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 .     

Development of a combined PCR-culture technique for the rapid detection of Arcobacter spp. in chicken meat

A combined PCR-culture technique was developed to detect Arcobacter spp. in fresh chicken meat. Following a short selective enrichment of chicken samples, bacterial DNA was extracted and amplified using primers targeted at the genes encoding 16S rRNA of Arcobacter spp. The selected primers amplify a 181-bp fragment from all Arcobacter spp., whereas no PCR product is generated for other bacteria, including the closely related Campylobacter and Helicobacter species. The assay was used to screen 96 retail-purchased chicken samples for the presence of Arcobacter spp. Fifty-three percent of the samples analysed were positive for this micro-organism. The assay is simple and sensitive and reduces the amount of time required to positively detect Arcobacter spp. in poultry meat.     

Prevalence of <Emphasis Type="Italic">Arcobacter</Emphasis> species in market-weight commercial turkeys

The prevalence of Arcobacter in live market weight turkeys was determined for six Midwestern commercial flocks at three intervals. Samples (n = 987) were collected from cloaca, feathers, ceca, crop, drinkers and environmental samples on farms and from carcasses at slaughter. Initially, EMJH-P80 and CVA isolated Arcobacter from 7.1% (40 of 564) of samples, while Arcobacter enrichment broth and selective agar recovered the microbe in 4.7% of samples (23 of 489 samples). Although EMJH-P80 coupled with CVA yielded Arcobacter more frequently, the selectivity of the modified Arcobacter agar enhanced the recognition of Arcobacter colonies. A multiplex PCR was used to identify all Arcobacter species and to differentiate Arcobacter butzleri. The low prevalence of Arcobacter detected in cloacal swab (2.0%, 6 of 298 samples) and cecal contents (2.1%, 3 of 145 samples) suggests that Arcobacter infrequently colonizes the intestinal tract. Despite its low prevalence in live turkeys, Arcobacter spp. were identified in 93% of carcass swabs (139 of 150 samples). The overall prevalence of Arcobacter in drinker water decreased from 67% (31 of 46 samples) in the summer of 2003 to 24.7% (18 of 73 samples) during resampling in the spring of 2004 and was inversely related to the chlorination level.     

Survival capacity in water of Arcobacter species under different temperature conditions

Aims:  To assess the survival capacity in vitro of arcobacters in water at temperatures applied in the food industry.
Methods and Results:  Four strains of each Arcobacter species were inoculated in potable water and water with 1% organic material and stored at 4, 7, 20, 52, 56 and 60°C. Samples were taken at known time points and the numbers of bacteria were determined on Arcobacter -selective medium. All Arcobacter species remained viable for a temperature-dependent period of time, although Arcobacter butzleri displayed a significant longer survival and heat resistance . No significant intraspecies differences were detected, resulting in no definite identification of origin or strain dependency. The survival period for all species was prolonged in the presence of the organic material only for the low temperatures.
Conclusions:  The present study demonstrates that water can act as a reservoir and as a potential source of Arcobacter contamination to humans and animals.
Significance and Impact of the Study:  This study assessed for the first time the survival of all human-related Arcobacter species in water. Particularly A. butzleri showed to be the most robust species with regard to temperature which is interesting as that species is often found in human clinical specimens.     

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.     

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.     

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.     

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

AIMS: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. METHODS AND RESULTS: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 x 10(5) to 1.1 x 10(8) MPN 100 ml(-1) and in freshly irrigated soils from 9.5 x 10(2) to 2.8 x 10(4) MPN g(-1) in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. CONCLUSIONS: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.     

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.     

Complete genome sequence of Arcobacter nitrofigilis type strain (CI)

Arcobacter nitrofigilis (McClung et al. 1983) Vandamme et al. 1991 is the type species of the genus Arcobacter in the family Campylobacteraceae within the Epsilonproteobacteria. The species was first described in 1983 as Campylobacter nitrofigilis [1] after its detection as a free-living, nitrogen-fixing Campylobacter species associated with Spartina alterniflora Loisel roots [2]. It is of phylogenetic interest because of its lifestyle as a symbiotic organism in a marine environment in contrast to many other Arcobacter species which are associated with warm-blooded animals and tend to be pathogenic. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a type stain of the genus Arcobacter. The 3,192,235 bp genome with its 3,154 protein-coding and 70 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Comparison of Arcobacter isolation methods, and diversity of Arcobacter spp. in Cheshire, United Kingdom

The aims of this study were, firstly, to compare five published methods for the isolation of Arcobacter spp. from animal feces in order to determine the most sensitive and specific method. Second, we analyzed the resulting isolates by multilocus sequence typing (MLST) in order to investigate the diversity of the isolates recovered. Third, we investigated the ability to recover Arcobacter spp. from frozen fecal samples. Seventy-seven fecal samples from cattle, sheep, and badgers were subjected to five isolation methods, based on published methods for the isolation of Arcobacter and Campylobacter spp. Thirty-nine Arcobacter butzleri isolates were analyzed using a multilocus sequence typing scheme. The survival of Arcobacter spp. in frozen samples was investigated by freezing the fecal samples at -80°C for 7 days and then applying the same five isolation methods. The most sensitive and specific method used an Arcobacter-specific broth in conjunction with modified charcoal cefoperazone deoxycholate agar (mCCDA) with added antibiotics. Freezing of fecal samples led to a reduction in the recovery of Arcobacter spp. by approximately 50%. The 39 allelic profiles obtained by MLST could be divided into 11 sequence types (STs). We have identified the most sensitive and specific method for the isolation of Arcobacter spp. from animal feces and demonstrated that the freezing of fecal samples prior to isolation reduces arcobacter recovery. MLST analysis of the isolates revealed a high level of diversity.     

Arcobacter in Lake Erie beach waters: an emerging gastrointestinal pathogen linked with human-associated fecal contamination

The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. To better characterize the health risk posed by this emerging waterborne pathogen, we investigated the occurrence of Arcobacter spp. in Lake Erie beach waters. During the summer of 2010, water samples were collected 35 times from the Euclid, Villa Angela, and Headlands (East and West) beaches, located along Ohio's Lake Erie coast. After sample concentration, Arcobacter was quantified by real-time PCR targeting the Arcobacter 23S rRNA gene. Other fecal genetic markers (Bacteroides 16S rRNA gene [HuBac], Escherichia coli uidA gene, Enterococcus 23S rRNA gene, and tetracycline resistance genes) were also assessed. Arcobacter was detected frequently at all beaches, and both the occurrence and densities of Arcobacter spp. were higher at the Euclid and Villa Angela beaches (with higher levels of fecal contamination) than at the East and West Headlands beaches. The Arcobacter density in Lake Erie beach water was significantly correlated with the human-specific fecal marker HuBac according to Spearman's correlation analysis (r = 0.592; P < 0.001). Phylogenetic analysis demonstrated that most of the identified Arcobacter sequences were closely related to Arcobacter cryaerophilus, which is known to cause gastrointestinal diseases in humans. Since human-pathogenic Arcobacter spp. are linked to human-associated fecal sources, it is important to identify and manage the human-associated contamination sources for the prevention of Arcobacter-associated public health risks at Lake Erie beaches.     

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.     

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%).     

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.     

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs

Aims:  To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs.
Methods and Results:  Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property.
Conclusions:  HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants.
Significance and Impact of the Study:  Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.     

Interaction of Arcobacter spp. with human and porcine intestinal epithelial cells

Little is known about the pathogenic mechanisms or potential virulence factors of Arcobacter spp. The aim of the study described here was to obtain more insights in the pathogenicity mechanisms of Arcobacter spp. by testing their ability to adhere to, invade and induce interleukin-8 expression in human Caco-2 and porcine IPI-2I cell lines. Eight Arcobacter strains were tested. Four strains were obtained from a culture collection, and represent the four Arcobacter spp. known to be associated with animals and humans. The other four strains were field isolates from the amniotic fluid of sows and from newborn piglets. All eight Arcobacter strains were able to adhere to both cell lines, and induced interleukin-8 production as early as 2 h after a 1h incubation period. This production was still increased 6 h postinfection. Differences in the cell association of the eight strains were obvious, with A. cibarius showing the highest adhesion ability. Invasion of intestinal epithelial cells was only observed for A. cryaerophilus strains. No correlation between invasiveness or strong adhesion of the tested strains and the level of interleukin-8 induction was observed.     

Arcobacter population dynamics in pigs on farrow-to-finish farms

Healthy pigs are an important reservoir for the emerging human pathogen Arcobacter which can result in contamination of porcine carcasses and pork and the spread of arcobacters into the environment. Up to now, the excretion of arcobacters by pigs has been studied, but information about the transmission routes in fattening pigs is lacking. The present study aimed to elucidate the Arcobacter population dynamics in pigs during the fattening period on four farrow-to-finish farms. On each farm, 30 clinically healthy, 12-week-old piglets were selected. Fecal samples were collected on 10 sampling occasions until a slaughter age of 30 weeks was reached. Arcobacter spp. were isolated by a selective method and identified by multiplex PCR. The genetic diversity was examined by amplified fragment length polymorphism and enterobacterial repetitive intergenic consensus PCR. The Arcobacter presence in the fecal samples on the four farms ranged from 11.3 to 50.0%, with excretion levels of up to 10(4) CFU/g feces. The ratio in which Arcobacter species were isolated varied between the farms and over time. Characterization revealed a high degree of genotypic diversity among the isolates. Arcobacter strains persisted and spread within the finishing unit during the fattening period. The occurrence of both unique and shared genotypes in pigs in adjacent and nonadjacent pens demonstrates that transmission routes other than fecal-oral transmission occur.     

Arcobacter bivalviorum sp. nov. and Arcobacter venerupis sp. nov., new species isolated from shellfish

A group of ten Arcobacter isolates (Gram negative, slightly curved motile rods, oxidase positive) was recovered from mussels (nine) and from clams (one). These isolates could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR). The aim of this study is to establish the taxonomic position of these isolates. The 16S rRNA gene sequence similarity of mussel strain F4(T) to the type strains of all other Arcobacter species ranged from 91.1% to 94.8%. The species most similar to the clams' strain F67-11(T) were Arcobacter defluvii (CECT 7697(T), 97.1%) and Arcobacter ellisii (CECT 7837(T), 97.0%). On the basis of phylogenetic analyses with 16S rRNA, rpoB, gyrB and hsp60 genes, the mussel and clam strains formed two different, new lineages within the genus Arcobacter. These data, together with their different phenotypic characteristics and MALDI-TOF mass spectra, revealed that these strains represent two new species, for which the names Arcobacter bivalviorum (type strain F4(T)=CECT 7835(T)=LMG 26154(T)) and Arcobacter venerupis (type strain F67-11(T)=CECT 7836(T)=LMG 26156(T)) are proposed.