P = 0.003and P = 0.033, respectively).

To our knowledge, this is the first report of the detection ofC. concisus and C. ureolyticus in Chile. However, these species are 2of the most reported emergent campylobacteria in developedcountries (Bullman et al., 2012; Inglis et al., 2011; Man et al.,2010). In relation to C. concisus, our findings agree with the study ofInglis et al. (2011), because we found a slight difference in theprevalence of C. concisus and C. jejuni by PCR detection. However,these authors reported a higher proportion of this species in healthystool samples compared to diarrheic samples, which could bebecause 2 genomospecies with different pathogenic potential havebeen found in C. concisus (Kalischuk and Inglis, 2011).

On the other hand, we found C. ureolyticus and A. butzleri to be thethird and fourth most prevalent species in diarrheic samples,respectively (Table 2), agreeing with similar recent studies in bothEurope and Africa (Bullman et al., 2012; Samie et al., 2007;Vandenberg et al., 2006). All these new data suggest that C. concisus,C. ureolyticus, and A. butzleri are emergent species that still have anunderestimated clinical importance.

olecular detection in human samples.

althy groups, based on the indicated methods

Healthy (n = 116)

Total Isolation Molecular detection Total

15 (10.7)b 1 (0.9)a 1 (0.9) 1 (0.9)0 (0.0) 1 (0.9)a 1 (0.9) 1 (0.9)

16 (11.4)b 0 (0.0) 4 (3.4) 4 (3.4)5 (3.6) 0 (0.0) 2 (1.7) 2 (1.7)2 (1.4) 0 (0.0) 0 (0.0) 0 (0)

38 (27.1) 2 (1.7) 8 (6.9) 8 (6.9)

and the filtration method (P N 0.05).

436 L. Collado et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 434–436

Since an effective national Campylobacter surveillance programdoes not exist in Chile (Fernández, 2000), our results contribute to theunderstanding of the local epidemiology of campylobacteria.

References

Bullman S, O'Leary J, Corcoran D, Sleator RD, Lucey B. Molecular-based detection of non-culturable and emerging campylobacteria in patients presenting with gastroen-teritis. Epidemiol Infect 2012;140:684–8.

Collado L, Figueras MJ. Taxonomy, epidemiology, and clinical relevance of the genusArcobacter. Clin Microbiol Rev 2011;24:174–92.

Douidah L, De Zutter L, Vandamme P, Houf K. Identification of five human and mammalassociated Arcobacter species by a novel multiplex-PCR assay. J Microbiol Methods2010;80:281–6.

Fernández H. Emergence of antimicrobial resistance in Campylobacter: theconsequences for incidence, clinical course, epidemiology and control. Theincreasing incidence of human campylobacteriosis. Report and Proceedings of aWHO Consultation of Experts. Copenhagen: WHO/CDS/CSR/APH/2001.7; 2000.p. 67–72.

Fernández H. Campylobacter and campylobacteriosis: a view from South America. RevPeru Med Exp Salud Publica 2011;28:121–7.

Figueras MJ, Collado L, Guarro J. A new 16S rDNA-RFLP method for thediscrimination of the accepted species of Arcobacter. Diagn Microbiol InfectDis 2008;62:11–5.

Harmon KM, Wesley IV. Multiplex PCR for the identification of Arcobacter anddifferentiation of Arcobacter butzleri from other arcobacters. Vet Microbiol 1997;58:215–27.

Inglis GD, Boras VF, Houde A. Enteric campylobacteria and RNA viruses associated withhealthy and diarrheic humans in the Chinook health region of southwesternAlberta, Canada. J Clin Microbiol 2011;49:209–19.

Kalischuk LD, Inglis GD. Comparative genotypic and pathogenic examination ofCampylobacter concisus isolates from diarrheic and non-diarrheic humans. BMCMicrobiol 2011;11:53.

Lastovica AJ, Allos BM. Clinical significance of Campylobacter and related species otherthan Campylobacter jejuni and Campylobacter coli. In: Nachamkin CM Szymanski,Blaser MJ, editors. Campylobacter. 3rd edn. Washington, DC: American Society forMicrobiology; 2008. p. 123–49.

Levican A, Collado L, Figueras MJ. Arcobacter cloacae sp. nov. and Arcobacter suissp. nov., two new species isolated from food and sewage. Syst Appl Microbiol,in press.

Linton D, Owen RJ, Stanley J. Rapid identification by PCR of the genus Campylobacter andof five Campylobacter species enteropathogenic for man and animals. Res Microbiol1996;147:707–18.

Man SM. The clinical importance of emerging Campylobacter species. Nat RevGastroenterol Hepatol 2011;8:669–85.

Man SM, Zhang L, Day AS, Leach ST, Lemberg DA, Mitchell H. Campylobacter concisusand other Campylobacter species in children with newly diagnosed Crohn's disease.Inflamm Bowel Dis 2010;16:1008–16.

Marshall SM, Melito PL, Woodward DL, Johnson WM, Rodgers FG, Mulvey MR. Rapididentification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene. J ClinMicrobiol 1999;37:4158–60.

Samie A, Obi CL, Barrett LJ, Powell SM, Guerrant RL. Prevalence of Campylobacterspecies, Helicobacter pylori and Arcobacter species in stool samples from the Vendaregion, Limpopo, South Africa: studies using molecular diagnostic methods. J Infect2007;54:558–66.

Vandenberg O, Houf K, Douat N, Vlaes L, Retore P, Butzler JP, et al. Antimicrobialsusceptibility of clinical isolates of non-jejuni/coli campylobacters and arcobactersfrom Belgium. J Antimicrob Chemother 2006;57:908–13.

Yamazaki-Matsune W, Taguchi M, Seto K, Kawahara R, Kawatsu K, Kumeda Y, et al.Development of a multiplex PCR assay for identification of Campylobacter coli,Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylo-bacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J Med Microbiol2007;56:1467–73.