Arcobacter species in diarrhoeal faeces from humans in New Zealand

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.1,2Three 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.8Occasionally A. butzleri9-11 and A. cryaerophilus12,13have been isolated from patients with bacteraemia but Arcobacter species have also been isolated from faecal samples from healthy humans.7,14-16Arcobacter 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.Materials and Methods 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 andAeromonas 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),19except that loading buffer was omitted, the MgCl2 concentration was increased from 1.3 to1.5 mmol L-1and 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). Results From 1380 diarrhoeal faecal samples, 16 isolates were presumptively identified as Arcobacterspp. but only 12 (0.9%) were positive by multiplex PCR. Table 1. Details of patients whose faeces yielded Arcobacter spp Isolate Age (yrs) Sex Symptoms Appearance of faeces Arcobacter spp isolated Other enteric pathogens detected 1 32 M Diarrhoea lasting 1 week Diarrhoeic butzleri None 2 32 M NR1 NR butzleri None 3 46 F Persistent diarrhoea lasting 1 week Loose butzleri None 4 53 M Persistent diarrhoea Loose butzleri Helicobacter pyloriantigen positive 5 72 M NR Semi-formed butzleri None 6 76 M Diarrhoea and vomiting Soft butzleri Aeromonas hydrophila 7 78 M ?Diarrhoea Loose butzleri None 8 2 F Diarrhoea Loose cryaerophilus None 9 31 F NR Watery cryaerophilus None 10 40 F NR Loose cryaerophilus Blastocystis hominis 11 56 F ?Diarrhoea Semi-formed cryaerophilus Helicobacter pyloriantigen positive 12 71 F ?Diarrhoea Semi-formed cryaerophilus None 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 andAeromonas 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 Isolate Arcobacterspecies Ciprofloxacin (mg/L) Sens≤11 Erythromycin (mg/L) Sens≤81 Tetracycline (mg/L) Sens≤41 Ampicillin (mg/L) Sens≤81 1 2 3 4 5 6 7 8 9 10 11 12 butzleri butzleri butzleri butzleri butzleri butzleri butzleri cryaerophilus cryaerophilus cryaerophilus cryaerophilus cryaerophilus 0.12 0.06 0.06 0.25 0.12 0.25 0.25 0.12 0.06 0.12 0.12 0.25 4 4 2 8 8 16 4 8 1 2 1 2 4 4 2 8 8 16 4 8 1 2 1 2 4 8 32 8 64 32 64 8 64 8 4 16