Arcobacter A. butzleri infections report diarrhea associated with abdominal pain; nausea and vomiting or fever also occur Species in (10,11). A third species, A. skirrowii, has recently been iso- lated from a person with chronic diarrhea (12). Despite Humans1 these occasional reports, the contribution of Arcobacter species to human diarrhea is still unknown. The aim of our study was to compare the prevalence and the clinical fea- Olivier Vandenberg,*† Anne Dediste,* tures of A. butzleri isolated from stools with those of Kurt Houf,‡ Sandra Ibekwem,§ C. jejuni. Hichem Souayah,* Sammy Cadranel,¶ Nicole Douat,#** G. Zissis,* J.-P. Butzler,§ The Study and P. Vandamme‡ From January 1995 to December 2002, all stool sam- During an 8-year study period, Arcobacter butzleri was ples submitted to two hospital laboratories serving the the fourth most common Campylobacter-like organism iso- Brugmann, Queen Fabiola, and Saint-Pierre University lated from 67,599 stool specimens. Our observations sug- Hospitals in Brussels, Belgium, were examined macro- gest that A. butzleri displays microbiologic and clinical scopically for consistency, gross blood, and mucus and features similar to those of Campylobacter jejuni; however, microscopically for parasites, leukocytes, and erythro- A. butzleri is more frequently associated with a persistent, cytes. These samples were also cultured for common bac- watery diarrhea. terial pathogens. Stool samples of patients <2 years of age were also evaluated for rotavirus and enteric adenovirus C ampylobacter is the most common cause of acute bac- terial enteritis in the United States and many other industrialized countries (1,2). When the diagnosis of infec- since viral diarrhea is mainly seen in young children. A specific culture protocol for the recovery of Campylobacter spp. and related organisms consisting of tion is based exclusively on culturing on selective media, one solid selective medium and a filtration method was >95% of Campylobacter infections are caused by systematically applied. In the selective agar method, the Campylobacter jejuni or C. coli. However, with refine- fecal suspension (approximately 1 g/mL of saline) was ments in isolation and identification methods, other related plated onto Butzler’s medium comprising Mueller-Hinton species such as C. upsaliensis, C. jejuni subsp. doylei, C. agar (Oxoid Ltd, Basingstoke, United Kingdom) contain- fetus subsp fetus, C. concisus, Arcobacter butzleri, ing 5% sheep blood and the antimicrobial supplement cef- Helicobacter fennelliae, and H. cinaedi, have emerged as operazone 30 mg/L, rifampicin 10 mg/L, and amphotericin potential enteric human pathogens (3). Since most labora- B 2 mg/L (Institute Virion, Rüschlikon, Switzerland) (13). tories do not use appropriate culture conditions to detect all The plates were incubated for 48 h at 42°C in a microaer- Campylobacter spp. and related organisms or do not iden- obic atmosphere (5% O2, 6% H2, 10% CO2, and 79% N2). tify isolates to species level, data on the incidence and clin- The plates were examined daily for growth of ical importance of these non–C. jejuni/coli organisms are Campylobacter species. scarce. The membrane filtration method was performed During the past decade, improvements in isolation tech- according to Lopez (14). Stool samples were diluted 1:5 in niques in veterinary medicine have led to the discovery of Brucella broth. Cellulose acetate filters (50 mm in diame- Arcobacter spp. as animal pathogens. Members of the ter) with a pore size of 0.45 µm were placed on the surface genus Arcobacter are aerotolerant Campylobacter-like of Mueller-Hinton agar plates containing 5% sheep blood. organisms. They were first isolated from aborted bovine Eight drops of the fecal suspension were placed on the top fetuses by Ellis in 1977 (4). Arcobacter spp. differ from of the membrane and allowed to filter passively for 30 min Campylobacter spp. by their ability to grow at lower tem- at 37°C in air. After filtration, the filters were removed, peratures and in air (5). Later, Arcobacter infections in and the plates were incubated at 37°C in a microaerobic humans were also described. Two species, A. butzleri and, atmosphere for up to 10 days. more rarely, A. cryaerophilus, have been associated with Two specific procedures for isolating Arcobacter were enteritis and occasionally bacteremia (6–9). Patients with used successively. Until April 1995, we used a membrane filtration technique (15). Subsequently, we switched to *Saint-Pierre University Hospital, Brussels, Belgium; †Free University of Brussels, Brussels, Belgium; ‡Ghent University, 1Presented in part at the 11th International Congress on Infectious Ghent, Belgium; §Vrije Universiteit Brussel, Brussels, Belgium; Diseases, Cancun, Mexico, Mar 4–8, 2004. (Abstract P09.030); ¶Queen Fabiola University Hospital, Brussels, Belgium; and at the 12th International Workshop on Campylobacter, #Brugmann University Hospital, Brussels, Belgium; and **Queen Helicobacter and Related Organisms. Aarhus, Denmark, Sept Fabiola University Hospital, Brussels, Belgium 6–10, 2003 (Abstract K-07). Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 10, October 2004 1863 DISPATCHES direct plating on Arcobacter selective medium (16), for and patients from a foreign country referred for surgery), which 0.5 g of stool samples was injected into an enrich- and underlying disease. Diarrhea was defined as at least ment broth (Brucella broth + antimicrobial supplement three unformed or liquid stools per day for at least 3 days. consisting of piperacillin 75 mg/L, cefoperazone 25 mg/L, Because of the study’s retrospective nature, we could only amphotericin B 10 mg/L, trimethoprim 20 mg/L, and classify diarrhea as acute (duration <15 days) or chronic cycloheximide 100 mg/L + 5% laked horse blood) and (duration >15 days). Treatment history included antimicro- incubated for 24 h at 25°C in a microaerobic atmosphere. bial history and use of intravenous fluid therapy, and, final- After incubation, 40 µL of enriched broth was plated onto ly, clinical status after 1 month of treatment (cure or Arcobacter selective medium (16). The plates were then persistent diarrhea). incubated for 3 days at 25°C in a microaerobic atmosphere All parameters were compared between index patients and examined daily. and matched controls. Comparisons were made by Pearson Gram-negative, motile, spiral, or curved rods were iden- χ2 test for 1:3 control data. Odds ratios (OR) and 95% con- tified as Campylobacter by morphologic and biochemical fidence intervals (CI) were calculated. characteristics consistent with the genus Campylobacter, as From January 1995 to December 2002, a total of 67,599 recommended by Vandamme et al. (5). A complementary stool specimens from 40,995 patients were submitted for distinction between A. butzleri and A. cryaerophilus was bacteriologic stool culture. These cultures yielded achieved by using sodium dodecyl sulfate–polyacrylamide Campylobacter and related organisms in 1,906 patients, gel electrophoresis of whole-cell proteins and by a multi- Salmonella species in 1,720 patients, and Shigella species plex polymerase chain reaction assay (17). in 244 patients. Other identified pathogens include To assess the pattern of clinical disease, patients with A. enteropathogenic Escherichia coli (137 patients), Yersinia butzleri infection were matched against three randomly enterocolitica (87 patients), Plesiomonas spp. (22 selected control patients with C. jejuni infection. To patients), and Aeromonas spp. (21 patients). achieve this, all charts of patients with A. butzleri infection Among the 1,906 Campylobacter and related organ- and controls were reviewed retrospectively by combining isms isolated during the study period, 77.2% were C. jeju- the records of the medical and microbiology departments. ni, 11.4% were C. coli, and 4.5% were C. upsaliensis A structured, close-ended questionnaire was used to collect (Table 1). Ninety-seven Arcobacter isolates were obtained the patient’s history, age, sex, status (outpatient or hospi- from 77 patients. Among them, A. butzleri was the most talized for >48 h) and history of international travel. The frequently isolated species (84 isolates from 67 patients), clinical history included diarrhea within the preceding 3 accounting for 3.5 % of the Campylobacter and related months, duration of symptoms, nature of the diarrhea organisms bacterial group. Thirteen A. cryaerophilus iso- (watery or bloody and presence of cells), intensity of fever, lates from 10 patients were obtained, but no A. skirrowii nausea or vomiting, abdominal pains, asymptomatic car- isolates were found. Other species, such as C. concisus, C. riage (routine screening of enteric pathogens in stools of fetus, C. curvus, C. lari, and C. hyointestinalis, were also HIV-infected persons, other immunocompromised states, found in small numbers (Table 1). 1864 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 10, October 2004  Arcobacter Species in Humans Medical records were available for 61 of the 67 patients Rectal bleeding, inflammatory exudates, or both were with A. butzleri infection. Ages of patients with A. butzleri significantly less common in A. butzleri than in C. jejuni infection were 30 days–90 years; there were slightly more infection (OR 0.15, 95% CI 0.05–0.46). A concomitant female than male patients. Fourteen patients were hospital- infection with another enteric pathogen occurred in a high- ized for >48 h. Four patients had traveled abroad before er proportion of patients with A. butzleri infection than onset of symptoms. Ten patients had underlying disease: 4 patients with C. jejuni infection, but the difference was not were HIV seropositive, and 3 were immunocompromised significant. Twelve patients with A. butzleri infection had (postrenal graft, celiac disease, and chemotherapy for cere- a coinfection with one of the following enteric pathogens: bellar astrocytoma). Other chronic illnesses included Salmonella enterica (n = 4) (2 ser. Enteritidis, 1 ser. dementia (n = 1), insulin-dependent diabetes mellitus Typhimurium and 1 ser. Virchow), Rotavirus (n = 3), C. (n = 1), and hepatitis C (n = 1). jejuni (n = 2), Giardia lamblia (n = 2), and Clostridium dif- Thirty-one patients complained of acute diarrhea (>24 ficile toxin B positive (n = 1). watery stools) lasting for 3 to 15 days, and 10 had persist- In 24 patients with Campylobacter jejuni infection, we ent or recurrent diarrhea lasting >2 weeks–2 months. Six found a coinfection with one another enteric pathogen: S. patients had abdominal pain without diarrhea. Twelve enterica (7) (3 ser. Enteritidis, 3 ser. Typhimurium, and 1 patients were asymptomatic. Sixteen patients received ser.Virchow), Rotavirus (5), Adenovirus (4), Giardia lam- antimicrobial therapy, but only 7 were treated empirically blia (2), Shigella flexneri (2), Yersinia enterocolitica (1), with an antimicrobial agent for which the strain was sus- and Clostridium difficile toxin B positive (1). In two addi- ceptible. The symptoms resolved in all patients except one, tional cases of campylobacteriosis, we found a coinfection regardless of the antimicrobial agent used. Among patients with two other enteric organisms: one patient was infected treated symptomatically, three patients had persistent or with S. dysenteriae and Hymenolepis nana and the other recurrent symptoms. patient with Rotavirus and Salmonella enterica serotype Sixty-seven patients with A. butzleri infection were Typhimurium. matched against 201 patients with C. jejuni infection. The To assess the prevalence of A. butzleri and age and sex distributions were similar for patients colo- Campylobacter jejuni in diarrheic stool specimens, we nized by each species. No significant differences in inter- considered only stools with loose or liquid macroscopic national travel were observed. However, proportionally aspect as diarrhea. Among the 67,599 stool specimens fewer patients with A. butzleri (79.1%) were treated as out- received, 12,413 were solid stools, and 55,186 were diar- patients than those with C. jejuni (90.5%) (OR 0.40, 95% rheic. Among the diarrheic stool specimens, we isolated A. CI 0.17–0.90) (Table 2). butzleri and C. jejuni in 77 (0.14%) and 3,209 (5.81%) Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 10, October 2004 1865 DISPATCHES stools, respectively. Among the nondiarrheic stool speci- Wenzel, and Jennifer Frost for their critical corrections; mens, we isolated A. butzleri and C. jejuni in 7 (0.06%) Dominique Monnet for his help in performing statistical analysis; and 205 (1.65%) stools, respectively. A. butzleri was more and Patricia Retore, Chantal Vandenborre, and Linda Vlaes for frequently isolated from diarrheic stool specimens than their skilled technical assistance. from nondiarrheic stools specimens. This difference was This work was supported in part by grants from the significant (OR 2.48, 95% CI 1.10–5.86) (p = 0.0175). We Fondation Vesale Research fellowship (foundation for medical observed a similar result for the recovery of C. jejuni from research) to Olivier Vandenberg. diarrheic stool specimens compared with nondiarrheic stools specimens. This difference was even more signifi- Dr. Vandenberg is a staff microbiologist at Saint Pierre cant (OR 3.68, 95% CI 3.18–4.25) (p < 0.0001). University Hospital in Brussels, Belgium. He is responsible for Because medical records were unavailable for 6 the National Reference Center for Enteric Campylobacter. He is patients with A. butzleri and 10 patients with C. jejuni, we also an assistant at the Infectious Diseases Epidemiology Unit of compared the clinical features of 61 patients with A. butz- the Public Health School of the Free University of Brussels. leri matched against those of 191 patients with C. jejuni. Although diarrhea was a common clinical feature of both References groups, it was more frequent in the C. jejuni–infected patients. The characteristics of the diarrhea differed signif- 1. Altekruse SF, Stern NJ, Fields PI, Swerdlow DL. Campylobacter jejuni—an emerging foodborne pathogen. Emerg Infect Dis. icantly, however. Patients with A. butzleri were more like- 1999;5:28–35. ly to have persistent diarrhea (OR 3.97, 95% CI 1.4–11.3), 2. Tauxe R. Incidence, trends and source of campylobacteriosis in or watery diarrhea (OR 2.20, 95% CI 1.18–4.13) than developed countries: an overview. In: The increasing incidence of those with C. jejuni infection, but they were less likely to campylobacteriosis in humans. Report and proceedings of a WHO consultation of experts. Geneva: World Health Organization; 2001. p. have acute diarrhea (OR 0.38 95%, CI 0.20–0.79). Other 42–3. clinical features did not differ significantly. Asymptomatic 3. Lastovica AJ, Engel ME, Blaser MJ. Atypical campylobacters and carriage was more frequently encountered in patients with related organisms. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg A. butzleri infection than in those with C. jejuni infection, HB, Guerrant RL, editors. Infections of the gastrointestinal tract. 2nd ed. Philadelphia: Lippincott Williams and Wilkins; 2003. p. 741–61. but not significantly (OR 2.22, 95% CI 0.94–5.21). 4. Ellis WA, Neill SD, O’Brien JJ, Ferguson HW, Hanna J. Isolation of Proportionally more patients with C. jejuni infections Spirillum/Vibrio-like organisms from bovine fetuses. Vet Rec. were treated with antimicrobial agents (41.4%) than 1977;100:451–2. patients with A. butzleri infections (26.2%) (OR 0.50, 95% 5. Vandamme P, Vancanneyt M, Pot B, Mels L, Hoste B, Dewettinck D, et al. Polyphasic taxonomic study of the emended genus Arcobacter CI 0.25–1.0). Among them, only 43.8% of patients with A. with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. butzleri infections were treated empirically with an antimi- nov., an aerotolerant bacterium isolated from veterinary specimens. crobial agent for which the strain was susceptible, where- Int J Syst Bacteriol. 1992;42:344–56. as 79.5% of patients with C. jejuni received an appropriate 6. Kiehlbauch JA, Brenner DJ, Nicholson MA, Baker CN, Patton CM, Steigerwalt AG, et al Campylobacter butzleri sp. nov. isolated from antimicrobial drug. humans and animals with diarrheal illness. J Clin Microbiol. 1991;29:376–85. Conclusions 7. Lerner J, Bruberger V, Preac-Mursic V. Severe diarrhea associated In this study, Arcobacter was the fourth most common with Arcobacter butzleri. Eur J Clin Microbiol Infect Dis. 1994;13:660–2. Campylobacter or Campylobacter-like organism isolated 8. Hsueh PR, Teng LJ, Yang PC, Wang SK, Chang SC, Ho SW, et al. from stool specimens in our laboratories. Our observations Bacteremia caused by Arcobacter cryaerophilus 1B. J Clin Microbiol. suggest that A. butzleri display similar microbiologic and 1997;35:489–91. clinical features as C. jejuni. However, compared with C. 9. On SL, Stacey A, Smyth J. Isolation of Arcobacter butzleri from a neonate with bacteraemia. J Infect. 1995;31:225–7. jejuni, A. butzleri are more frequently associated with a 10. Dediste A, Aeby A, Ebraert A, Vlaes L, Tridiani R, Vandenberg O, et persistent and watery diarrhea and less associated with al. Arcobacter in stools: clinical features, diagnosis and antibiotic bloody diarrhea. This first study on Arcobacter in humans susceptibility. In: Lastovica AJ, Newell DG, Lastovica EE, editors. could be the beginning of future research to better under- Campylobacter, Helicobacter and related organisms. Cape Town, South Africa: Institute of Child Health, University of Cape Town; stand the pathogenesis and epidemiology of these 1998. p. 436–9. non–jejuni/coli Campylobacter. 11. Vandamme P, Pugina P, Benzi G, Van Etterijck R, Vlaes L, Kersters K, et al. Outbreak of recurrent abdominal cramps associated with Acknowledgments Arcobacter butzleri in an Italian school. J Clin Microbiol. 1992;30:2335–7. We thank Regine De Koster and Françoise Mengal for their 12. Wybo I, Breynaert J, Lindenburg F, Houf K, Lauwers S. Isolation of secretarial services; the personnel of the Department of Arcobacter skirrowii from a patient with chronic diarrhea. J Clin Microbiology for their daily technical assistance; Jeanne-Marie Microbiol. 2004;42:1851–2. Devaster for help preparing the article; Martin Skirrow, Richard 1866 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 10, October 2004  Arcobacter Species in Humans 13. Goossens H, De Boeck M, Coignau H, Vlaes L, Van den Borre C, 16. de Boer E, Tilburg JJ, Woodward DL, Lior H, Johnson WM. A selec- Butzler JP. Modified selective medium for isolation of tive medium for the isolation of Arcobacter from meats. Lett Appl Campylobacter spp. from faeces: comparison with Preston medium, Microbiol. 1996;23:64–6. a blood-free medium, and a filtration system. J Clin Microbiol. 17. Houf K, Tutenel A, De Zutter L, Van Hoof J, Vandamme P. 1986;24:840–3. Development of a multiplex PCR assay for the simultaneous detec- 14. Lopez L, Castillo FJ, Clavel A, Rubio MC. Use of a selective medi- tion and identification of Arcobacter butzleri, Arcobacter um and a membrane filter method for isolation of Campylobacter cryaerophilus and Arcobacter skirrowii. FEMS Microbiol Lett. species from Spanish paediatric patients. Eur J Clin Microbiol Infect 2000;193:89–94 Dis. 1998;17:489–92. 15. Marinescu M, Collignon A, Squinazi F, Derimay R, Woodward D, Address for correspondence: Olivier Vandenberg. Department of Lior H. Two cases of persistent diarrhoea associated with Arcobacter Microbiology, Saint-Pierre University Hospital, Rue Haute 322, B-1000 species. In: Newell DG, Ketley JM, Feldman RA, editors. Campylobacter, Helicobacter and related organisms. New York: Brussels, Belgium; fax: 32-2-535.4656; email: olivier_vandenberg@ Plenum;1996. p. 521–3. stpierre-bru.be Search past issues Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 10, October 2004 1867