Genetic characterization and biofilm formation of potentially pathogenic foodborne Arcobacter isolates.

Various species of the genus Arcobacter are regarded as emerging food pathogens and can be cause of human gastroenteric illness, among others. In order to gain knowledge on the risk associated with the presence of arcobacters in retail foods, this study aimed to determine their presence in a variety of products; to evaluate the genetic diversity and the occurrence of virulence and biofilm-associated genes in the isolated strains; and to assess their biofilm activity on polystyrene, borosilicate and stainless steel. Arcobacters were detected in the 22.3% of the analysed samples and the 83 recovered isolates were identified as A. butzleri (n = 53), A. cryaerophilus (n = 24), A. skirrowii (n = 2), A. thereius (n = 3) and A. vitoriensis (n = 1). They were isolated from virtually all tested food types, but mostly from squids and turkey meat (contamination levels of 60% and 40%, respectively). MLST differentiated 68 STs, most of which were novel (89.7%) and represented by a single strain (86.9%). Five novel STs were detected in various isolates derived from seafood, and the statistical analysis revealed their potential association with that type of food product (p < 0,001). All the isolates except one harboured virulence-associated genes and the highest incidence was noted for A. butzleri. Nineteen isolates (23.5%) were able to form biofilms on the different surfaces tested and, of note; glass enhanced the adhesion ability of the majority of them (84.2%). The results highlight the role that common food products can have in the transmission of Arcobacter spp., the pathogenic potential of the different species, and the survival and growth ability of several of them on different food contact surfaces. Therefore, the study provides interesting information regarding the risk arcobacters may pose to human health and the food industry.

Aliarcobacter vitoriensis sp. nov., isolated from carrot and urban wastewater.

Two isolates, one recovered from a carrot and another one from urban wastewater, were characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates clustered together, and were most closely related to Aliarcobacter lanthieri. Multilocus phylogenetic analysis (MLPA) using the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) suggested that these isolates formed a distinct phylogenetic lineage among the genera derived from the former genus Arcobacter. Whole-genome sequence, in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) value between the genome of strain F199T and those of related species confirmed that these isolates represent a novel species. These strains can be differentiated from its phylogenetically closest species A. lanthieri by its inability to growth on 1% glycine and by their enzyme activity of esterase lipase (C8) and acid phosphatase. Our results, by the application of a polyphasic analysis, confirmed that these two isolates represent a novel species of the genus Aliarcobacter, for which the name Aliarcobacter vitoriensis sp. nov. is proposed. The type strain is F199T (=CECT 9230T=LMG 30050T).

Variation in Campylobacter jejuni culturability in presence of Acanthamoeba castellanii Neff.

Free-living amoebae (FLA) are protozoa that are widely distributed in the environment mainly in water and soil related habitats. These amoebae have also been reported to be associated with some bacterial pathogens for humans such as Campylobacter spp. The species C. jejuni is the causative agent of about 90% of human campylobacteriosis cases worldwide and this disease may even end up in severe autoimmune sequelae as Guillain-Barré syndrome (GBS). In this study, the interactions between the strain Acanthamoeba castellanii Neff and Campylobacter jejuni was investigated. Campylobacter jejuni was coincubated with Acanthamoeba castellanii Neff trophozoites at different temperatures, in order to evaluate the C. jejuni ability to grow in presence A. castellanii culture and Acanthamoeba Conditioned Medium (ACM). C. jejuni was coincubated with A. castellanii axenic culture at different temperatures in aerobic conditions. Our results revealed that bacteria were still cultivable (Blood Agar medium, at 37 °C, in microaerophilic atmosphere) after a 14 days C. jejuni - A. castellanii coculture, comparing with C. jejuni alone, which was only cultivable for 24 h.

Study of Biofilm Formation Ability of Foodborne Arcobacter butzleri under Different Conditions.

The transmission of Arcobacter butzleri, an emerging food- and waterborne pathogen, is possibly favored by its ability to adhere to abiotic surfaces. In this study, we assessed the biofilm formation ability of 42 A. butzleri isolates recovered from different food products. Overall, nine isolates (21.4%) were able to adhere to polystyrene. Among them, a chicken-derived isolate was classified as strongly adherent. Based on the chi-square test, no relation was found between the adhesive abilities of the isolates and their source (P > 0.05). An aerobic atmosphere enhanced the adhesion ability of the majority of the adherent isolates (66.7%), because when tested in microaerobic conditions, a t test indicated that only three isolates increased their biofilm formation ability significantly (P < 0.05). In addition, seven (77.8%) of these nine isolates were able to adhere to glass surfaces, and viable cells were recovered from all the stainless steel coupons tested. Therefore, our results confirm the biofilm formation ability of A. butzleri, which may be influenced by the incubation atmosphere and the abiotic surface.

LL37 loaded nanostructured lipid carriers (NLC): A new strategy for the topical treatment of chronic wounds.

The LL37 is a human antimicrobial peptide which not only has a broad spectrum of antimicrobial activity, but it has also been proved to modulate wound healing by participating in angiogenesis, epithelial cell migration and proliferation, and immune response. In this work, LL37 has been encapsulated in nanostructured lipid carriers (NLCs), produced by the melt-emulsification method, in order to improve its effectiveness. The characterisation of the NLC-LL37 showed a mean size of 270nm, a zeta potential of -26mV and an encapsulation efficiency of 96.4%. The cytotoxicity assay performed in Human Foreskin Fibroblasts demonstrated that the NLC-LL37 did not affect cell viability. Moreover, the in vitro bioactivityassay evidenced that the peptide remained active after the encapsulation, since the NLC-LL37 reversed the activation of the macrophages induced by LPS in the same way as the LL37 in solution. In addition, the in vitro antimicrobial assay revealed the NLC-LL37 activity against Escherichia coli. The effectiveness of the nanoparticles was assessed in a full thickness wound model indb/dbmice. The data demonstrated that NLC-LL37 significantly improved healing compared to the same concentration of the LL37 solution in terms of wound closure, reepithelisation grade and restoration of the inflammatory process. Overall, these findings suggest a promising potential of the NLC-LL37 formulation for chronic wound healing.

Prevalence of ten putative virulence genes in the emerging foodborne pathogen Arcobacter isolated from food products.

Arcobacter spp. are considered to be emerging food- and waterborne pathogens for both humans and animals. However, their virulence mechanisms are still poorly understood. In this study the presence of ten virulence genes (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, tlyA and iroE) was assessed in a set of 47 strains of Arcobacter butzleri, 10 of Arcobacter cryaerophilus and 1 Arcobacter skirrowii strain recovered from different food products (pork, chicken, beef, milk, clams and mussels). Overall, the genes cadF, ciaB, cj1349, mviN, pldA and tlyA were detected in all A. butzleri and A. skirrowii strains. Lower detection rates were observed for irgA, iroE, hecA and hecB. The genes hecB and iroE were detected neither in A. cryaerophilus nor in A. skirrowii. The genes hecA and irgA were not detected in A. skirrowii. It was noteworthy that the genes hecA and hecB were significantly (P < 0.05) highly detected in A. butzleri strains isolated from clams compared with strains isolated from milk and chicken. Therefore, our findings underline clams as a source of A. butzleri strains with high prevalence of putative virulence genes. This could be hazardous to human health, especially because these bivalves are usually consumed raw or undercooked.

Spread of an Enterococcus faecalis sequence type 6 (CC2) clone in patients undergoing selective decontamination of the digestive tract.

Enterococcus faecalis (E. faecalis) is a common cause of nosocomial infection in immunocompromised patients. The presence and dissemination of high-risk clonal complexes, such as CC2, is an ongoing problem in hospitals. The aim of this work was to characterize 24 E. faecalis isolates from ICU patients undergoing selective decontamination of the digestive tract (SDD) by phenotypical (antimicrobial susceptibility) and genotypical (presence of virulence genes, RAPD-PCR and MLST) methods. Our results showed high prevalence of the ST6 E. faecalis clone (91.6%), especially adapted to the hospital environment, with a multidrug resistance pattern and a multitude of putative virulence genes. In addition, ST179 (4.2%) and ST191 (4.2%) were detected. By RAPD-PCR analysis, the 22 isolates identified as ST6 showed six different DNA patterns, while the two remaining isolates, ST179 and ST191, showed two additional profiles. CC2 is a known clonal complex with high adaptability to hospital environment and worldwide distribution. The high prevalence of the ST6 clone in the studied population could be related to the presence of gentamicin in the SDD mixture since most strains were gentamicin resistant. Consequently, strict surveillance should be applied for rapid detection and control of this clone to prevent future spread outside the ICU.

Multilocus sequence typing reveals genetic diversity of foodborne Arcobacter butzleri isolates in the North of Spain.

The emerging pathogen Arcobacter butzleri is being increasingly isolated from different animal food products but the routes of its transmission to human are not well established yet. Typing methods would be useful in gaining such knowledge. Here we report the great genetic diversity observed among A. butzleri isolates from different food products. Forty-five isolates were analyzed by Multilocus Sequence Typing (MLST). A total of 157 alleles were identified across all seven loci, ranging from 16 alleles at glnA to 31 at glyA. MLST differentiated the isolates into 34 sequence types (STs), with the majority of isolates containing a unique sequence type. Seventy-four new alleles were identified, which resulted in the assignment of 33 new STs. No association of alleles or STs with food source was observed. For the first time, lateral gene transfer from Arcobacter skirrowii to A. butzleri at the glyA locus is also reported.

Prevalence and genetic diversity of arcobacter in food products in the north of Spain.

The bacterial contamination of food products can cause serious public health problems. Interest in Arcobacter contamination has increased due to the relationship between these bacteria and human enteritis. We studied the prevalence and genetic diversity of Arcobacter species at the retail level in the province of Alava in Basque Country, Spain. The results showed a high genetic diversity and indicated the regular presence of the main Arcobacter spp. associated with human enteric illness in food products. Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii were detected with an overall prevalence close to 40% and were isolated from 15 (42.8%) fresh cow's milk samples, 12 (73.3%) shellfish samples, 11 (55%) chicken samples, 2 (10%) pork samples, and 1 (5%) beef sample. The results indicate the need to investigate the impact of Arcobacter spp. on public health.

Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR.

A multiplex PCR was developed for simultaneous detection of the cytolethal distending toxin (cdt) genes of Campylobacter jejuni. Three primer pairs targeting each one of the cdtA, cdtB and cdtC genes were designed and combined in the same PCR reaction. The assay was evaluated with 100 C. jejuni strains recovered from humans and animals and it was found to be rapid and specific. Two isolates presented several deletions affecting both cdtA and cdtB genes. High prevalence (98%) of the three cdt genes was found among isolates of different geographic origins.

MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains.

Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.

PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli.

A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.