Butzleri Strains Research Articles

Pathogenic Potential Abilities of Seafood and Environmental Derived Arcobacter butzleri Strains on HeLa and Vero Cell Lines

Pathogenic Potential Abilities of Seafood and Environmental Derived Arcobacter butzleri Strains on HeLa and Vero Cell Lines

Exploring multi-antibiotic resistance in Arcobacter butzleri isolates from a poultry processing plant in northern Italy: An in-depth inquiry

Arcobacter butzleri is a foodborne pathogen that has been identified in various animal-derived foods, notably in poultry meat. The prevalence of this bacterium in poultry underscores the need for comprehensive investigations into its dissemination within poultry meat processing plants. Such assessments are critical due to the potential cross-contamination that may occur, impacting the safety of the final food product. This study endeavours to evaluate the genomic similarity of 56 A. butzleri isolates obtained from chicken carcasses (specifically neck skins and caecum, post-slaughter) and equipment surfaces following cleaning and sanitizing procedures. All samples originate from a poultry slaughterhouse in Northern Italy. The genomic analysis includes single nucleotide polymorphism, average nucleotide identity, and core genome multi-locus sequence typing analysis, aiming to discern potential cross-contamination between carcasses and equipment. The study evaluates antibiotic resistance, biofilm production, and host cell colonization of A. butzleri strains through in vitro assays. Genomic de-replication reveals the presence of 31 distinct strains. Results elucidate that a portion of these strains is evident in multiple sources, indicative of cross-contamination even post-cleaning and sanitization. The in vitro tests underline multi-antibiotic resistance of A. butzleri with genes associated with antibiotic resistance, among them mexAB-oprM, showing a correlation to the observed resistance. This study establishes the colonization capability of A. butzleri strains on a human gut mucus-secreting cell model, suggesting a potential virulence factor. Detection of various putative virulence genes further supports the hypothesis of strains virulence potential. This study holds importance of A. butzleri as it unveils the antibiotic resistance and pathogenic capabilities inherent in this species, identifying associated genetic traits. Through comprehensive genomic analysis, it was conclusively validated instances of cross-contamination between various sources and equipment surfaces, underscoring the pervasive dissemination of this bacterium within a food processing plant and its persistence. This research serves as an exploration into the intricate dynamics of A. butzleri within poultry processing environments. It accentuates the imperative for stringent hygiene protocols and ongoing surveillance to curtail the risk of cross-contamination, thereby ensuring the safety of poultry products for discerning consumers.

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.

Pathogenicity assessment of Arcobacter butzleri isolated from Canadian agricultural surface water

BackgroundWater is considered a source for the transmission of Arcobacter species to both humans and animals. This study was conducted to assess the prevalence, distribution, and pathogenicity of A. butzleri strains, which can potentially pose health risks to humans and animals. Cultures were isolated from surface waters of a mixed-use but predominately agricultural watershed in eastern Ontario, Canada. The detection of antimicrobial resistance (AMR) and virulence-associated genes (VAGs), as well as enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) assays were performed on 913 A. butzleri strains isolated from 11 agricultural sampling sites.ResultsAll strains were resistant to one or more antimicrobial agents, with a high rate of resistance to clindamycin (99%) and chloramphenicol (77%), followed by azithromycin (48%) and nalidixic acid (49%). However, isolates showed a significantly (p < 0.05) high rate of susceptibility to tetracycline (1%), gentamycin (2%), ciprofloxacin (4%), and erythromycin (5%). Of the eight VAGs tested, ciaB, mviN, tlyA, and pldA were detected at high frequency (> 85%) compared to irgA (25%), hecB (19%), hecA (15%), and cj1349 (12%) genes. Co-occurrence analysis showed A. butzleri strains resistant to clindamycin, chloramphenicol, nalidixic acid, and azithromycin were positive for ciaB, tlyA, mviN and pldA VAGs. ERIC-PCR fingerprint analysis revealed high genetic similarity among strains isolated from three sites, and the genotypes were significantly associated with AMR and VAGs results, which highlight their potential environmental ubiquity and potential as pathogenic.ConclusionsThe study results show that agricultural activities likely contribute to the contamination of A. butzleri in surface water. The findings underscore the importance of farm management practices in controlling the potential spread of A. butzleri and its associated health risks to humans and animals through contaminated water.

Widespread Multidrug Resistance of Arcobacter butzleri Isolated from Clinical and Food Sources in Central Italy.

The Arcobacter genus comprises a group of bacteria widely distributed in different habitats that can be spread throughout the food chain. Fluoroquinolones and aminoglycosides represent the most common antimicrobial agents used for the treatment of Arcobacter infections. However, the increasing trend of the antimicrobial resistance of this pathogen leads to treatment failures. Moreover, the test implementation and interpretation are hindered by the lack of reference protocols and standard interpretive criteria. The purpose of our study was to assess the antibiotic resistance pattern of 17 A. butzleri strains isolated in Central Italy from fresh vegetables, sushi, chicken breast, and clinical human samples to provide new and updated information about the antimicrobial resistance epidemiology of this species. Antimicrobial susceptibility testing was carried out by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)'s disc diffusion method. All the strains were multidrug resistant, with 100% resistance to tetracyclines and cefotaxime (third generation cephalosporins). Some differences were noticed among the strains, according to the isolation source (clinical isolates, food of animal origin, or fresh vegetables), with a higher sensitivity to streptomycin detected only in the strains isolated from fresh vegetables. Our data, together with other epidemiological information at the national or European Union (EU) level, may contribute to developing homogeneous breakpoints. However, the high prevalence of resistance to a wide range of antimicrobial classes makes this microorganism a threat to human health and suggests that its monitoring should be considered by authorities designated for food safety.

Genomic Characterization of Arcobacter butzleri Strains Isolated from Various Sources in Lithuania.

Arcobacter (A.) butzleri, the most widespread species within the genus Arcobacter, is considered as an emerging pathogen causing gastroenteritis in humans. Here, we performed a comparative genome-wide analysis of 40 A. butzleri strains from Lithuania to determine the genetic relationship, pangenome structure, putative virulence, and potential antimicrobial- and heavy-metal-resistance genes. Core genome single nucleotide polymorphism (cgSNP) analysis revealed low within-group variability (≤4 SNPs) between three milk strains (RCM42, RCM65, RCM80) and one human strain (H19). Regardless of the type of input (i.e., cgSNPs, accessory genome, virulome, resistome), these strains showed a recurrent phylogenetic and hierarchical grouping pattern. A. butzleri demonstrated a relatively large and highly variable accessory genome (comprising of 6284 genes with around 50% of them identified as singletons) that only partially correlated to the isolation source. Downstream analysis of the genomes resulted in the detection of 115 putative antimicrobial- and heavy-metal-resistance genes and 136 potential virulence factors that are associated with the induction of infection in host (e.g., cadF, degP, iamA), survival and environmental adaptation (e.g., flagellar genes, CheA-CheY chemotaxis system, urease cluster). This study provides additional knowledge for a better A. butzleri-related risk assessment and highlights the need for further genomic epidemiology studies in Lithuania and other countries.

Antimicrobial Resistance and Pathogenicity of Aliarcobacter butzleri Isolated from Poultry Meat

Aliarcobacter butzleri (A. butzleri) is an emergent zoonotic food-related pathogen that can be transmitted through the consumption of poultry meat. Data regarding the pathogenicity and resistance of A. butzleri are still scarce, and the presence of virulent MDR strains of this zoonotic pathogen in poultry meat is an issue of particular concern to public health. This study aimed to characterize the pathogenicity and antimicrobial resistance profiles of A. butzleri strains isolated from poultry meat sold at retail markets in São Paulo, Brazil. The minimum inhibitory concentrations of 27 strains were determined using the broth microdilution method. The results showed that 77.7% of the isolates were resistant to clindamycin, 62.9% to florfenicol, 59.2% to nalidixic acid, 11.1% to azithromycin, 7.4% to ciprofloxacin and telithromycin, and 3.7% to erythromycin and tetracycline, although all were susceptible to gentamicin. Moreover, 55.5% of the virulent isolates were also multidrug-resistant (MDR). Three strains were selected for pathogenicity tests in vitro and in vivo. The tested strains expressed weak/moderate biofilm production and showed a diffuse adhesion pattern (3 h) in HeLa cells and toxicity in Vero cells (24 h). Experimental inoculation in 11-week-old chicks induced a transitory inflammatory enteritis. Intestinal hemorrhage and destruction of the intestinal crypts were observed in the rabbit ileal loop test. Considering the fact that Brazil is a major exporter of poultry meat, the data from this study point to the need of improvement of the diagnostic tools, as well as of the adoption of surveillance guidelines and more specific control strategies to ensure food safety, reducing the presence of pathogenic MDR strains in broilers.

Genetic characteristics, antimicrobial resistance, and prevalence of Arcobacter spp. isolated from various sources in Shenzhen, China.

Arcobacter spp. is a globally emerging zoonotic and foodborne pathogen. However, little is known about its prevalence and antimicrobial resistance in China. To investigate the prevalence of Arcobacter spp. isolated from various sources, 396 samples were collected from human feces, chicken cecum, and food specimens including chicken meat, beef, pork, lettuce, and seafood. Arcobacter spp. was isolated by the membrane filtration method. For 92 strains, the agar dilution method and next-generation sequencing were used to investigate their antimicrobial resistance and to obtain whole genome data, respectively. The virulence factor database (VFDB) was queried to identify virulence genes. ResFinder and the Comprehensive Antibiotic Resistance Database (CARD) were used to predict resistance genes. A phylogenetic tree was constructed using the maximum likelihood (ML) method with core single-nucleotide polymorphisms (SNPs). We found that 27.5% of the samples (n = 109) were positive for Arcobacter spp., comprising Arcobacter butzleri (53.0%), Arcobacter cryaerophilus (39.6%), and Arcobacter skirrowii (7.4%). Chicken meat had the highest prevalence (81.2%), followed by seafood (51.9%), pork (43.3%), beef (36.7%), lettuce (35.5%), chicken cecum (8%), and human fecal samples (0%, 0/159). Antimicrobial susceptibility tests revealed that 51 A. butzleri and 40 A. cryaerophilus strains were resistant to streptomycin (98.1, 70%), clindamycin (94.1, 90%), tetracycline (64.7, 52.5%), azithromycin (43.1%, 15%), nalidixic acid (33.4, 35%), and ciprofloxacin (31.3, 35%) but were susceptible to erythromycin, gentamicin, chloramphenicol, telithromycin, and clindamycin (≤10%). A. skirrowii was sensitive to all experimental antibiotics. The virulence factors tlyA, mviN, cj1349, ciaB, and pldA were carried by all Arcobacter spp. strains at 100%, and the following percentages were cadF (95.7%), iroE (23.9%), hecB (2.2%), hecA, and irgA (1.1%). Only one A. butzleri strain (F061-2G) carried a macrolide resistance gene (ereA). One A. butzleri and one A. cryaerophilus harbored resistance island gene clusters, which were isolated from pork and chicken. Phylogenetic tree analysis revealed that A. butzleri, A. cryaerophilus, and A. skirrowii were separated from each other. To our knowledge, this is the first report of the isolation of Arcobacter spp. from vegetables and seafood in China. The resistance island gene cluster found in pork and chicken meat and the presence of virulence factors could be a potential risk to human health.

Genotyping, antibiotic resistance and prevalence of Arcobacter species in milk and dairy products.

Background Arcobacter spp. has been considered an emerging foodborne pathogen and a hazard to human health. The dairy chain has been isolated from different sources; nevertheless, data on Arcobacter occurrence in raw milk and dairy products in Iran are still scant.ObjectiveThe present study investigates the prevalence, antimicrobial susceptibility and the presence of virulence genes of Arcobacters species isolated from milk and dairy products.MethodsThen, a total of 350 raw milk samples and 400 dairy product samples were collected from dairy supply centers in Isfahan, Iran. Presumptive Arcobacter strains were obtained by enriching samples in Oxoid Arcobacter enrichment broth (AEB) followed by the filtration of enrichment product through 0.45‐μm pore size membrane filters laid onto non‐selective blood at 30°C under microaerophilic conditions. Molecular identification of Arcobacter cryaerophilus and A. butzleri was performed by Polymerase chain reaction (PCR) amplification of the 16S rRNA gene, followed by sequencing. The disc diffusion method was used to determine the antimicrobial susceptibility of isolates. Targeted resistance and virulence genes were detected using multiplex PCR.ResultsThe results show a low recovery rate of Arcobacter spp. in milk. Arcobacters were found in all types of milk, except raw camel milk, but were absent from all dairy products. Arcobacter butzleri was the predominant species in raw milk. Detection of virulence genes shows that all virulence genes targeted were found among A. butzleri, and six (cadF, cj1349, irgA, mviN, pldA, tlyA) were found among A. cryaerophilus. All A. butzleri strains and some A. cryaerophilus strains isolated from milk were resistant to amoxicillin‐clavulanic acid and tetracycline. All A. cryaerophilus isolates from milk were susceptible to gentamycin, streptomycin, erythromycin and ciprofloxacin. The distribution of resistance genes in Arcobacter strains in milk shows that all isolates carried tet(O) and blaOXA‐61 genes.ConclusionsIn conclusion, the results indicate a low recovery rate of Arcobacter spp. in milk and milk products. However, a significant number of Arcobacter strains with putative virulence genes may be potential pathogens for humans and an overall increase in Arcobacter resistance to first‐line antibiotics. These results highlight the need for regular surveillance of Arcobacter strains in milk and milk products in Iran.

Natural Transformation as a Mechanism of Horizontal Gene Transfer in Aliarcobacter butzleri.

Aliarcobacter butzleri is an emergent enteropathogen, showing high genetic diversity, which likely contributes to its adaptive capacity to different environments. Whether natural transformation can be a mechanism that generates genetic diversity in A. butzleri is still unknown. In the present study, we aimed to establish if A. butzleri is naturally competent for transformation and to investigate the factors influencing this process. Two different transformation procedures were tested using exogenous and isogenic DNA containing antibiotic resistance markers, and different external conditions influencing the process were evaluated. The highest number of transformable A. butzleri strains were obtained with the agar transformation method when compared to the biphasic system (65% versus 47%). A. butzleri was able to uptake isogenic chromosomal DNA at different growth phases, and the competence state was maintained from the exponential to the stationary phases. Overall, the optimal conditions for transformation with the biphasic system were the use of 1 μg of isogenic DNA and incubation at 30 °C under a microaerobic atmosphere, resulting in a transformation frequency ~8 × 10−6 transformants/CFU. We also observed that A. butzleri favored the transformation with the genetic material of its own strain/species, with the DNA incorporation process occurring promptly after the addition of genomic material. In addition, we observed that A. butzleri strains could exchange genetic material in co-culture assays. The presence of homologs of well-known genes involved in the competence in the A. butzleri genome corroborates the natural competence of this species. In conclusion, our results show that A. butzleri is a naturally transformable species, suggesting that horizontal gene transfer mediated by natural transformation is one of the processes contributing to its genetic diversity. In addition, natural transformation can be used as a tool for genetic studies of this species.

The ability of Aliarcobacter butzleri strains isolated from foods of animal origin in Costa Rica to form biofilm.

Aliarcobacter butzleri is a zoonotic emerging food and waterborne pathogen widely distributed in nature. It is present in food processing environments and can easily be spread through the food industry because of its ability to form biofilm. The aim of this work was to determine the ability of strains isolated in Costa Rica from different food matrixes of animal origin to form biofilm. Thirty-eight A. butzleri strains previously isolated and identified from animal origin products were analyzed using the method described by Stepmovic et al. (2000), in three culture broths, brain heart infusion broth, Boer broth and Houf broth.Results showed that 67% of poultry origin strains, 62.5% of meat origin strains and just 8% of milk origin strains showed ability to form biofilm.The findings of this study confirm the adherence ability of A. butzleri to form biofilm, a characteristic that can promote dispersion and cross contamination along food industry processing lines.

Functional pangenome analysis reveals high virulence plasticity of Aliarcobacter butzleri and affinity to human mucus

Aliarcobacter butzleri is an emerging pathogen that may cause enteritis in humans, however, the incidence of disease caused by this member of the Campylobacteriaceae family is still underestimated. Furthermore, little is known about the precise virulence mechanism and behavior during infection. Therefore, in the present study, through complementary use of comparative genomics and physiological tests on human gut models, we sought to elucidate the genetic background of a set of 32 A. butzleri strains of diverse origin and to explore the correlation with the ability to colonize and invade human intestinal cells in vitro.The simulated infection of human intestinal models showed a higher colonization rate in presence of mucus-producing cells. For some strains, human mucus significantly improved the resistance to physical removal from the in vitro mucosa, while short time-frame growth was even observed. Pangenome analysis highlighted a hypervariable accessory genome, not strictly correlated to the isolation source. Likewise, the strain phylogeny was unrelated to their shared origin, despite a certain degree of segregation was observed among strains isolated from different segments of the intestinal tract of pigs. The putative virulence genes detected in all strains were mostly encompassed in the accessory fraction of the pangenome. The LPS biosynthesis and in particular the chain glycosylation of the O-antigen is harbored in a region of high plasticity of the pangenome, which would indicate frequent horizontal gene transfer phenomena, as well as the involvement of this hypervariable structure in the adaptive behavior and sympatric evolution of A. butzleri.Results of the present study deepen the current knowledge on A. butzleri pangenome by extending the pool of genes regarded as virulence markers and provide bases to develop new diagnostic approaches for the detection of those strains with a higher virulence potential.

Antimicrobial Resistance and in silico Virulence Profiling of Aliarcobacter butzleri Strains From German Water Poultry.

Aliarcobacter butzleri is an emerging foodborne and zoonotic pathogen that is usually transmitted via contaminated food or water. A. butzleri is not only the most prevalent Aliarcobacter species, it is also closely related to thermophilic Campylobacter, which have shown increasing resistance in recent years. Therefore, it is important to assess its resistance and virulence profiles. In this study, 45 Aliarcobacter butzleri strains from water poultry farms in Thuringia, Germany, were subjected to an antimicrobial susceptibility test using the gradient strip diffusion method and whole-genome sequencing. In the phylogenetic analysis, the genomes of the German strains showed high genetic diversity. Thirty-three isolates formed 11 subgroups containing two to six strains. The antimicrobial susceptibility testing showed that 32 strains were resistant to erythromycin, 26 to doxycycline, and 20 to tetracycline, respectively. Only two strains were resistant to ciprofloxacin, while 39 strains were resistant to streptomycin. The in silico prediction of the antimicrobial resistance profiles identified a large repertoire of potential resistance mechanisms. A strong correlation between a gyrA point mutation (Thr-85-Ile) and ciprofloxacin resistance was found in 11 strains. A partial correlation was observed between the presence of the bla3 gene and ampicillin resistance. In silico virulence profiling revealed a broad spectrum of putative virulence factors, including a complete lipid A cluster in all studied genomes.

Antimicrobial resistance and virulence genes profiles of Arcobacter butzleri strains isolated from back yard chickens and retail poultry meat in Chile.

This research aims to investigate the presence and pathogenic potential of Arcobacter in poultry meat samples purchased in the retail market of Valdivia (South of Chile) as well as in faecal samples from backyard chickens from rural areas around this city. The isolates obtained were identified by molecular methods. Furthermore, putative virulence genes were assessed by PCR and the antimicrobial resistance was tested by phenotypic methods. Arcobacter was present in 41·6% of the samples, with the highest value in retail poultry meat (55·7%) followed by backyard production (28·0%). Arcobacter butzleri was the most prevalent species (75·6%) followed by Arcobacter skirrowii (14·8%) and Arcobacter cryaerophilus (9·6%). An 8·5% of A. butzleri strains from meat were resistant to both ciprofloxacin and tetracycline and 6·1% were resistant to erythromycin, while none was resistant to gentamycin, unlike strains from domestic chickens, which showed no resistance. Furthermore, A. butzleri strains from chicken meat presented a higher prevalence of virulence genes than strains from domestic chickens. In fact, in this last group, some genes (hecA, hecB and irgA) were completely absent. Therefore, this study provides insight on the epidemiology of Arcobacter in Chilean poultry and suggests that under traditional breeding conditions strains are, apparently, less pathogenic and drug resistant.

Phenotype and genomic background of Arcobacter butzleri strains and taxogenomic assessment of the species

In this study the phenotypic and genomic characterization of two Arcobacter butzleri (Ab) strains (Ab 34_O and Ab 39_O) isolated from pre-cut ready-to-eat vegetables were performed. Results provided useful data about their taxonomy and their overall virulence potential with particular reference to the antibiotic and heavy metal susceptibility. These features were moreover compared with those of two Ab strains isolated from shellfish and a genotaxonomic assessment of the Ab species was performed.The two Ab isolated from vegetables were confirmed to belong to the Aliarcobacter butzleri species by 16S rRNA gene sequence analysis, MLST and genomic analyses. The genome-based taxonomic assessment of the Ab species brought to the light the possibility to define different subspecies reflecting the source of isolation, even though further genomes from different sources should be available to support this hypothesis. The strains isolated from vegetables in the same geographic area shared the same distribution of COGs with a prevalence of the cluster “inorganic ion transport and metabolism”, consistent with the lithotrophic nature of Arcobacter spp. None of the Ab strains (from shellfish and from vegetables) metabolized carbohydrates but utilized organic acids and amino acids as carbon sources. The metabolic fingerprinting of Ab resulted less discriminatory than the genome-based approach. The Ab strains isolated from vegetables and those isolated from shellfish endowed multiple resistance to several antibiotics and heavy metals.

Identification of virulence and antibiotic resistance factors in Arcobacter butzleri isolated from bovine milk by Whole Genome Sequencing.

Arcobacter butzleri is a pathogenic aerobic bacterium responsible for diarrhea and septicemia in humans. It is frequently isolated from food products of animal origin, including milk and dairy products. To data, few reports are currently available on the genetic characteristics and virulence profiles of A. butzleri. The aim of this study was to investigate the genetic diversity and to characterize the virulence and antibiotic resistance profiles of 10 A. butzleri strains isolated from bovine milk samples by Whole Genome Sequence (WGS). Multi-Locus Sequence Typing (MLST) revealed that three isolates belonged to the ST66, two to the ST420 and the remaining five strains to the ST627, ST629, ST630, ST633 and ST637, respectively. The 100% of the strains carried cadF, pldA, ciaB, cj1349, mviN and tlyA virulence factors genes; 60% iroE; 50% irgA; 10% hecB. Resistome prediction showed a multidrug resistance: 100% of isolates resulted resistant to fluoroquinolones and tetracycline; 90% of strains to rifampicin and cephalosporins and a minor percentage to other antibiotics. Furthermore, the 50% of strains harbored four mutations in Mycobacterium tuberculosis katG gene conferring resistance to isoniazid. The study provided interesting data on the virulence characteristics and on the genetic endowment related to the antimicrobial resistance of A. butzleri isolates from milk. The determination of the STs also added information concerning the genetic variability of this microorganism. To date, a very limited number of studies have been published on the typing of A. butzleri using WGS, so this paper proposes an innovative methodological approach that allows a rapid and complete characterization of pathogenic microorganisms.

Genomic Characterization of Arcobacter butzleri Isolated From Shellfish: Novel Insight Into Antibiotic Resistance and Virulence Determinants.

Arcobacter (A.) butzleri is an emerging pathogenic microorganism, whose taxonomy has been recently suggested to be emended to the Aliarcobacter (Al.) butzleri comb. nov. Despite extensive taxonomic analysis, only few fragmented studies have investigated the occurrence and the prevalence of virulence and antibiotic resistance determinants of this species in strains isolated from shellfish. Herein we report for the first time the whole genome sequencing and genomic characterization of two A. butzleri strains isolated from shellfish, with particular reference to the antibiotic, heavy metals and virulence determinants. This study supported the taxonomic assignment of these strains to the Al. butzleri species, and allowed us to identify antibiotic and metal resistance along with virulence determinants, also additional to those previously reported for the only two A. butzleri strains from different environments genomically characterized. Moreover, both strains showed resistance to β-lactams, vanocomycin, tetracycline and erythromycin and susceptibility to aminoglycosides and ciprofloxacin. Beside enlarging the availability of genomic data to perform comparative studies aimed at correlating phenotypic differences associated with ecological niche and geographic distribution with the genetic diversity of A. butzleri spp., this study reports the endowment of antibiotic and heavy metal resistance and virulence determinants of these shellfish-isolated strains. This leads to hypothesize a relatively high virulence of A. butzleri isolated from shellfish and prompt the need for a wider genomic analysis and for in vitro and in vivo studies of more strains isolated from this and other ecological niches, to unravel the mechanism of pathogenicity of this species, and the potential risk associated to their consumption.

Arcobacter spp. in bovine milk: An emerging pathogen with potential zoonotic risk.

The aim of the present study was to assess the prevalence and genetic characteristics of Arcobacter spp. in bovine bulk tank milk produced in Apulia Region (Italy). Samples collected from 396 dairy farms, after enrichment in a selective broth, were subjected to an Arcobacter genus - specific Real Time PCR. Positive broths, previously filtered, were seeded on Karmali, MCCD and Columbia Blood Agar plates; presumptive Arcobacter spp. colonies were identified using an amplification and sequencing method and then characterized by Multi-Locus Sequence Typing (MLST). Prevalence of Arcobacter spp. in bovine milk samples was 5% (20/396); A. butzleri was the only isolated species, in agreement with previous studies that reported A. butzleri as the most commonly recovered species in milk and dairy products. MLST analysis of the 20 A. butzleri strains identified 81 alleles and 16 STs. Consistent with previous studies, MLST revealed a high level of heterogeneity between the A. butzleri isolates and confirmed the high discriminatory power of this method and its suitability for epidemiological investigations. This study confirmed the importance of raw milk as a possible source of Arcobacter spp. for humans.

Arcobacter butzleri strains isolated from different sources display adhesive capacity to epithelial cells in vitro

Background: The genus Arcobacter, belonging to the family Campylobacteraceae, includes polar flagellated, curved or spiral rod shaped bacteria firstly described as Vibrio/Spirillum organisms and later as aerotolerant Campylobacter-like microorganisms. Currently, this genus comprises nine validated species and a candidatus. Among Arcobacter species, Arcobacter butzleri is recognized as a zoonotic agent and the most common species of the genus isolated from environmental water, food and clinical samples. Recently, this species was considered a serious hazard to human health. However, their pathological properties, potential virulence factors as well as their clinical significance remain uncertain or not completely defined. The aim of this study was to establish the ability of 78 A. butzleri strains isolated from different sources to adhere to HEp-2 cells in vitro. Materials, Methods & Results: All the strains were isolated using first an enrichment medium incubated aerobically at 26oC for 48h. After that, 100 µL of the broth were streaked onto Arcobacter selective agar plates and incubated at 26°C for 72h. All the strains were identified phenotypically using standard assays. Definitive identification was achieved using a multiplex polymerase chain reaction. Adhesive capacity was determined infecting HEp-2 cells with the strains under study following Skaletsky et al. protocol, with slight modifications. An adherent Escherichia coli isolate was included as a positive control, whereas uninoculated cell lines were used as negative controls. Adhesion results were expressed as the percentage of HEp-2 cells showing adhering bacteria and the number of bacteria (± SD) adhered to cells was determined. Strains were considered as adherent, if at least 20% of HEp-2 cells showed one ore more adhered bacteria. Adherence was also demonstrated by scanning electronic microscopy. All the strains showed adhesive capacity to HEp-2 cells in vitro. The lowest adhesion percentage (27%) was observed in a strain isolated from dog feces. In at least one strain per source it was possible to observe 100% adhesion. The lowest number of adhered bacteria (1.03 ± 1.53) was observed in a strain isolated from river water and the highest number (76.6 ± 5.59), in a strain isolated from duck feces. Discussion: Regardless of their isolation source, the 78 Arcobacter butzleri strains tested in the present study were able to adhere to HEp-2 cells in vitro. This property was demonstrated in different proportions by other authors in strains isolated from environmental, meat and human fecal samples. Electronic scanning microscopy shows curved bacilli adhering to HEp-2 cells forming a microcolony-like arrangement. This may occur in a stepwise mechanism. First, A. butzleri adhere to HEp-2 cells and then, due to their capacity to form biofilms, interbacterial adherence can be expressed. The capacity of A. butzleri to form biofilms attaching to stainless steel, copper and glass has been demonstrated in previous studies. Further studies are necessary to elucidate the molecular and cellular mechanisms accounting for this biological property; and hence, its actual role in the colonization of human and animal intestinal epithelium by A. butzleri.

Application of MALDI-TOF MS for the subtyping of Arcobacter butzleri strains and comparison with their MLST and PFGE types

For the first time, this study evaluated the use of MALDI-TOF as a typing tool for Arcobacter butzleri. A total of 104 A. butzleri strains isolated from different sources in an artisanal dairy plant in Italy were identified and typed using MALDI-TOF and compared with their multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) profiles found in previous studies. MALDI-TOF correctly identified all the isolates to species level. No clearly delineated clusters appeared on dendrograms based on either the complete spectra or the significant peaks, but nine clusters were defined using the cophenetic correlation. Interestingly, MALDI-TOF proved able to discriminate A. butzleri strains below species level, confirming its potential use for epidemiological surveys. As expected, the comparative analysis with PFGE and MLST showed that the discriminatory index was lower for MALDI-TOF but roughly comparable to sequence types and pulsotypes. MALDI-TOF appears to be a relatively low cost answer to the urgent need for more rapid, less expensive typing tools suitable for source attribution studies, readily allowing multiple typing methods to be combined. This study provides insights into MALDI-TOF as potential epidemiological tool. Its application in healthcare surveillance systems awaits further exploration to encourage interaction and convergence studies between primary care in humans and animal and food veterinary authorities as part of the One Health concept.