Nucleotide sequence of the gyrA gene of Arcobacter species and characterization of human ciprofloxacin-resistant clinical isolates

The nucleotide sequence of the gyrA gene of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter skirrowii was determined. The deduced GyrA proteins are closely related to those of Wolinella succinogenes and Helicobacter pullorum, whereas those of Campylobacter species showed less sequence identity. The phylogenetic analysis of GyrA sequences provides a result similar to 16S rRNA gene sequence phylogenetic analysis and allows the discrimination among A. butzleri species. In addition, a Thr-->Ile mutation at amino acid 85 in the quinolone resistance-determining region was associated with ciprofloxacin resistance for two A. butzleri and one A. cryaerophilus ciprofloxacin-resistant strains.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Differentiation of Arcobacter species by numerical analysis of AFLP profiles and description of a novel Arcobacter from pig abortions and turkey faeces

AIMS: To evaluate the efficacy of amplified fragment length polymorphism (AFLP)-based genetic profiling for taxonomic and epidemiological analyses of diverse Arcobacter species. METHODS AND RESULTS: Seventy-two isolates of A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis, and a previously unclassified porcine abortion strain were studied. AFLP profiling was performed using a BglII-Csp6I-based protocol previously used to characterize Campylobacter species. Duplicate profiles of 20 isolates were 93.25% similar, indicating high reproducibility. Numerical analysis of all 72 strains revealed five phenons at the 29% similarity level, four of which represented each of the known species studied. The remaining phenon was further characterized by phenotypic and 16S rDNA sequence analyses, the results of which indicated it to be a novel Arcobacter species. The genetically distinct subgroups of A. cryaerophilus were differentiated at the 39.5% similarity level. For strain typing, 62 distinct types were defined, with evidence of clonal lineages within A. butzleri, A. cryaerophilus and A. skirrowii. CONCLUSIONS: AFLP profiling is an effective means of determining taxonomic and strain relationships for arcobacters. SIGNIFICANCE AND IMPACT OF THE STUDY: First use of AFLP profiling for diverse Arcobacter species; indication of clonality in A. butzleri, A. cryaerophilus and A. skirrowii; potentially novel Arcobacter taxon identified.     

Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR

AIMS: To determine the prevalence of Arcobacter in various food, animal and water sources in Turkey and to subtype the isolated strains using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS AND RESULTS: A total of 806 samples consisting of chicken (100) and turkey meat (100); minced beef (27); rectal swabs from cattle (173), sheep (68) and dogs (62); cloacal swabs of broilers (100) and layers (100); gall bladders of cattle (50) and drinking water samples (26) were examined. A previously described membrane filtration method was used for the isolation. Isolates were identified at species level using multiplex-PCR and discriminated by ERIC-PCR for subtyping. Ninety-eight (12.1%) of the samples examined were found positive for arcobacters. Arcobacter spp. were isolated from 68%, 4%, 6.9%, 8% and 37% of chicken and turkey meats, rectal swabs and gall bladders of cattle and minced beef, respectively. No arcobacters were obtained from the rectal swabs of sheep and dogs, cloacal swabs of broilers and layers, and water samples examined. In total, 99 Arcobacter isolates were obtained. Of these isolates, 92 were identified as Arcobacter butzleri, five were Arcobacter skirrowii and two were Arcobacter cryaerophilus. Thirteen distinct DNA profiles among A. butzleri isolates were obtained by the ERIC-PCR. Of these profiles, eight were from chicken carcass, three from cattle rectal swab and two from minced beef meat isolates. Some of the isolates originated from different sources gave the same DNA profiles. All isolates of A. skirrowii and A. cryaerophilus gave different DNA profiles. CONCLUSIONS: Poultry carcasses, minced beef meat, rectal swabs and gall bladders of cattle were found to be positive for Arcobacter spp. A. butzleri was the predominant species isolated. In addition, large heterogeneity among the Arcobacter isolates was determined. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of the poultry carcasses and minced beef meat, rectal and gall bladder samples of cattle with arcobacters poses a risk for both human and animal infections. Detection of several different Arcobacter strains may suggest multiple sources for contamination and infection.     

Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.     

Isolation of Arcobacter spp. from a brackish environment

Arcobacter spp. were isolated from water and mussels of two brackish lakes near Messina (Italy). The isolates were phenotypically characterized on the basis of a large battery of cultural and biochemical tests. By comparison with the reference strains Arcobacter butzleri ATCC 49616 and A. cryaerophilus ATCC 43157 they may be considered Arcobacter butzleri-like bacteria. The current isolation suggests that the brackish environment may play an important role in the survival and transmission of Arcobacter spp. also by seafood cultured in the examined waters.     

Diversity and prevalence of Arcobacter spp. in broiler chickens

Ninety-nine strains of Arcobacter spp., isolated from 10 chicken carcasses purchased from a supermarket and 15 chicken carcasses collected from a poultry abattoir, were speciated using a variety of phenotypic identification methods. All were tested using API Campy test strips and the 16-test (Preston) identification scheme developed for campylobacters. Fifty strains were selected for examination using a more comprehensive probabilistic identification scheme, and the identity of representative strains confirmed by protein profiling using SDS-PAGE. All 25 carcasses yielded Arcobacter butzleri . Three supermarket and 10 abattoir carcasses also carried A. cryaerophilus , and two abattoir carcasses carried A. skirrowii . The API Campy scheme proved unsatisfactory for identifying these strains: only 20 of 99 strains were accurately identified, all of which were A. cryaerophilus , the only Arcobacter sp. included in the database. Moreover, 76 of 99 strains were misidentified. The 16-test scheme identified all the arcobacter strains as A. cryaerophilus, since neither A. butzleri nor A. skirrowii had been described when the scheme was developed. The computer-assisted probabilistic scheme succeeded in identifying all but one strain, the identity of which was clarified by the use of SDS-PAGE. To our knowledge this is the first time that arcobacters other than A. butzleri have been reported in poultry meat or any other food of animal origin. Their high prevalence in poultry products may be of significance to public health.     

Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution

We investigated the presence of Arcobacter spp. in 205 water samples of freshwater, seawater and sewage in Spain. These bacteria were present in 55.1% of the samples (113/205) and were significantly associated for the first time with bacterial indicators of fecal pollution. The dominant species in the positive samples was Arcobacter butzleri (94%) followed by Arcobacter cryaerophilus (30%) and Arcobacter skirrowii (1.8%).     

Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

Identification of Arcobacter isolates by PCR

K.M. HARMON AND I.V. WESLEY. 1996. A polymerase chain reaction (PCR) assay was developed for the identification of the three species of Arcobacter which have been recovered from clinically ill or healthy humans and/or livestock, namely Arcobacter butzleri, Arcobacter skirrowii and Arcobacter cryaerophilus . The assay utilizes primers targeted to the genes encoding 16S rRNA of Arcobacter spp. The assay reduces the amount of time required to positively identify strains of Arcobacter .     

Microbiological status of piggery effluent from 13 piggeries in the south east Queensland region of Australia

AIMS: To assist in the development of safe piggery effluent re-use guidelines by determining the level of selected pathogens and indicator organisms in the effluent ponds of 13 south-east Queensland piggeries. METHODS AND RESULTS: The numbers of thermotolerant coliforms, Campylobacter jejuni/coli, Erysipelothrix rhusiopathiae, Escherichia coli, Salmonella and rotavirus were determined in 29 samples derived from the 13 piggeries. The study demonstrated that the 13 final effluent ponds contained an average of 1.2 x 10(5) colony-forming units (CFU) 100 ml(-1) of thermotolerant coliforms and 1.03 x 10(5) CFU 100 ml(-1) of E. coli. The Campylobacter level varied from none detectable (two of 13 piggeries) to a maximum of 930 most probable number (MPN) 100 ml(-1) (two of 13 piggeries). Salmonella was detected in the final ponds of only four of the 13 piggeries and then only at a low level (highest level being 51 MPN 100 ml(-1)). No rotavirus and no Erysip. rhusiopathiae were detected. The average log10 reductions across the ponding systems to the final irrigation pond were 1.77 for thermotolerant coliforms, 1.71 for E. coli and 1.04 for Campylobacter. CONCLUSIONS: This study has provided a baseline knowledge on the levels of indicator organisms and selected pathogens in piggery effluent. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge gained in this study will assist in the development of guidelines to ensure the safe and sustainable re-use of piggery effluent.     

Detection and enumeration of Arcobacter spp. in the coastal environment of the Straits of Messina (Italy)

Seawater and plankton samples from the Straits of Messina, Italy, were analysed to confirm the occurrence of potentially pathogenic Arcobacter butzleri, A. cryaerophilus and A. skirrowii. Both classical cultural methods and molecular techniques were used as confirmative steps of the growth in enrichment broth to enumerate and differentiate these bacteria. Only A. butzleri was isolated from seawater and plankton samples and was more abundant when associated with plankton than free-living. A. cryaerophilus was occasionally detected by PCR assay from environmental samples. The PCR procedure, used in a combined method, was useful in enumerating Arcobacter spp. in marine environment.     

Specific detection of Arcobacter spp. in estuarine waters of Southern Italy by PCR and fluorescent in situ hybridization

Aim:  To evaluate the reliability of culture-independent methods in comparison with culture-dependent ones for the detection of Arcobacter spp. in estuarine waters of Southern Italy.
Methods and Results:  PCR and fluorescent in situ hybridization (FISH) procedures were used to detect arcobacters directly in water samples and after enrichment cultures. The samples totally were positive by molecular methods (PCR and FISH) but only 75% were culture positive, confirming the limitation of these latter to detect Arcobacter spp. in natural samples. Culturable arcobacters were retrieved in all times except in July, and isolated species were ascribed only to Arcobacter cryaerophilus .
Conclusions:  Culturable and nonculturable forms of Arcobacter in the estuarine environment were present. PCR assays were more sensitive than traditional culture in detecting Arcobacter butzleri and A. cryaerophilus . FISH comparatively to PCR technique may provide information about cell morphology and viability of single cells.
Significance and Impact of the Study:  Our investigation indicates the existence of an environmental reservoir of potential pathogenic arcobacters in an estuarine Italian area, which may survive under a viable but not culturable state.     

Ecology of Arcobacter species in chicken rearing and processing

AIMS: To investigate whether Arcobacter spp. colonize the poultry-rearing environment or whether they are contaminants acquired during transportation and/or from the processing plant. METHODS AND RESULTS: Samples were collected on poultry farms and in the processing plant during slaughter and dressing. Two cultural methods of detection were used. Isolates were identified to species level using a multiplex-polymerase chain reaction (m-PCR) method, either on the initial suspensions, or after enrichment, or on pure cultures of isolates. Of the 62 samples examined from poultry farms, arcobacters were found only outside the rearing sheds (in effluent sludge and stagnant water). Thirty-four samples were examined from the processing plant and 26 were positive for arcobacters. All the isolates were Arcobacter butzleri. Arcobacters were not found in any sample by direct plating nor by m-PCR on the initial suspensions, thus it was concluded that numbers were very low. CONCLUSIONS: Arcobacter spp. were not found in samples from the live birds and their immediate environment, but A. butzleri was found in effluent sludge and stagnant water outside the rearing sheds. However, A. butzleri is common in poultry abattoirs, and it appears that poultry carcasses are contaminated during processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacters are not found inside poultry-rearing sheds, but are contaminants in the processing environment.     

Assessment of the genetic diversity among arcobacters isolated from poultry products by using two PCR-based typing methods

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.     

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.     

Pet cats as carriers of Arcobacter spp. in Southern Italy

Aims:  To evaluate the presence of Arcobacter spp. in different biological samples from domestic cats in Southern Italy by using a species-specific PCR assay and thus to elucidate their potential significance as sources of human infection.
Methods and Results:  We investigated the prevalence of Arcobacter DNA in oral swabs, in peripheral blood samples and fine needle lymph node aspirate specimens from 85 cats of which 17 were clinically healthy and 68 had clinical signs of oral disease or lymphadenomegaly. Overall, molecular analysis has shown that Arcobacter -specific DNA was found in 78·8% (67 of 85) of all the cats. In the 67 Arcobacter -positive cats, 66 (77·6%) and 29 (34·1%) were found positive for Arcobacter butzleri and Arcobacter cryaerophilus, respectively. None of the examined samples gave a PCR product for Arcobacter skirrowii .
Conclusions:  This study demonstrates that pet cats commonly carry Arcobacter in the oral cavity. According to the clinical data, the Arcobacter detection results showed no significant difference between cats with oral pathology and those suffering from other different pathologies.
Significance and Impact of the Study:  Pet cats harbour Arcobacter spp. and may play a role in their dissemination in the domestic habitat. The high prevalence in a limited number of cat samples in this study may be of significance.     

The isolation and characterization of a rumen chitinolytic bacterium

J.S. DICKSON, T.R. MANKE, i.v. WESLEY AND A.L. BAETZ. 1996. Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm² tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log₁₀ cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus , which was non-culturable in broth culture, attained population densities of 10⁹ cells ml^-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm² resulted in increased cell densities.     

Prevalence of campylobacters and arcobacters in ducks at the abattoir

Ten duck carcasses, five from each of two different flocks, and four pairs of pooled duck caecal contents, each pair from a separate flock, were examined by a variety of techniques for arcobacters and campylobacters. Campylobacter coli, C. jejuni ssp. jejuni , C. upsaliensis, Arcobacter cryaerophilus and A. butzleri were isolated from duck caecal contents. Campylobacter coli, C. jejuni ssp. jejuni, A. cryaerophilus, A. butzleri and A. skirrowii were isolated from carcasses. The most effective methods for isolating these bacteria from carcasses involved selective enrichment in campylobacter enrichment broth, containing a cefoperazone, amphotericin, teicoplanin supplement, followed by plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA), or plating onto non-selective blood agar after filtration through a 0·65 μm pore size cellulose acetate filter. In contrast, recovery from caecal contents was most effective by direct plating onto mCCDA. API test strips performed poorly, failing to identify A. skirrowii or A. butzleri (which are not included in the scheme), or even many common campylobacters. The Preston biochemical characterization scheme was more helpful, though it did not distinguish between Arcobacter species. The species of most isolates of campylobacter, identified using the Preston scheme, was confirmed by the use of SDS-PAGE of whole cell proteins and this technique was also used successfully to speciate arcobacters.     

Molecular fingerprinting of Campylobacter and Arcobacter isolated from chicken and water.

The potential of a fingerprinting method based on the single-enzyme amplified fragment length polymorphism (s-AFLP) technique was evaluated for its efficacy in detecting foodborne Campylobacter and Arcobacter species. Campylobacter and Arcobacter isolates from chicken and water samples were subjected to s-AFLP and pulsed-field gel electrophoresis (PFGE) profiling. Molecular typing revealed a high degree of heterogeneity. AFLP was found to be appropriate for differentiating minimal genomic variations, which makes this technique a valuable tool for the identification of isolates. PFGE was effective in showing epidemiological relationships among closely related isolates. Either technique allowed the discrimination of A. butzleri from A. cryaerophilus and A. skirrowii. When used together, s-AFLP and PFGE can be applied to determine taxonomic and epidemiological relationships among campylobacteria.