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| Journal of the New Zealand Medical Association,
20-April-2012, Vol 125 No 1353
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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.
Materials and MethodsAll 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).
ResultsFrom 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
DiscussionThe 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
References:
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