Characterization of Arcobacter spp. Isolated from human diarrheal, non-diarrheal and food samples in Thailand.

Arcobacter butzleri is an emerging zoonotic food-borne and water-borne pathogen that can cause diarrhea in humans. The global prevalence of A. butzleri infection is underestimated, and little is known about their phenotypic and genotypic characterization. The aim of this study was to determine antimicrobial susceptibility (AST) profiles, detect related virulence genes, and classify sequence type (ST) of A. butzleri isolates obtained from human stool and food samples. A total of 84 A. butzleri isolates were obtained from human diarrheal (n = 25), non-diarrheal (n = 24) stool, and food (n = 35) samples in Thailand. They were evaluated for phenotypic identification by conventional microbiological procedures and AST by Kirby-Bauer disc diffusion method as well as virulence genes detection. Representative isolates from each origin were selected based on the presence of virulence genes and AST profiles to analyze genetic diversity by multilocus sequence typing (MLST). All isolates showed resistance to nalidixic acid 40.5% (34/84), ciprofloxacin 11.9% (10/84), azithromycin 8.3% (7/84), and erythromycin 3.6% (3/84). Regarding the ten virulence genes detected, cj1349, mviN and pldA had the highest prevalence 100% (84/84), followed by tlyA 98.8% (83/84), cadF 97.6% (82/84), ciaB 71.4% (60/84), hecA and hecB 22.6% (19/84), iroE 15.5% (13/84) and irgA 10.7% (9/84), respectively. Three virulence genes were present among A. butzleri isolates of human diarrheal stool and food samples, with a significant difference observed among isolates; hecB [36% (9/25) and 8.6% (3/35)], hecA [36% (9/25) and 5.7% (2/35)], and irgA [24% (6/25) and 2.9% (1/35)] (p < 0.05), respectively. The hecA and hecB virulence genes functions are related to the mechanism of hemolysis, while irgA supports a bacterial nutritional requirement. MLST analysis of 26 A. butzleri isolates revealed that 16 novel STs exhibited high genetic diversity. The results of this study is useful for understanding potentially pathogenic and antimicrobial-resistant A. butzleri in Thailand. The pathogenic virulence markers hecB, hecA, and irgA have the potential to be developed for rapid diagnostic detection in human diarrheal stool. No significant relationships among STs and sources of origin were observed. Little is known about A. butzleri, the mechanism of action of these virulence genes, is a topic that needs further investigation.

Arcobacter butzleri: Up-to-date taxonomy, ecology, and pathogenicity of an emerging pathogen.

Arcobacter butzleri, recently emended to the Aliarcobacter butzleri comb. nov., is an emerging pathogen causing enteritis, severe diarrhea, septicaemia, and bacteraemia in humans and enteritis, stillbirth, and abortion in animals. Since its recognition as emerging pathogen on 2002, advancements have been made in elucidating its pathogenicity and epidemiology, also thanks to advent of genomics, which, moreover, contributed in emending its taxonomy. In this review, we provide an overview of the up-to-date taxonomy, ecology, and pathogenicity of this emerging pathogen. Moreover, the implication of A. butzleri in the safety of foods is pinpointed, and culture-dependent and independent detection, identification, and typing methods as well as strategies to control and prevent the survival and growth of this pathogen are provided.

Arcobacter butzleri, un agente zoonótico en leche bovina / Arcobacter butzleri, a zoonotic agent in bovine milk / Arcobacter butzleri, um agente zoonótico em leite Bovino

Para la búsqueda de especies de Arcobacter fueron estudiadas 50 muestras de leche bovina obtenidas de los centros de acopio de la empresa láctea Ecolac, de las provincias de Loja y Zamora Chinchipe, Ecuador, y se aisló un total de ocho cepas de Arcobacter butzleri (16%). Esta frecuencia de aislamiento concuerda y está dentro de los rangos descriptos en la literatura. Todas las cepas aisladas fueron sensibles a gentamicina. Se encontró alta frecuencia de resistencia a tetraciclina (6/8 cepas) y a ciprofloxacina (4/8 cepas). Se verificó la ocurrencia de multirresistencia en tres de las ocho cepas aisladas.

A total of 50 samples of bovine milk obtained from bulk tanks milk of the collection centers belonging to the company ECOLAC, of the provinces of Loja and Zamora Chinchipe, Ecuador, were studied for Arcobacter species diagnosis, being isolated 8 strains of Arcobacter butzleri (16%). This frequency of isolation agrees and falls within the ranges described in the literature. All the isolated strains were susceptible to gentamicin. High resistance levels to tetracycline and ciprofloxacin were found with 6/8 and 4/8 resistant strains respectively. Multi-resistance was found in three of the eight isolated strains.

Foram estudadas, para a pesquisa de espécies de Arcobacter, 50 amostras de leite bovino, obtidas dos centros de coleta da empresa de laticínios ECOLAC, das províncias de Loja e Zamora Chinchipe, Equador, sendo isoladas em total 8 cepas de Arcobacter butzleri (16%). Esta frequência de isolamento concorda e está dentro dos níveis descritos na literatura. Todas as cepas isoladas foram sensíveis à gentamicina. Foi encontrada alta frequência de resistência à tetraciclina (6/8 cepas) e à ciprofloxacina (4/8 cepas), sendo verificada a ocorrência de multirresistência em três das oito cepas isoladas.

Occurrence, virulence gene and antimicrobial susceptibility profiles of Arcobacter sp. isolated from catla (Catla catla) in India.

In the present study, a total of 100 catla (Catla catla-major South Asian carp, local name botcha) collected from local fish markets and aquaculture ponds were subjected for isolation and characterization of Arcobacter sp. In all, 21 Arcobacter sp. were isolated, of which 18 (85·7%) were Arcobacter butzleri and three (14%) were A. cryoaerophilus as identified by multiplex PCR. All 18 A. butzleri isolates were positive for mviN, ciaB and tlyA virulence genes, three of A. cryoaerophilus isolates carried mviN gene and none of the isolates were positive for cadF, irgA, cj1349, hecA and hecB genes. All isolates (n = 21) were resistant to penicillin (100%). Meanwhile, 71·43, 23·81, 23·81, 14·29 and 9·52% of the isolates showed resistance towards vancomycin, nalidixic acid, erythromycin, cefixime and kanamycin, respectively. Multidrug resistance was observed in 23·81% of the Arcobacter sp. isolates and none of the isolates were positive for any of the extended spectrum beta-lactamases either by phenotypic or by molecular identification genes (blaOXA , blaSHV , blaTEM , blaCTX-M1 , blaCTX-M2 and blaCTX-M9 groups). The results emphasize the need to implement specific control procedures to reduce the use of antibiotics in aquaculture particularly the ones which are very important in human medicine. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter species are emerging food- and water-borne human pathogens. In this study, Arcobacter butzleri was predominant in fish compared to A. cryoaerohilus and A. skirrowii. Higher incidence of arcobacters in fish market samples suggests cross contamination and unhygienic handling of fish in markets. Virulence genes profile and antibiotics resistance of the Arcobacter sp. isolated in current study indicate pathogenic potential of Arcobacter sp. to humans. Occurrence of multidrug-resistant Arcobacter sp. in fish is a major concern in food safety. To our knowledge, this is the first report of Arcobacter sp. from freshwater fish, catla (Catla catla) in India.

Virulence and antibiotic resistance plasticity of Arcobacter butzleri: Insights on the genomic diversity of an emerging human pathogen.

Arcobacter butzleri is a foodborne emerging human pathogen, frequently displaying a multidrug resistant character. Still, the lack of comprehensive genome-scale comparative analysis has limited our knowledge on A. butzleri diversification and pathogenicity. Here, we performed a deep genome analysis of A. butzleri focused on decoding its core- and pan-genome diversity and specific genetic traits underlying its pathogenic potential and diverse ecology. A. butzleri (genome size 2.07-2.58 Mbp) revealed a large open pan-genome with 7474 genes (about 50% being singletons) and a small but diverse core-genome with 1165 genes. It presents a plastic virulome (including newly identified determinants), marked by the differential presence of multiple adaptation-related virulence factors, such as the urease cluster ureD(AB)CEFG (phenotypically confirmed), the hypervariable hemagglutinin-encoding hecA, a type I secretion system (T1SS) harboring another agglutinin and a novel VirB/D4 T4SS likely linked to interbacterial competition and cytotoxicity. In addition, A. butzleri harbors a large repertoire of efflux pumps (EPs) and other antibiotic resistant determinants. We unprecedentedly describe a genetic mechanism of A. butzleri macrolides resistance, (inactivation of a TetR repressor likely regulating an EP). Fluoroquinolones resistance correlated with Thr-85-Ile in GyrA and ampicillin resistance was linked to an OXA-15-like ß-lactamase. Remarkably, by decoding the polymorphism pattern of the main antigen PorA, we show that A. butzleri is able to exchange porA as a whole and/or hypervariable epitope-encoding regions separately, leading to a multitude of chimeric PorA presentations that can impact pathogen-host interaction during infection. Ultimately, our unprecedented screening of short sequence repeats indicates that phase variation likely modulates A. butzleri key adaptive functions. In summary, this study constitutes a turning point on A. butzleri comparative genomics revealing that this human gastrointestinal pathogen is equipped with vast and diverse virulence and antibiotic resistance arsenals that open a multitude of phenotypic fingerprints for environmental/host adaptation and pathogenicity.

Campylobacter and Arcobacter species in food-producing animals: prevalence at primary production and during slaughter.

The Campylobacter and Arcobacter genera encompass closely related species that are ubiquitous in nature and are harboured in the gastrointestinal tract of many animals, including food-producing animals (cattle, sheep, pigs and poultry). In humans Campylobacter spp. is the cause of most of the gastroenteritis cases worldwide and in more severe cases the infection can result in Guillian Barré syndrome. Similarly, Arcobacter species can cause gastroenteritis as well as bacteraemia. Infections in humans can be induced by the consumption of contaminated vegetables, meat, milk and water. However, food originating from animals, especially meat, has been recognised as a source of infection, in fact, poultry meat and meat products have been globally reported as the main source of infection. It is clear that food-producing animals are important reservoirs for Campylobacter and Arcobacter species, which implies successful colonisation of the gastrointestinal tract at primary production and contamination during the slaughter process. During slaughter the evisceration step has been recognised as the most likely point of contamination, as accidental spillage of intestinal fluid and rapture of gastrointestinal tract can occur. Therefore, improper hygienic practices can ultimately allow for the contamination of finished/retail products intended for human consumption. This literature review will seek to explore the infection of food-producing animals with Campylobacter and Arcobacter species at primary production and contamination during the slaughter of food-producing animals.

Characteristics and exposure risks of potential pathogens and toxic metal(loid)s in aerosols from wastewater treatment plants.

Aerosols from wastewater treatment plants (WWTPs) are considered to be potentially hazardous to on-site employees and surrounding residents. However, their harmful components and their effects remain poorly understood. In this study, the characteristics, responsible factors, sources and exposure risks of potential pathogens and toxic metal(loid)s in aerosols from four WWTPs were investigated. There were 21 potential pathogens and 15 toxic metal(loid)s detected in the aerosols. Arcobacter and Fe were the dominant taxa responsible for the dissimilarity of the potential pathogen population and toxic metal(loid) composition between the aerosols and the wastewater/sludge, respectively. Both meteorological factors and sources affected pathogen and toxic metal(loid) composition. The potential pathogens and toxic metal(loid)s in indoor aerosols mainly originated from wastewater/sludge, while those in outdoor aerosols originated from wastewater/sludge and ambient air. The highest respirable fraction (<3.30 µm) concentrations and proportions were detected at the aeration units. Non-carcinogenic and carcinogenic risks of toxic metal(loid)s for both adults and children were found within and/or around WWTPs, and non-carcinogenic risks of bacteria for children were found at downwind, suggesting the need for active safeguard procedures, such as that employees wear masks and work clothes, covering the main emission sites, and collecting and destroying of aerosols.

Transcriptional analysis of flagellar and putative virulence genes of Arcobacter butzleri as an endocytobiont of Acanthamoeba castellanii.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Novel virulence, antibiotic resistance and toxin gene-specific PCR-based assays for rapid pathogenicity assessment of Arcobacter faecis and Arcobacter lanthieri.

BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.

Occurrence of virulence-associated genes in Arcobacter butzleri and Arcobacter cryaerophilus isolates from foodstuff, water, and clinical samples within the Czech Republic.

Bacteria of the Arcobacter (A.) genus, originating mainly from food and water, are dreaded germs for humans as well as animals. However, the virulence of these bacteria has not been fully elucidated yet. This study looked at the occurrence of eight virulence-associated factors (ciaB, cj1349, pldA, irgA, hecA, tlyA, mviN, hecB) in a total of 80 isolates of Arcobacter butzleri and 22 isolates of A. cryaerophilus. The isolates were derived from food, water, and clinical samples. A polymerase chain reaction using specific primers was used to detect these virulence-associated genes. The presence of all genes in the isolates of A. butzleri (98.8% ciaB, 95.0% cj1349, 98.8% pldA, 22.5% irgA, 31.3% hecA, 95.0% tlyA, 97.5% mviN, 38.8% hecB) and A. cryaerophilus (95.5% ciaB, 0.0% cj1349, 9.1% pldA, 0.0% irgA, 0.0% hecA, 31.8% tlyA, 90.9% mviN, 0.0% hecB) was monitored. Among the tested isolates, there were 13 isolates (12.7%) of A. butzleri, in which the presence of all eight virulence-associated genes was recorded in the genome. In contrast, in one A. cryaerophilus strain, none of the observed genes were detected. The presence of ciaB and mviN genes was significantly more frequent in A. cryaerophilus isolates than other genes (P < 0.05). In general, more virulence-associated genes have been detected in A. butzleri isolates compared to A. cryaerophilus. The most common gene combination (ciaB, cj1349, pldA, tlyA, mviN) was detected in case of 39 isolates. In 50.0% of A. butzleri isolates derived from clinical samples, all eight virulence-associated genes were significantly more frequently detected (P < 0.05). The tlyA gene occurred significantly more frequent in A. butzleri isolates from meat and water samples and irgA and hecB genes in clinical samples. Therefore, our study provides information about occurrence of virulence-associated genes in genome of Arcobacter isolates. These findings could be hazardous to human health, because the presence of virulence-associated genes is the assumption for potential dangerousness of these bacteria. Our results indicate high incidence of virulence-associated genes in Arcobacter genomes and hence potentially pathogenic properties of the studied strains.

Prevalence of pathogenic Arcobacter species in South Korea: Comparison of two protocols for isolating the bacteria from foods and examination of nine putative virulence genes.

Contamination of foodstuffs by potentially enteropathogenic Arcobacter spp. is becoming a concern worldwide. However, few studies have examined virulence-associated genes in isolates of Arcobacter spp. from food. Here, we investigated the prevalence of three pathogenic Arcobacter species, A. butzleri, A. cryaerophilus, and A. skirrowii, in chicken, pork, and leafy green vegetables (n = 323) in South Korea. Samples were examined using two different protocols selected from a literature review: Acrobacter selective broth (ASB) II + Arcobacter selective medium (ASM) II (protocol A), and ASB II + modified charcoal cefoperazone deoxycholate agar supplemented with CAT (protocol B). Overall, Arcobacter spp. were detected in 45.8% of food samples, and the recovery rate of protocol B (37.8%) was significantly higher than that of protocol A (30.7%) (p < 0.05). Refrigerated chicken gizzard samples showed the highest detection rate (100%), followed by refrigerated chicken wing (79.5%), intestine (77.3%), neck skin (63.3%), pork (55.6%), frozen chicken legs (5.0%), and leafy green vegetables (4.4%) (p < 0.05). All isolates from chicken and leafy green vegetables were identified as A. butzleri, whereas A. cryaerophilus and A. skirrowii were mainly detected in pork. Most samples (95.8%) harbored more than one of nine putative virulence factors (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, and tlyA), and 91.3% harbored more than two. Isolates harboring all nine putative virulence genes were obtained from 1.9% of samples: five pork and one chicken. This study provides comprehensive and de facto evidence regarding prevalence of an emerging pathogen, Arcobacter spp., in various foods, along with their virulence potential. The results justify further research with respect to their role in food safety.

Filling the gaps in clinical proteomics: a do-it-yourself guide for the identification of the emerging pathogen Arcobacter by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Arcobacters are considered emerging gastrointestinal pathogens. Rapid, reliable and species-specific identification of these bacteria is important. Biochemical tests commonly yield negative or variable results. Molecular methods prove more reliable but are time consuming and lack specificity. Matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a fast, cheap and robust technique that has revolutionized genus and species identification in clinical microbiology. The performance of an in vitro diagnostic (RUO) spectral database of MALDI-TOF MS for the identification of human clinically relevant Arcobacter isolates was validated and compared to an in house created Reference Spectral database (RS) containing a representative set of deposited Arcobacter strains of zoonotic interest. A challenge panel of clinical, human and veterinary, unique Campylobacteraceae strains was used to test accuracy. Using direct colony transfer, sensitivity with RS was significantly better than with RUO for A. butzleri and A. cryaerophilus identification (100% and 92% versus 74% and 16%). For A. skirrowii, sensitivity remained low (21% versus 0%). Reanalysis using formic acid overlay (on-target extraction) augmented sensitivity for the latter species to 64%. Specificity of RS database remained excellent without any misidentifications of human clinical strains including Campylobacter fetus and C. jejuni/coli. The use of an enriched database for MALDI-TOF MS identification of Arcobacter spp. of human interest produced high-confidence identifications to species level resulting in a significantly improved sensitivity with conservation of excellent specificity. Misidentifications, which can have therapeutic and public health consequences, were not encountered.

First isolation report of Arcobacter cryaerophilus from a human diarrhea sample in Costa Rica.

Arcobacter cryaerophilus is an emerging enteropathogen and potential zoonotic agent transmitted by food and water. In Costa Rica, this bacterium has not been associated with cases of human gastroenteritis, even though it has been isolated from farm animals, especially poultry. This paper reports the first isolation of A. cryaerophilus from a human case of bloody watery diarrhea and the virulence genes associated with this isolate.

Virulence genotypes and antimicrobial susceptibility patterns of Arcobacter butzleri isolated from seafood and its environment.

Arcobacter butzleri is an emerging pathogen isolated from animals, food and the environment. In this study, 147 A. butzleri isolated from seafood and the coastal environment were tested for the presence of ten putative virulence genes (cadF, cj1349, ciaB, mviN, pldA, tlyA, hecA, hecB, irgA, iroE) and antimicrobial susceptibilities. Majority of the isolates harbored mviN (100%), cj1349 (97.2%), ciaB (95.9%), tlyA (91.8%), pldA (91.1%) and cadF (89.7%). Lower detection rates were observed for hecA (10.8%), hecB (19%), iroE (12.9%) and irgA (17.6%). Three A. butzleri isolates harbored all ten virulence genes. The occurrence of cj1349, ciaB, pldA, tlyA and hecA genes was significantly different (P≤0.05) among the isolates from different sources. All (100%) A. butzleri isolates were resistant to vancomycin, cephalothin, cefoxitin and sulphamethizole and susceptible to polymyxin-B, kanamycin, streptomycin, gentamicin, tetracycline and imipenem. Resistance to clinically important antibiotics such as cefotaxime (99.3%), ceftazidime (87.7%), nalidixic acid (70.7%), ampicillin (72.1%), ertapenem and amoxicillin-clavulanic acid (41.9%) was observed in A. butzleri from the environment. The isolates were highly susceptible to norfloxacin (97.9%) and colistin (97.2%), followed by ciprofloxacin (88.4%), meropenem (74.8%), chloramphenicol (72.7%) and erythromycin (69.3%). A. butzleri from different sources were not significantly different with respect to their antimicrobial susceptibility patterns. Multidrug resistance was observed in 66 (81.4%) isolates from fish, 29 (72.5%) isolates from shellfish and 17 (65.3%) isolates from coastal water. A. butzleri harboring virulence genes and resistance to multiple antibiotics found in seafood could be a potential health risk to seafood handlers and consumers. Continuous monitoring of seafood for potentially pathogenic A. butzleri is important to understand the evolution of antibiotic resistance in this emerging food pathogen and to determine the antimicrobial therapy regimen in the event of food-borne A. butzleri infections.

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.

Arcobacter: an emerging food-borne zoonotic pathogen, its public health concerns and advances in diagnosis and control - a comprehensive review.

Arcobacter has emerged as an important food-borne zoonotic pathogen, causing sometimes serious infections in humans and animals. Newer species of Arcobacter are being incessantly emerging (presently 25 species have been identified) with novel information on the evolutionary mechanisms and genetic diversity among different Arcobacter species. These have been reported from chickens, domestic animals (cattle, pigs, sheep, horses, dogs), reptiles (lizards, snakes and chelonians), meat (poultry, pork, goat, lamb, beef, rabbit), vegetables and from humans in different countries. Arcobacters are implicated as causative agents of diarrhea, mastitis and abortion in animals, while causing bacteremia, endocarditis, peritonitis, gastroenteritis and diarrhea in humans. Three species including A. butzleri, A. cryaerophilus and A. skirrowii are predominantly associated with clinical conditions. Arcobacters are primarily transmitted through contaminated food and water sources. Identification of Arcobacter by biochemical tests is difficult and isolation remains the gold standard method. Current diagnostic advances have provided various molecular methods for efficient detection and differentiation of the Arcobacters at genus and species level. To overcome the emerging antibiotic resistance problem there is an essential need to explore the potential of novel and alternative therapies. Strengthening of the diagnostic aspects is also suggested as in most cases Arcobacters goes unnoticed and hence the exact epidemiological status remains uncertain. This review updates the current knowledge and many aspects of this important food-borne pathogen, namely etiology, evolution and emergence, genetic diversity, epidemiology, the disease in animals and humans, public health concerns, and advances in its diagnosis, prevention and control.

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.

Occurrence of putative virulence genes on Arcobacter butzleri isolated from three different environmental sites throughout the dairy chain.

AIMS: This comparative study investigated the occurrence of cadF, cj1349, ciaB, pldA, tlyA, hecA, hecB, mviN, irgA and IroE genes in 212 Arcobacter butzleri isolated from three different environmental sites linked to the dairy chain (farms, industrial and artisanal dairy plants) located in three Italian regions (Lombardy, Emilia-Romagna and Calabria). METHODS AND RESULTS: According to the presence of these genes, different pathotypes (P-types) were determined. The main genes detected were ciaB, mviN, tlyA, cj1349, pldA and cadF, while the least common genes were iroE, hecA, hecB and irgA. TlyA, irgA, hecA, hecB and iroE, which were significantly more frequent in isolates recovered in industrial dairy plants. Twelve P-types were detected. The occurrence of the most frequently detected P-types (P-types 1, 2, 3 and 5) differed significantly (P < 0·001) in relation to both the environmental site and geographical area of isolation. The highest diversity in P-types was observed in industrial dairy plants and in the Calabria region. CONCLUSIONS: The results of this study show a correlation between the occurrence of putative virulence genes and virulence genotype variability depending on the environmental site and geographical origin of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides insights into the similar distribution of putative virulence genes in a dairy chain and other sources' isolates and also into a geographical distribution of some P-types. We have shown that industrial dairy plants may represent an environmental site favouring a selection of the isolates with a higher pathogenetic pattern.

The Immunopathogenic Potential of Arcobacter butzleri - Lessons from a Meta-Analysis of Murine Infection Studies.

BACKGROUND: Only limited information is available about the immunopathogenic properties of Arcobacter infection in vivo. Therefore, we performed a meta-analysis of published data in murine infection models to compare the pathogenic potential of Arcobacter butzleri with Campylobacter jejuni and commensal Escherichia coli as pathogenic and harmless reference bacteria, respectively. METHODOLOGY / PRINCIPAL FINDINGS: Gnotobiotic IL-10-/- mice generated by broad-spectrum antibiotic compounds were perorally infected with A. butzleri (strains CCUG 30485 or C1), C. jejuni (strain 81-176) or a commensal intestinal E. coli strain. Either strain stably colonized the murine intestines upon infection. At day 6 postinfection (p.i.), C. jejuni infected mice only displayed severe clinical sequelae such as wasting bloody diarrhea. Gross disease was accompanied by increased numbers of colonic apoptotic cells and distinct immune cell populations including macrophages and monocytes, T and B cells as well as regulatory T cells upon pathogenic infection. Whereas A. butzleri and E. coli infected mice were clinically unaffected, respective colonic immune cell numbers increased in the former, but not in the latter, and more distinctly upon A. butzleri strain CCUG 30485 as compared to C1 strain infection. Both, A. butzleri and C. jejuni induced increased secretion of pro-inflammatory cytokines such as IFN-γ, TNF, IL-6 and MCP-1 in large, but also small intestines. Remarkably, even though viable bacteria did not translocate from the intestines to extra-intestinal compartments, systemic immune responses were induced in C. jejuni, but also A. butzleri infected mice as indicated by increased respective pro-inflammatory cytokine concentrations in serum samples at day 6 p.i. CONCLUSION / SIGNIFICANCE: A. butzleri induce less distinct pro-inflammatory sequelae as compared to C. jejuni, but more pronounced local and systemic immune responses than commensal E. coli in a strain-dependent manner. Hence, data point towards that A. butzleri is more than a commensal in vertebrate hosts.

Occurrence of emerging food-borne pathogenic Arcobacter spp. isolated from pre-cut (ready-to-eat) vegetables.

Given that changes in consumer food behaviours have led to an increase in the demand for pre-cut ready-to-eat (RTE) vegetables, and that few data are currently available on the occurrence of Arcobacter spp. in such foods, the aim of the present study was to assess the occurrence of Arcobacter spp. that carry virulence-associated genes on pre-cut RTE vegetables, using cultural and molecular methods. Arcobacter was detected using biomolecular identification methods in 44/160 (27.5%) of the samples, of which 40/44 (90.9%) isolates corresponded to A. butzleri and 4/44 (9.1%) to A. cryaerophilus. Studying the incidence of 9 virulence-associated genes revealed the widespread distribution of these genes among the Arcobacter isolates tested. The results obtained in our research provided plenty of information on the health risks associated with the direct consumption of raw vegetables, and highlight the need to implement further studies at each level of the production chain, in order to obtain further information to help protect human health.