Arcobacter species isolated from various seafood and water sources; virulence genes, antibiotic resistance genes and molecular characterization.

Arcobacter spp. has gained clinical significance as an emerging diarrheagenic pathogen associated with water reservoirs in recent years. The complete clinical significance of Arcobacter remains rather speculative due to the virulence and antibiotic susceptibility of individual strains. This study aimed to assess the prevalence of Arcobacter spp. in fish, water, and shellfish. A total of 150 samples were collected from the Adana, Kayseri and Kahramanmaras provinces in Turkey. Arcobacter spp. was isolated from 32 (21%) of the 150 samples. The most prevalent species was A. cryaerophilus, 17 (56%), A. butzleri 13 (37%) and A. lacus 2 (6%). As a result, the ratios of the mviN, irgA, pldA, tlyA and hecA target genes were found as 17 (51%), 1 (3%), 7 (23%), 7 (23%), 1 (3%), respectively. While bla OXA-61, tetO and tetW were positive in all isolates, were found as mcr1/2/6, mcr3/7, and mcr5, genes %37.5, %25, and %34.3, respectively. Although in A. butzleri was found 10 (58%), 1 (3%), 3 (43%), 2 (28%) (mviN, irgA, pldA, and tlyA, respectively) virulence genes 7 (42%), 4 (57%), 5 (72%), 1 (3%) was found (mviN, irgA, tlyA, and hecA, respectively) virulence genes in A. cryoaerophilus. Moreover, was found for the mcr 1/2/6 7 (58%) genes, for the mcr 3/7 genes 3 (38%) in A. butzleri. In A. cryoaerophilus was found for the mcr 1/2/6 genes 5 (42%), for the mcr 3/7 genes 5 (62%), and for the mcr 5 gene 10 (100%). Thus, the current study indicated that the existence of Arcobacter spp. isolated from fish and mussel samples may pose a potential risk to public health.

Prevalence and antibacterial susceptibilities of Arcobacter spp. and Campylobacter spp. from fresh vegetables.

This study was aimed at the isolation and identification of Arcobacter spp. and Campylobacter spp. from fresh vegetables sold at district markets in the Kayseri province, and at the determination of the antibacterial susceptibility of the recovered isolates. For this purpose, a total of 175 vegetable samples, including 35 spinach, 35 lettuce, 35 parsley, 35 arugula, and 35 radish samples, were collected. While the pre-enrichment and membrane filtration techniques were used for the isolation of Arcobacter spp., the pre-enrichment and direct inoculation methods were used for the isolation of Campylobacter spp. The isolates were identified by means of phenotypic tests and the polymerase chain reaction (PCR), using genus- and species-specific primers. In addition, the susceptibilities of the isolates to amoxicillin-clavulanic acid, enrofloxacin, erythromycin, gentamicin, neomycin, streptomycin, and tetracycline were determined by the disk diffusion method. Out of the 175 vegetable samples tested, 93 (53.14%) were found to be positive for Arcobacter spp., and 119 Arcobacter spp. isolates were recovered from these 93 positive samples. All of the samples examined were found to be negative for Campylobacter spp. One hundred one (86%) and 14 (10%) of the 119 Arcobacter isolates obtained were identified as A. butzleri and A. cryaerophilus, respectively, but four isolates could not be identified at the species level by mPCR. Mixed contamination with more than one species and/or genotypes of Arcobacter was detected in 24 of the positive samples. While all of the Arcobacter isolates were susceptible to erythromycin, gentamicin, streptomycin, and tetracycline, 2 (1.68%), 2 (1.68%), and 5 (4.20%) isolates were resistant to amoxicillin/clavulanic acid, enrofloxacin, and neomycin, respectively. Consequently, the determination of a high prevalence of arcobacters and mixed contamination with more than one species and/or genotypes of arcobacters in vegetables often consumed raw by humans demonstrated that the consumption of raw vegetables may be a risk to the public health.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species.

AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg µl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.

Isolation, molecular identification and quinolone-susceptibility testing of Arcobacter spp. isolated from fresh vegetables in Spain.

Some species of the Arcobacter genus are considered emerging foodborne and waterborne enteropathogens. However, the presence of Arcobacter spp. in vegetables very little is known, because most studies have focused on foods of animal origin. On the other hand, quinolones are considered as first-line drugs for the treatment of infection by campylobacteria in human patients, but few data are currently available about the resistance levels to these antibiotics among Arcobacter species. Therefore, the aim of this study was to investigate the presence and diversity of arcobacters isolated from fresh vegetables such as lettuces, spinaches, chards and cabbages. Resistance to quinolones of the isolates was also investigated. One hundred fresh vegetables samples purchased from seven local retail markets in Valencia (Spain) during eight months were analysed. The study included 41 lettuces, 21 spinaches, 34 chards and 4 cabbages. Samples were analysed by culture and by molecular methods before and after enrichment. By culture, 17 out of 100 analysed samples were Arcobacter positive and twenty-five isolates were obtained from them. Direct detection by PCR was low, with only 4% Arcobacter spp. positive samples. This percentage increased considerably, up 20%, after 48 h enrichment. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 17 out of the 25 isolates were identified as A. butzleri and 8 as A. cryaerophilus. Only two A. butzleri isolates showed resistance to levofloxacin and ciprofloxacin. The sequencing of a fragment of the QRDR region of the gyrA gene from the quinolones-resistant isolates revealed the presence of a mutation in position 254 of this gene (C-T transition). This study is the first report about the presence of pathogenic species of Arcobacter spp. in chards and cabbages and confirms that fresh vegetables can act as transmission vehicle to humans. Moreover, the presence of A. butzleri quinolone resistant in vegetables could pose a potential public health risk.

Determination of the presence and antimicrobial resistance of Arcobacter species in broiler carcasses at different stages of slaughter line.

In this study, to investigate Arcobacter spp. contamination post-scalding and de-feathering, post-evisceration, post-chilling, and packaged products, which are the most essential contamination stages of broiler slaughter, a total of 108 samples were taken from three different broiler slaughterhouses at different times. Isolates obtained by cultural methods in 104 of 108 samples were analyzed by mPCR method to identify pathogen Arcobacter spp. Arcobacter butzleri, Arcobacter cryaerophilus, and mixed contamination of both Arcobacter species were detected in 51 samples. Of the 51 isolates, 27 (52.9%) were A. butzleri, 16 (31.4%) were A. cryaerophilus, and 8 (15.7%) were mixed contamination of A. butzleri and A. cryaerophilus, while Arcobacter skirrowii was not detected. A. butzleri and A. cryaerophilus contamination was 59.2% post-scalding and de-feathering, 43.4% post-evisceration, 44.4% and 48.1% post-chilling and in packaged products, respectively. All A. butzleri strains were found to be 100% resistant to cefoperazone and penicillin and sensitive to tetracycline. A. cryaerophilus strains were 100% resistant to cefoperazone, penicillin, and cloxacillin and susceptible to tetracycline and erythromycin. In the study, it was determined that Arcobacter spp. caused a very intense contamination (85.18%-100%) and also contamination rates of identified pathogen strains (A. butzleri and A. cryaerophilus) were very high (59.2% and 43.4%) in broiler slaughtering stages. Considering that each step in broiler slaughter could contaminate the next stage, developing a safe slaughter and minimizing the risk toward the final product, it was concluded that critical control points could not be well managed in broiler slaughterhouses, and broiler meat may pose a significant risk to public health.

Arcobacteraceae: An Exploration of Antibiotic Resistance Featuring the Latest Research Updates.

The Arcobacteraceae bacterial family includes species isolated from animals and related food products. Moreover, these species have been found in other ecological niches, including water. Some species, particularly Arcobacter butzleri and Arcobacter cryaerophilus, have been isolated from human clinical cases and linked to gastrointestinal symptoms. The presence of antibiotic-resistant strains is a concern for public health, considering the possible zoonoses and foodborne infections caused by contaminated food containing bacteria resistant to antibiotic treatments. This review aims to highlight the importance of antibiotic resistance in Arcobacter spp. isolates from several sources, including information about antibiotic classes to which this bacterium has shown resistance. Arcobacter spp. demonstrated a wide spectrum of antibiotic resistance, including several antibiotic resistance genes. Antibiotic resistance genomic traits include efflux pumps and mutations in antibiotic target proteins. The literature shows a high proportion of Arcobacter spp. that are multidrug-resistant. However, studies in the literature have primarily focused on the evaluation of antibiotic resistance in A. butzleri and A. cryaerophilus, as these species are frequently isolated from various sources. These aspects underline the necessity of studies focused on several Arcobacter species that could potentially be isolated from several sources.

Microbiological and Toxicological Investigations on Bivalve Molluscs Farmed in Sicily.

Bivalves can concentrate biological and chemical pollutants, causing foodborne outbreaks whose occurrence is increasing, due to climatic and anthropic factors that are difficult to reverse, hence the need for improved surveillance. This study aimed to evaluate the hygienic qualities of bivalves sampled along the production and distribution chain in Sicily and collect useful data for consumer safety. Bacteriological and molecular analyses were performed on 254 samples of bivalves for the detection of enteropathogenic Vibrio, Arcobacter spp., Aeromonas spp., Salmonella spp., and beta-glucuronidase-positive Escherichia coli. A total of 96 out of 254 samples, collected in the production areas, were processed for algal biotoxins and heavy metals detection. Bacterial and algal contaminations were also assessed for 21 samples of water from aquaculture implants. Vibrio spp., Arcobacter spp., Aeromonas hydrophila, Salmonella spp., and Escherichia coli were detected in 106/254, 79/254, 12/254, 16/254, and 95/254 molluscs, respectively. A total of 10/96 bivalves tested positive for algal biotoxins, and metals were under the legal limit. V. alginolyticus, A. butzleri, and E. coli were detected in 5, 3, and 3 water samples, respectively. Alexandrium minutum, Dinophysis acuminata, Lingulodinium polyedra, and Pseudonitzschia spp. were detected in water samples collected with the biotoxin-containing molluscs. Traces of yessotoxins were detected in molluscs from water samples containing the corresponding producing algae. Despite the strict regulation by the European Commission over shellfish supply chain monitoring, our analyses highlighted the need for efficiency improvement.

Occurrence and Antibiotic Resistance of Arcobacter Species Isolates from Poultry in Tunisia.

ABSTRACT: Arcobacter is considered an emergent foodborne enteropathogen. Despite the high prevalence of this genus in poultry, the occurrence of Arcobacter spp. contamination in Tunisia remains unclear. The objectives of this study were (i) to isolate Arcobacter species (A. butzleri and A. cryaerophilus) by the culture method from different species of raw poultry meat, (ii) to verify the isolates by multiplex PCR (m-PCR) assay and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and (iii) to determine the antibiotic resistance profiles of the isolates. A total of 250 poultry product samples (149 chicken and 101 turkey) were collected from various supermarkets in Sfax. The samples consisted of breasts, wings, legs, and neck skins. The overall isolation frequency of Arcobacter spp. was 10.4%. Arcobacter spp. were found in 13.42% of the chicken samples and in 5.49% of the turkey samples. All the acquired isolates were subject to detailed confirmation with subsequent species classification using m-PCR and MALDI-TOF MS. A. butzleri was found in 22 samples (84.61%) and A. cryaerophilus in 4 samples (15.38%). Thus, m-PCR and MALDI-TOF MS were able to detect A. butzleri significantly better than the conventional method (χ2 = 49.1 and P < 0.001). Arcobacter was isolated from poultry in every season, at contamination levels of 30.76, 23.07, 19.23, and 26.92% in summer, spring, autumn, and winter, respectively. The disk diffusion method was used to determine the susceptibility of Arcobacter isolates to six antimicrobial drugs. All A. butzleri isolates (n = 24) were significantly resistant to erythromycin (P = 0.0015), ampicillin (P = 0.001), and ciprofloxacin (P = 0.05). All tested A. cryaerophilus strains (n = 4) were susceptible to ampicillin, gentamicin, and amoxicillin-clavulanic acid. Multidrug resistance was observed in 83% of the Arcobacter spp. isolates. Our study detected Arcobacter spp. in Tunisian poultry; because of their multidrug resistance, these species may constitute a public health problem.

Arcobacter spp. in raw milk from vending machines in Piedmont and occurrence of virulence genes in isolates.

Arcobacter spp. has been recognized as an emerging foodborne pathogen and a hazard to human health. In the dairy chain, it has been isolated from different sources, nevertheless data on Arcobacter occurrence in raw milk provided by vending machines are few. This study aimed to identify potentially pathogenic Arcobacter spp. in raw milk intended for human consumption sold through vending machines located in Piedmont. In an 8-month period, 37 raw milk samples were collected from 24 dairy farms: 12 (32,4%) were collected directly in farm from bulk tank milk and 25 (67,6%) from vending machines. Eight (21,6%) out of the 37 milk samples and 7 (29,2%) out of the 24 dairy farms were positive for Arcobacter spp. by culture examination. Four (16%) out of the 25 samples from vending machines and 4 (33,3%) out of the 12 samples from bulk tank milk were positive. All 8 isolates were identified as A. butzleri both by MALDI-TOF MS and multiplex end-point PCR. According to the detection of virulence genes, a total of four Patho-types were highlighted: 5 isolates in P-type 1 and only one isolate for each of the P-types 2-3-4. A. butzleri isolates carrying encoding virulence factors genes were isolated from raw milk intended for human consumption: these findings strengthen the compulsory consumption after boiling as required by current legislation and suggest the need of enlarging the analytical investigations to other microorganisms not yet included in the food safety criteria.

Detection of Arcobacter spp. in food products collected from Sicilia region: A preliminary study.

The aim of the study was to evaluate the occurrence of Arcobacter spp. in food samples collected from Sicilia region. A total of 91 food products of animal origin (41 meat, 17 fresh milk, 18 shellfish) and 15 samples of fresh vegetables, were examined by cultural method and confirmed by biochemical analysis and PCR methods. The detection of Arcobacter spp. was performed, after selective enrichment, on two selective agar plates: Arcobacter agar and mCCD (modified charcoal cefoperazone deoxycholate) agar supplemented with CAT (Cefoperazone, Amphotericin B and Teicoplanin). Arcobacter species were isolated using the membrane filtration technique. In 13 (14.3%) out of the 91 tested samples, the presence of Arcobacter spp. was found: the isolates were confirmed by multiplex PCR and identified as belonging to the species A. butzleri and A. cryaerophilus. The highest prevalence rate was observed in chicken meat (8.8%) followed by shellfish (3.3%). Negative results have been obtained for raw milks and vegetables samples. The preliminary study highlights the importance of this emerging pathogen and the need for further studies on its prevalence and distribution in different types of food for human consumption.

Development of a Quantitative PCR Assay for Arcobacter spp. and its Application to Environmental Water Samples.

Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction-1 and sometimes detected as few as 3 copies reaction-1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3-9.1 log copies 100 mL-1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.

Susceptibility to 18 drugs and multidrug resistance of Arcobacter isolates from different sources within the Czech Republic.

OBJECTIVES: Arcobacter spp. are considered to be potential foodborne pathogens, and consumption of contaminated food containing these bacteria could endanger human and animal health. Arcobacter butzleri and Arcobacter cryaerophilus are the species most frequently isolated from food of animal origin and from other samples. The aim of this study was to evaluate the susceptibility of arcobacters isolated in the Czech Republic. No information about antibiotic susceptibility and multidrug resistance of arcobacters isolated in the Czech Republic is available in the literature before now. METHODS: The antimicrobial resistance of A. butzleri (n=80) and A. cryaerophilus (n=20) isolated from meat of animal origin, water sources and clinical samples was examined by the disk diffusion method. RESULTS: Arcobacters were resistant to one or more antimicrobial agents in 99% (99/100) of tested isolates. Most of the Arcobacter isolates were resistant to ß-lactam antibiotics, i.e. ampicillin (81.0%), amoxicillin/clavulanic acid (28.0%), cefalotin (73.0%) and aztreonam (93.0%). Arcobacters were also frequently resistant to lincosamides, i.e. clindamycin (98.0%). Of the aminoglycosides, amikacin, gentamicin and tobramycin were evaluated to be the most effective antibiotics among those tested against arcobacters. CONCLUSIONS: These results demonstrate substantial resistance in Arcobacter isolates to 18 antimicrobial agents commonly used in medical and veterinary medicine. Multidrug resistance was found in 93.8% (75/80) of A. butzleri isolates and 70.0% (14/20) of A. cryaerophilus isolates.

Prevalence of Putative Virulence Genes in Campylobacter and Arcobacter Species Isolated from Poultry and Poultry By-Products in Tunisia.

Campylobacter and Arcobacter spp. are common causes of gastroenteritis in humans; these infections are commonly due to undercooked poultry. However, their virulence mechanism is still poorly understood. The aim of this study was to evaluate the presence of genotypic virulence markers in Campylobacter and Arcobacter species using PCR. The prevalence of virulence and cytolethal distending toxin (CDT) genes was estimated in 71 Campylobacteraceae isolates. PCR was used to detect the presence of virulence genes (iam, cadF, virB1, flaA, cdtA, cdtB, and cdtC) using specific primers for a total of 45 Campylobacter isolates, including 37 C. jejuni and 8 C. coli. All the Campylobacter isolates were positive for the cadF gene. The plasmid gene virB11 was not detected in any strain. The invasion associated marker was not detected in C. jejuni. Lower detection rates were observed for flaA, cdtA, cdtB, and cdtC. The presence of nine putative Arcobacter virulence genes (cadF, ciaB, cj1349, mviN, pldA, tlyA, irgA, hecA, and hecB) was checked in a set of 22 Arcobacter butzleri and 4 Arcobacter cryaerophilus isolates. The pldA and mviN genes were predominant (88.64%). Lower detection rates were observed for tlyA (84.76%), ciaB (84.61%), cadF and cj1349 (76.92%), IrgA and hecA (61.53%), and hecB (57.69%). The findings revealed that a majority of the Campylobacteraceae strains have these putative virulence genes that may lead to pathogenic effects in humans.

Prevalence and antimicrobial susceptibility of Arcobacter species in cow milk, water buffalo milk and fresh village cheese.

In this study, the presence of Arcobacter spp. was examined in cow milk (n=50), water buffalo (WB) milk (n=50) and fresh village cheese (n=50) samples. The 16S rDNA-RFLP method was used for the identification of Arcobacter spp. The disc diffusion method was used to investigate the susceptibility of all strains identified to 18 different antimicrobial substances. The most commonly isolated Arcobacter species were found to be Arcobacter butzleri (38.89%), Arcobacter cryaerophilus (22.23%) and Arcobacter skirrowii (11.12%) in cow milk; A. cryaerophilus (33.33%), Arcobacter cibarius (20.83%) and A. butzleri (12.50%) in WB milk; and A. skirrowii (28.57%), A. butzleri (21.43%) and A. cryaerophilus (14.29%) in fresh village cheese. This is the first study to identify the presence of Arcobacter nitrofigilis, Arcobacter cloacae, Arcobacter halophilus, Arcobacter bivalviorum and A. cibarius species in analyzed samples. It was found that all of the A. cryaerophilus (n:16) isolates were resistant to cefoperazone, cloxacillin and penicillin G; all of the A. skirrowii (n:12) and A. butzleri (n:10) isolates were resistant to cefoperazone, tetracycline, ampicillin, erythromycin, cloxacillin and penicillin G. It was concluded that cow milk, WB milk and fresh village cheese samples are an important source of Arcobacter species and pose a risk to public health.