Microbial contamination pathways in a poultry abattoir provided clues on the distribution and persistence of Arcobacter spp.

The consumption of contaminated poultry meat is a significant threat for public health, as it implicates in foodborne pathogen infections, such as those caused by Arcobacter. The mitigation of clinical cases requires the understanding of contamination pathways in each food process and the characterization of resident microbiota in the productive environments, so that targeted sanitizing procedures can be effectively implemented. Nowadays these investigations can benefit from the complementary and thoughtful use of culture- and omics-based analyses, although their application in situ is still limited. Therefore, the 16S-rRNA gene-based sequencing of total DNA and the targeted isolation of Arcobacter spp. through enrichment were performed to reconstruct the environmental contamination pathways within a poultry abattoir, as well as the dynamics and distribution of this emerging pathogen. To that scope, broiler's neck skin and caeca have been sampled during processing, while environmental swabs were collected from surfaces after cleaning and sanitizing. Metataxonomic survey highlighted a negligible impact of fecal contamination and a major role of broiler's skin in determining the composition of the resident abattoir microbiota. The introduction of Arcobacter spp. in the environment was mainly conveyed by this source rather than the intestinal content. Arcobacter butzleri represented one of the most abundant species and was extensively detected in the abattoir by both metataxonomic and enrichment methods, showing higher prevalence than other more thermophilic Campylobacterota. In particular, Arcobacter spp. was recovered viable in the plucking sector with high frequency, despite the adequacy of the sanitizing procedure.IMPORTANCEOur findings have emphasized the persistence of Arcobacter spp. in a modern poultry abattoir and its establishment as part of the resident microbiota in specific environmental niches. Although the responses provided here are not conclusive for the identification of the primary source of contamination, this biogeographic assessment underscores the importance of monitoring Arcobacter spp. from the early stages of the production chain with the integrative support of metataxonomic analysis. Through such combined detection approaches, the presence of this pathogen could be soon regarded as hallmark indicator of food safety and quality in poultry slaughtering.

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.

Evaluation of a Harmonized Undergraduate Catalog for Veterinary Public Health and Food Hygiene Pedagogy in Europe.

Current and emerging veterinary public health (VPH) challenges raised by globalization, climate change, and industrialization of food production require the veterinarian's role to evolve in parallel and veterinary education to adapt to reflect these changes. The European Food Hygiene catalog was developed to provide a list of topics relevant to Day One Competencies in VPH. A study was undertaken to ensure that the catalog and teaching practices were pertinent to the work of public health veterinarians. Relevant stakeholders were consulted using questionnaires and semi-structured interviews. A long questionnaire was distributed to 49 academics teaching VPH in European veterinary schools to review topics listed in the catalog. Eighteen responses were received (36.7%), representing 12 European countries. There was general agreement that most topics were appropriate for the undergraduate VPH curriculum. A short questionnaire was distributed to 348 European veterinarians working in the industry. Twenty-four questionnaires (6.7%) were received, representing eight European countries. Despite the low participation rate, topics needing greater emphasis in the undergraduate curriculum included Hazard Analysis Critical Control Points (HACCP), food microbiology, and audits. Seven semi-structured interviews with public health veterinarians working in the UK identified the need for curricular changes including greater practical experience and a shift from a focus on meat inspection to risk management. This may be partly achieved by replacing traditional lectures with authentic case-based scenarios. The study findings can be used to inform the future direction to VPH education for veterinary students across Europe.

Arcobacter vandammei sp. nov., isolated from the rectal mucus of a healthy pig.

A study on the polyphasic taxonomic classification of an Arcobacter strain, R-73987T, isolated from the rectal mucus of a porcine intestinal tract, was performed. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain could be assigned to the genus Arcobacter and suggested that strain R-73987T belongs to a novel undescribed species. Comparative analysis of the rpoB gene sequence confirmed the findings. Arcobacter faecis LMG 28519T was identified as its closest neighbour in a multigene analysis based on 107 protein- encoding genes. Further, whole-genome sequence comparisons by means of average nucleotide identity and in silico DNA-DNA hybridization between the genome of strain R-73987T and the genomes of validly named Arcobacter species resulted in values below 95-96 and 70  %, respectively. In addition, a phenotypic analysis further corroborated the conclusion that strain R-73987T represents a novel Arcobacter species, for which the name Arcobacter vandammei sp. nov. is proposed. The type strain is R-73987T (=LMG 31429T=CCUG 75005T). This appears to be the first Arcobacter species recovered from porcine intestinal mucus.

Isolation, characterization and antibiotic resistance of Proteus mirabilis from Belgian broiler carcasses at retail and human stool.

Proteus mirabilis is an important pathogen involved in human urinary tract infections, and also more isolated from stools of patients with diarrheal disease than from healthy patients. The role of food, especially poultry products as source for human infection and multi-resistant strains remains unclear. As a resident in broilers' intestines, P. mirabilis can contaminate broiler carcasses due to slaughter practices, and be a risk for human infection. The present study evaluated the performance of five isolation media, and subsequently examined the presence of P. mirabilis on broiler carcasses at retail. Additionally, isolates were characterized by the Dienes' test, repetitive element PCR fingerprinting and pulsed-field gel electrophoresis, and their antibiotic resistance profile determined. Using a combined isolation protocol on blood agar, xylose lysine deoxycholate agar and violet red bile glucose agar, P. mirabilis was isolated from 29 out of 80 broiler carcasses (36.25%) with a mean contamination level of 2.25 ± 0.50 log10 CFU/g. A high strain heterogeneity was present in isolates from broilers and human stool. The same strains were not shared, but the antibiotic resistance profiling was similar. A role of poultry products as source for human infection should be taken into account.

Assessment of food microbiological indicators applied on poultry carcasses by culture combined MALDI-TOF MS identification and 16S rRNA amplicon sequencing.

Examination of the bacterial contamination on food products is still largely performed by standardized culture methods, though culture-independent methods are suggested as a more reliable approach. Knowledge of the diversity of bacteria isolated from food as well as the impact of the plate incubation conditions applied are still understudied. The impact of incubation at 7 °C and 30 °C on total aerobic bacterial count and diversity, and the performance of ISO methods generally applied in microbiological quality examination were assessed by culture combined MALDI-TOF MS identification and 16S rRNA amplicon sequencing. Examining breast skin of 16 chicken carcasses, no significant impact of the incubation temperature on the total aerobic bacteria level and diversity was detected, limiting the usefulness of additional psychrophilic examination. Bacteria phenotypically similar to Pseudomonas, were identified on selective CFC plates, and on MRS agar plates for lactic acid bacteria, Escherichia coli and Staphylococcus were commonly present. Application of 16S rRNA amplicon sequencing revealed a higher bacterial diversity, but the impact of the DNA extraction kit applied, and the detection of non-viable bacteria should be taken into account to interpret the final outcome.

Microbial Diversity and Putative Opportunistic Pathogens in Dishwasher Biofilm Communities.

Extreme habitats are not only limited to natural environments, but also exist in manmade systems, for instance, household appliances such as dishwashers. Limiting factors, such as high temperatures, high and low pHs, high NaCl concentrations, presence of detergents, and shear force from water during washing cycles, define microbial survival in this extreme system. Fungal and bacterial diversity in biofilms isolated from rubber seals of 24 different household dishwashers was investigated using next-generation sequencing. Bacterial genera such as Pseudomonas, Escherichia, and Acinetobacter, known to include opportunistic pathogens, were represented in most samples. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus, and Rhodotorula, also known to include opportunistic pathogenic representatives. This study showed how specific conditions of the dishwashers impact the abundance of microbial groups and investigated the interkingdom and intrakingdom interactions that shape these biofilms. The age, usage frequency, and hardness of incoming tap water of dishwashers had significant impact on bacterial and fungal community compositions. Representatives of Candida spp. were found at the highest prevalence (100%) in all dishwashers and are assumed to be one of the first colonizers in recently purchased dishwashers. Pairwise correlations in tested microbiomes showed that certain bacterial groups cooccur, as did the fungal groups. In mixed bacterial-fungal biofilms, early adhesion, contact, and interactions were vital in the process of biofilm formation, where mixed complexes of bacteria and fungi could provide a preliminary biogenic structure for the establishment of these biofilms.IMPORTANCE Worldwide demand for household appliances, such as dishwashers and washing machines, is increasing, as is the number of immunocompromised individuals. The harsh conditions in household dishwashers should prevent the growth of most microorganisms. However, our research shows that persisting polyextremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions and supported by the biofilm mode of growth. The significance of our research is in identifying the microbial composition of biofilms formed on dishwasher rubber seals, how diverse abiotic conditions affect microbiota, and which key microbial members were represented in early colonization and contamination of dishwashers, as these appliances can present a source of domestic cross-contamination that leads to broader medical impacts.

Impact of Acanthamoeba Cysts on Stress Resistance of Salmonella enterica Serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a, and Escherichia coli O:26.

The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H2O2, benzalkonium chloride, 55°C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i) Acanthamoeba castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34- to 4.72-log10 cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H2O2 (>92% decrease in median log10 CFU/ml reduction) and 70% ethanol (>99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of Yersinia enterocolitica (74% decrease in median log10 reduction), Escherichia coli (58%), and Salmonella enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%), and Listeria monocytogenes (99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria.IMPORTANCE The results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens.

Minimal standards for describing new species belonging to the families Campylobacteraceae and Helicobacteraceae: Campylobacter, Arcobacter, Helicobacter and Wolinella spp.

Ongoing changes in taxonomic methods, and in the rapid development of the taxonomic structure of species assigned to the Epsilonproteobacteria have lead the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Campylobacter and Related Bacteria to discuss significant updates to previous minimal standards for describing new species of Campylobacteraceae and Helicobacteraceae. This paper is the result of these discussions and proposes minimum requirements for the description of new species belonging to the families Campylobacteraceae and Helicobacteraceae, thus including species in Campylobacter, Arcobacter, Helicobacter, and Wolinella. The core underlying principle remains the use of appropriate phenotypic and genotypic methods to characterise strains sufficiently so as to effectively and unambiguously determine their taxonomic position in these families, and provide adequate means by which the new taxon can be distinguished from extant species and subspecies. This polyphasic taxonomic approach demands the use of appropriate reference data for comparison to ensure the novelty of proposed new taxa, and the recommended study of at least five strains to enable species diversity to be assessed. Methodological approaches for phenotypic and genotypic (including whole-genome sequence comparisons) characterisation are recommended.

Antimicrobial susceptibility testing of Arcobacter butzleri and Arcobacter cryaerophilus strains isolated from Belgian patients.

OBJECTIVES: To evaluate the feasibility of different methods for susceptibility testing of human Arcobacter isolates, to assess susceptibility to antibiotics commonly used to treat diarrhoeal illness and to obtain MIC distribution data. METHODS: One-hundred-and-six unique Arcobacter strains were collected during an epidemiological study on pathogens in gastroenteritis. Strains were identified by multiplex PCR and PCR-RFLP, and characterized by PFGE. Susceptibility to ampicillin, erythromycin, tetracycline, doxycycline, gentamicin and ciprofloxacin was determined using gradient strip and disc diffusion methodology. Optimal conditions for growth and incubation were tested. Azithromycin was tested with gradient strip diffusion on a subset of 40 strains. Sequence analysis of the quinolone resistance-determining region of gyrA was performed for a subset of 18 strains. RESULTS: Based on gradient diffusion results, most Arcobacter strains were susceptible to gentamicin (99%) and tetracycline (89%). Erythromycin (78%), ciprofloxacin (72%) and doxycycline (76%) retained moderate activity against Arcobacter spp. Only 9% of the strains were susceptible to ampicillin. Most Arcobacter butzleri strains were susceptible to ciprofloxacin (87%), whereas half of the Arcobacter cryaerophilus isolates (51%) showed high-level resistance (MIC >32 mg/L). MIC50 values were comparable for both macrolide antibiotics. Ciprofloxacin-resistant strains possessed an identical mutation in gyrA. Overall, categorical agreement between gradient and disc diffusion results was 60%. Gradient diffusion showed superior readability. CONCLUSIONS: Gradient diffusion methodology is preferred for routine susceptibility testing. Acquired resistance to fluoroquinolones was observed in A. cryaerophilus. Macrolides are not first-choice empirical antibiotics for Arcobacter infections. Tetracyclines can be suggested for treatment of documented Arcobacter-related gastrointestinal infections.

Taxonomic dissection of Achromobacter denitrificans Coenye et al. 2003 and proposal of Achromobacter agilis sp. nov., nom. rev., Achromobacter pestifer sp. nov., nom. rev., Achromobacter kerstersii sp. nov. and Achromobacter deleyi sp. nov.

The phenotypic and genotypic characteristics of a historical collection of strains identified as Achromobacter denitrificanswere examined. Sequence analysis of a 765 bp nrdA gene fragment revealed that eight of these strains belonged to the recently described Achromobacter aegrifaciens, Achromobacter mucicolens, and Achromobacter insolitus, and that one strain belonged to Achromobacter xylosoxidans. The analysis also suggested the presence of four novel species of the genus Achromobacter among the remaining strains. The latter was confirmed by multilocus sequence analysis of concatenated nusA, eno, rpoB, gltB, lepA, nuoL andnrdA gene fragments and extensive genotypic and phenotypic characterization. We propose to name these novel species as Achromobacter agilis sp. nov., nom. rev. (type strain LMG 3411T=CCUG 62454T), Achromobacter pestifer sp. nov., nom. rev. (type strain LMG 3431T=CCUG 61959T) , Achromobacter kerstersii sp. nov. (type strain LMG 3441T=CCUG 62449T) and Achromobacter deleyi sp. nov. (type strain LMG 3458T=CCUG 62433T).

Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria.

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 µg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed.

Novel lactic acid bacteria isolated from the bumble bee gut: Convivina intestini gen. nov., sp. nov., Lactobacillus bombicola sp. nov., and Weissella bombi sp. nov.

Twelve isolates of lactic acid bacteria (LAB) were obtained in the course of a bumble bee gut microbiota study and grouped into four matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry clusters. Comparative 16S rRNA gene sequence analysis revealed that cluster 1 isolates, represented by strain LMG 28288(T), are most closely related to Lactobacillus apis (97.0% sequence similarity to that of L. apis LMG 26964(T)). Cluster 2 isolates represented by strain LMG 28290(T) are most closely related to Weissella hellenica (99.6% sequence similarity to that of W. hellenica LMG 15125(T)). The single cluster 3 and 4 isolates had identical 16S rRNA gene sequences which were 94.8% similar to that of Leuconostoc mesenteroides subsp. mesenteroides LMG 6893(T), their nearest phylogenetic neighbour. A polyphasic taxonomic study additionally including comparative pheS sequence analysis, DNA-DNA hybridization experiments, DNA G+C content analysis, (GTG)5-PCR fingerprinting and a biochemical characterization, demonstrated that cluster 1 isolates represent a novel Lactobacillus species for which we propose the name Lactobacillus bombicola sp. nov. with LMG 28288(T) (= DSM 28793(T)) as the type strain; and that cluster 2 isolates represent a novel Weissella species for which we propose the name Weissella bombi sp. nov. with LMG 28290(T) (= DSM 28794(T)) as the type strain. Cluster 3 and 4 isolates, in contrast, represented a very distinct, novel taxon that could be distinguished from members of the genera Leuconostoc and Fructobacillus, its nearest phylogenetic neighbours, by its cellular morphology, non-fructophilic metabolism and DNA G+C content. We therefore classify both isolates into a novel species representing a novel LAB genus for which the name Convivina intestini gen. nov., sp. nov. is proposed with LMG 28291(T) (= DSM 28795(T)) as the type strain.

Assessment of Risk Factors for a High Within-Batch Prevalence of Yersinia enterocolitica in Pigs Based on Microbiological Analysis at Slaughter.

The purpose of the current study was to find farm-level factors influencing the bacteriological prevalence of Yersinia enterocolitica in pigs at time of slaughter. On 100 farms, data concerning a broad range of farm aspects (e.g., management and housing system, biosecurity, and hygiene measurements) were collected using a face-to-face questionnaire. At the slaughterhouse, tonsils of on average 70 slaughter pigs per batch were sampled to determine the infection status of pigs. After univariable mixed-effect logistic regressions, variables that were related to the Yersinia prevalence (p<0.05) were included in a multivariable model. In this model, the factors remaining positively associated with a higher Y. enterocolitica carriage in the tonsils (p<0.1) were an increasing number of piglet suppliers, a high density of pig farms in the area, and the use of semislatted floors in the fattening pig unit. The proper use of a disinfection bath before entering the stables and a poor biosecurity level were protective factors, although a higher prevalence was associated with a significant positive interaction between the presence of pets in the stables and a poor biosecurity level. Reducing the number of piglet suppliers, using a disinfection bath properly, and prohibiting pets inside the stables could be easily implemented by pig farmers to lower the prevalence of Y. enterocolitica in pigs at slaughter.

Prevalence of Arcobacter species among humans, Belgium, 2008-2013.

We examined fecal samples from 6,774 patients with enteritis in Belgium, 2008-2013. Members of the genus Arcobacter were the fourth most common pathogen group isolated, and the isolation rate was higher than previously reported. Culturing Arcobacter in a microbiology laboratory is feasible and should thus be tested for in cases of diarrheal disease.

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed.

In recent years, the frequent detection of the banned thyreostat thiouracil (TU) in livestock urine has been related to endogenous TU formation following digestion of glucosinolate-rich Brassicaceae crops. Recently, it was demonstrated that, upon in vitro digestion of Brassicaceae, fecal bacteria induce TU detection in livestock (porcine livestock > bovines). Therefore, the present study was intended to isolate and identify bacteria involved in this intestinal TU formation upon Brassicaceae digestion and to gain more insight into the underlying mechanism in porcine livestock. Twenty porcine fecal inocula (gilts and multiparous sows) were assessed through static in vitro colonic-digestion simulations with rapeseed. After derivatization and extraction of the fecal suspensions, TU was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS(2)). On average, lower TU concentrations were observed in fecal colonic simulations in gilts (8.35 ng g(-1) rapeseed ± 3.42 [mean ± standard deviation]) than in multiparous sows (52.63 ng g(-1) ± 16.17), which correlates with maturation of the gut microbial population with age. Further exploration of the mechanism showed cell-dependent activity of the microbial conversion and sustained TU-forming activity after subjection of the fecal inoculum to moderate heat over a time span of up to 30 min. Finally, nine TU-producing bacterial species were successfully isolated and identified by a combination of biochemical and molecular techniques as Escherichia coli (n = 5), Lactobacillus reuteri (n = 2), Enterococcus faecium (n = 1), and Salmonella enterica subsp. arizonae (n = 1). This report demonstrates that endogenous formation of TU is Brassicaceae induced and occurs under colonic conditions most likely through myrosinase-like enzyme activity expressed by different common intestinal bacterial species.

Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability.

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37°C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains.

Diversity of Campylobacter in retail meat and liver of lambs and goat kids.

The presence, genetic diversity, and antimicrobial susceptibility profile of Campylobacter spp. in retail lamb and goat kid carcasses were assessed. A total of 200 samples consisting of 100 meat and 100 liver surface swabs were collected from 47 lamb and 53 goat kid carcasses at 23 retail markets in Northern Greece, and 125 Campylobacter isolates were recovered from 32 meat surfaces (32%) and 44 liver surfaces (44%). Multiplex polymerase chain reaction and restriction fragment length polymorphism analysis specified Campylobacter coli as the most frequently detected species (59.2%) followed by C. jejuni (40.8%). Pulsed-field gel electrophoresis (PFGE) was applied in order to typify a subset of randomly selected isolates (n=80). SmaI-PFGE successfully clustered the 80 isolates in 38 SmaI-PFGE types, indicating high heterogeneity among the analyzed Campylobacter isolates, and provided data regarding the dissemination of Camplobacter among carcasses stored in the same retail market. Antimicrobial susceptibility profiles of Campylobacter isolates, assessed by the disk-diffusion method, indicated that 31 isolates (24.8%) were multidrug resistant, and the most common profile was the concurrent resistance to tetracycline and streptomycin. Overall, 56.8% of isolates (n=71, multidrug-resistant isolates included) exhibited resistance to at least one antimicrobial (tetracycline 34.4%, quinolones 27.2%, and streptomycin 20.8%). However, all isolates were susceptible to erythromycin and gentamicin. The findings of this study verify the contamination of retail lamb and goat kid carcasses with a heterogeneous population of thermotolerant campylobacters. These data underscore the fact that retail meat and liver of small ruminants could serve as vehicles for consumer contamination with Campylobacter and that further investigation is necessary in order to evaluate the risk imposed by such products within the epidemiology of human campylobacteriosis cases.

The involvement of Pseudoterranova decipiens fish infestation on the shelf-life of fresh Atlantic cod (Gadus morhua) fillet.

Zoonotic nematodes of the family Anisakidae are highly common in many marine fish species, which act as paratenic hosts for the third larval stage. In the fish, these parasites may migrate from the fish's gastro-intestinal tract (GI-tract) further to the coelomic cavity and muscles, making them a possible contamination source of bacteria they carry on their cuticle and in their GI-tract. A previous study revealed no apparent effect of Anisakis simplex on spoilage of fish, but the equally common anisakid species Pseudoterranova decipiens has a larger body surface potentially increasing the bacterial load brought into the fish muscle upon migration. As the presence of shelf-life reducing spoilage bacteria in the microbiome of this anisakid species has been demonstrated, the objective of the present study was to assess the potential shelf-life reducing effect of P. decipiens in fresh fish fillets stored in a domestic refrigerator. Atlantic cod was used as a model since members of the cod family are the third most consumed marine fish globally and it has the highest prevalence of P. decipiens infections. Infected and non-infected codfish fillet portions were collected and microbiologically analyzed at day 0 and day 4 of storage in a domestic fridge. Three isolation media were used to enhance maximum bacterial recovery and isolates were identified using MALDI-TOF MS and 16S rRNA gene sequencing. In parallel to the microbiological examination, sensory analysis was performed daily on the cod fillets to evaluate the freshness of the fish. Results revealed the presence of typical spoilage bacteria (e.g., Pseudomonas sp., Photobacterium sp.) in all fish, but based on the total viable counts, total H2S-producing bacteria, and sensory analysis, there were no objective indications to assume an increased fish spoilage rate by the presence and migration P. decipiens. Additionally, a beta-diversity comparison revealed no significant differences in microbiota composition between infected and non-infected fish parts, though individual heterogeneity in microbiome composition among Atlantic codfish individuals was found. As total viable counts did, however, exceed the guideline limits for fresh fish, further research should now focus on the role of the candling step as a potential source of post-harvest contamination. As such, anisakid infection might still accelerate fish spoilage, though now in an indirect way.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.