Journal of the New Zealand Medical Association, 20-April-2012, Vol 125 No 1353
Arcobacter species in diarrhoeal faeces from humans in New Zealand
Owen Mandisodza, Elizabeth Burrows, Mary Nulsen
Arcobacter species, formerly classified as aerotolerant Campylobacter species, are widely distributed in production animals, pets, wild animals, and the environment. Colonised animals, particularly poultry, frequently show no symptoms but, on occasions, Arcobacter spp. have been implicated in abortions, mastitis and diarrhoea.1,2 Arcobacter spp are also common in foods such as meats and shell fish, and fresh water.1,2
Three of the 12 species, A. butzleri, A. cryaerophilus and A. skirowii have been isolated from humans with diarrhoea or other gastrointestinal symptoms,1,3 in particular, watery or persistent diarrhoea.4–7 A. butzleri was the only pathogen detected in an outbreak of recurrent abdominal cramps in 10 children aged 3 to 7 years in an Italian school.8
Occasionally A. butzleri9-11 and A. cryaerophilus12,13 have been isolated from patients with bacteraemia but Arcobacter species have also been isolated from faecal samples from healthy humans.7,14-16
Arcobacter spp. have recently been detected in a high proportion of chicken meat samples purchased in Palmerston North, New Zealand17 so the aim of this study was to investigate their prevalence in the faeces of humans with diarrhoea in one region of New Zealand.
All faecal samples sent to a community laboratory in Hawke’s Bay, New Zealand, for diagnosis of gastrointestinal infection, between October, 2007 and June, 2008, were cultured for Arcobacter spp. after they had had been sampled for routine screening of pathogens.
For the initial enrichment, 1 g of faeces was emulsified in 9 mL of Arcobacter broth (Oxoid Ltd, UK) and incubated at 28°C for 48 hrs in a microaerobic atmosphere with gas packs (AnaeroPack SystemTM, Mitsubishi Gas Chemicals, Japan). This was then subcultured onto Arcobacter selective agar, containing Arcobacter broth (28g L-1), Oxoid No. 1 agar (12g L-1), plus the following antimicrobial agents supplied by Aldrich Sigma NZ: cefoperazone (16mg L-1), trimethoprim (64mg L-1), novobiocin (32mg L-1). amphotericin B (10mg L-1), 5-fluorouracil (100mg L-1).
Agar plates were incubated for 48 hrs in a microaerobic atmosphere. Preliminary identification was based on colony morphology and Gram reaction of the isolates from pure culture, by oxidase test using oxidase strips (Oxoid Ltd, UK), and by dark-field microscopy for darting motility. Presumptive isolates of Arcobacter spp, subcultured onto 5% sheep blood agar, were preserved on Microbank porous beads system (Pro-Lab Diagnostic) and stored at -80°C for later molecular characterization.
Routine faecal screening included culture for Salmonella, Shigella, Campylobacter, Yersinia and Aeromonas species. Selected stools were also examined for E. coli O157 and/or rotavirus. If requested, a Helicobacter pylori faecal antigen test, a Cryptosporidium plus Giardia species antigen test and microscopic examination for parasites were also done. Clinical data on positive samples was derived from laboratory records. Reference strains of A. butzleri (ATCC 49616) and A. cryaerophilus (ATCC 43158 and ATCC49942) were obtained from the Institute of Environmental Science and Research Limited (ESR), Wellington, New Zealand. Ethical approval was provided by the Central Ethical Committee (HDEC CEN/07/04/026).
The minimum inhibitory concentration (MIC) of ampicillin, tetracycline, ciprofloxacin and erythromycin was determined for Arcobacter spp. grown for 48 hrs on blood agar and suspended in saline to a density equivalent to 1.0 McFarland standard. For each antibiotic-isolate combination, a Mueller Hinton agar plate enriched with 5% sheep blood (Fort Richard, NZ) was spread with 100µL of the suspension, overlaid with an MIC Evaluator strip (Oxoid, UK) and incubated at 28°C for 48 hrs in a microaerobic environment. The MICs were classified as susceptible, intermediate or resistant according to the criteria used in the 1997-2006 NARMS report for Campylobacter for tetracycline, erythromycin and ciprofloxacin and for Salmonella, Shigella and E. coli O157 for ampicillin.18
Multiplex polymerase chain reaction (m-PCR) was performed as described by Houf et al (2000),19 except that loading buffer was omitted, the MgCl2 concentration was increased from 1.3 to 1.5 mmol L-1 and one to two colonies of suspected Arcobacter, grown for 48 h on 5% sheep blood agar plates at 27±2°C microaerobically, were added directly to the reaction mix which was then heated to 94°C for 3 min prior to amplification in a GeneAmp PCR System 2400 (Biosystems, Singapore) Amplified products were separated by electrophoresis in 1.5% agarose. Gels were stained with ethidium bromide and inspected visually under UV light. DNA from A. butzleri (ATCC 49616), and A. cryaerophilus (ATCC 43158) type strains were included as positive controls.
For PFGE, frozen-stored isolates of Arcobacter were streaked onto 5% sheep blood agar plates and grown microaerobically for 48–72 hours at 27±2°C. Colonies were suspended in 2 mL of phosphate buffered saline (PBS) to a final optical density (OD) of 1.00 ± 0.20. Suspended cells (400 mcL) were mixed with 20 mcL of proteinase K (20 mg mL-1) (Amresco, USA) and equal volumes of 1% Seakem Gold agarose (Cambrex Bioscience, USA) prepared in 0.5× TBE buffer. The mixture was transferred to Chef disposable plug moulds (Bio-Rad, USA) and allowed to solidify at room temperature. Plugs were incubated at 55°C in 5 mL of lysis buffer (50 mM Tris, 50 mM EDTA and 1% Sarcosyl) and 25 mcL of proteinase K for 3 hours.
Treated plugs were washed once with 10-15 mL of MilliQ (MQ) water and four times with 10-15 mL of TE buffer (10 mM Tris and 1 mM EDTA) for 10-15 min at 55°C. About 2 mm of the plug was digested with EagI (New England Biolabs, USA) at 37°C for four hours. The restriction fragments were separated by electrophoresis in 1% of Seakem Gold agarose (Cambrex Bioscience, USA) using a CHEF Mapper (Bio-Rad, USA).
The gels were run using the following conditions: Initial switch time 0.1 seconds, final switch time 90 seconds, run time 20 hours, angle 120°, gradient 6V/cm, temperature 14°C and ramping factor linear. The gels were stained for 10 minutes in ethidium bromide solution, destained with sterile water and visualised using the Gel-DOC 2000 software (Bio-Rad, USA).
From 1380 diarrhoeal faecal samples, 16 isolates were presumptively identified as Arcobacter spp. but only 12 (0.9%) were positive by multiplex PCR.
Table 1. Details of patients whose faeces yielded Arcobacter spp
1NR: none recorded
A. butzleri was cultured mainly from males and A. cryaerophilus from females (Table 1) and the difference between the two sexes is statistically significant (p=0.015). Four patients had an additional pathogen detected, namely Helicobacter pylori (two), Blastocystis hominis and Aeromonas hydrophila. All except one of the patients were adults, with ages ranging from 31 to 78 years. Three patients had persistent diarrhoea but, information was not provided for another four.
PFGE indicated that the Arcobacter isolates from diarrhoeal faeces were different from each other (data not shown) and also from those from poultry meat previously isolated in Palmerston North.17
All of the Arcobacter isolates were susceptible to ciprofloxacin and all but one susceptible to erythromycin. That A. butzleri isolate was resistant to ampicillin and tetracycline with intermediate resistance to erythromycin (Table 2). Three additional Arcobacter isolates showed intermediate resistance to tetracycline. Only half the isolates were susceptible to ampicillin.
Table 2. Antimicrobial susceptibility of Arcobacter spp. isolated from the faeces of patients with diarrhoea
1 The resistance break points were ≥4 mg/L for ciprofloxacin, ≥32 mg/L for erythromycin, ≥16 mg/L for tetracycline and ≥32 mg/L for ampicillin.18
The isolation of A. butzleri and A. cryaerophilus from 0.9% of diarrhoeal faecal samples collected in Hawke’s Bay, New Zealand is consistent with the 1% isolation rate of A. butzleri reported for diarrhoeal stools in France11 but higher than the 0.14% reported for A. butzleri and A. cryaerophilus in both Belgium7 and Denmark.20
Culture-based methods yielded A. butzleri from 2.4% of faecal samples collected from Thai children with diarrhoea21 but the use of PCR to detect Arcobacter spp. directly from faeces has generally yielded a higher proportion of positive results, e.g. 7.5% for A. butzleri , 3.5% for A. cryaerophilus and 2% for Arcobacter skirowii for patients hospitalised with diarrhoea or other gastrointestinal disorders in South Africa15 and 8% for A. butzleri from patients with travellers’ diarrhoea who had visited Mexico, Guatemala or India.22 By contrast, A. butzleri was detected in only 1.2% of diarrhoeal stools by means of PCR in another study in France.23
However the real significance of Arcobacter isolation is difficult to determine since several pathogens have been detected in a number of these patients. In the present study, one third had a second pathogen detected (Table 1) which is comparable with the 20% of patients with A. butzleri plus another enteric pathogen reported by Vandenberg et al (2004).7 The latter group also found that 16% of patients with A. butzleri in their faeces had an underlying disease and 20% of the A. butzleri isolates were from asymptomatic patients. Of 16 patients with travellers’ diarrhoea with A. butzleri detected, 15 also harboured either enterotoxigenic Escherichia coli (ETEC) or Campylobacter sp.22
Likewise 20 of 33 patients with Arcobacter spp. hospitalised in South Africa had one to three other gastrointestinal pathogens detected.15 Arcobacter spp. have also been detected in faeces collected from asymptomatic patients, including 7 abattoir workers in Switzerland14 and 26% of healthy subjects in Italy.16 Interestingly the latter group found an increased carriage rate of Arcobacter spp. (79%) in older people with type 2 diabetes but no gastrointestinal disorders.
Other bacterial species isolated from the 1380 diarrhoeal faecal samples examined for Arcobacter spp. in the current study were: Campylobacter (15.1%), Salmonella (2.6%), Aeromonas (2.2%), Yersinia (1.9%) and Shigella (0.1%) (S. Wallace, personal communication). Thus Arcobacter spp. (0.9%) were more common than Shigella, much less common than Campylobacter spp and roughly similar in frequency to the other enteric bacterial pathogens.
Two studies found that A. butzleri was more common in the faeces of females than males7,8 and one found the opposite15 but the differences in all studies were small. Another group isolated A. cryaerophilus from the faeces of 1.4% of healthy men who worked in abattoirs.14 Thus it is likely that the unequal distribution of the two Arcobacter species across the sexes shown in Table 1, although statistically significant, is not biologically meaningful.
Based on results from single isolates, Arcobacter spp. have been described as antibiotic resistant.9,24 However, the observation that all the isolates in this study were susceptible to ciprofloxacin (Table 2) is consistent with reports that 89 to 100% are susceptible to ciprofloxacin.11,25–27 Likewise, erythromycin susceptibility (92%, Table 2) and 87 to 100%27-29 is common among Arcobacter spp. By contrast, the relatively low proportion of isolates susceptible to tetracycline in this study (67%, Table 2) differs from the 100% susceptibility reported for isolates from the USA,27 Japan,29 and Thailand26 but resistance to ampicillin is common worldwide.9,28,30
We conclude that A. butzleri and A. cryaerophilus do occasionally cause diarrhoea in New Zealanders which may be persistent or watery. However their real significance as emerging enteric pathogens, both in New Zealand and overseas,1 is unclear. Their ability to colonise healthy animals and survive on meats1,17 and in the environment does mean human exposure is likely to be common but further studies would be useful to better establish the virulence of Arcobacter spp. for humans before recommending that laboratories routinely test for these bacteria.
Competing interests: None declared.
Author information: Owen Mandisodza, Medical Laboratory Scientist, Hawke’s Bay Hospital Laboratory, Hastings; Elizabeth Burrows, Technician, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North; Mary Nulsen, Associate Professor, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North
Acknowledgement: Owen Mandisodza received funding from the Hawke’s Bay Medical Research Foundation Inc. and the Institute of Veterinary, Animal and Biomedical Sciences, Massey University Postgraduate Student Fund.
Correspondence: Mary Nulsen, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand. Fax: +64 (0)6 3505714; email: M.F.Nulsen@massey.ac.nz
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