Prevalence, quantitative load and genetic diversity of Campylobacter spp. in dairy cattle herds in Lithuania

BACKGROUND: Campylobacteriosis is a zoonotic disease, and animals such as poultry, pigs and cattle may act as reservoirs for Campylobacter spp. Cattle shed Campylobacter spp. into the environment and they can act as a reservoir for human infection directly via contact with cattle or their faeces or indirectly by consumption of contaminated food. The aim of this study was to determine the prevalence, the quantitative load and the genetic strain diversity of Campylobacter spp. in dairy cattle of different age groups. RESULTS: Faecal samples of 200 dairy cattle from three farms in the central part of Lithuania were collected and examined for Campylobacter. Cattle herds of all three farms were Campylobacter spp. positive, with a prevalence ranging from 75% (farm I), 77.5% (farm II) to 83.3% (farm III). Overall, the highest prevalence was detected in calves (86.5%) and heifers (86.2%). In contrast, the lowest Campylobacter prevalence was detectable in dairy cows (60.6%). C. jejuni, C. coli, C. lari and C. fetus subsp. fetus were identified in faecal samples of dairy cattle. C. upsaliensis was not detectable in any sample. The high counts of Campylobacter spp. were observed in faecal material of dairy cattle (average 4.5 log10 cfu/g). The highest numbers of Campylobacter spp. were found in faecal samples from calves (average 5.3 log10 cfu/g), whereas, faecal samples from cows harboured the lowest number of Campylobacter spp. (average 3.7 log10 cfu/g). Genotyping by flaA PCR-RFLP analysis of selected C. jejuni isolates showed that some genotypes were present in all farms and all age groups. However, farm or age specific genotypes were also identified. CONCLUSIONS: Future studies are needed to investigate risk factors related to the degree of colonisation in cattle. Based on that, possible measures to reduce the colonisation and subsequent shedding of Campylobacter in cattle could be established. It is important to further investigate the epidemiology of Campylobacter in the cattle population in order to assess associated risks to public health

Overlap of Antibiotic Resistant Campylobacter jejuni MLST Genotypes Isolated From Humans, Broiler Products, Dairy Cattle and Wild Birds in Lithuania

Antimicrobial resistance was determined for 341 thermophilic Campylobacter jejuni isolates obtained from human clinical cases (n = 101), broiler products (n = 98), dairy cattle (n = 41) and wild birds (n = 101) with known multilocus sequence types (MLST) in Lithuania. The minimum inhibitory concentration (MIC) values for ciprofloxacin, tetracycline, gentamicin, ceftriaxone and erythromycin were determined with the agar dilution method. MIC values were compared with MLST types to find possible associations among isolation source, sequence type and resistance to antibiotics. The proportions of resistant strains were 94.2% (human), 95% (wild birds), 100% (broiler products) and 100% (dairy cattle) for one of the tested antibiotics. Most frequently, resistance to ciprofloxacin was observed (91.5%), followed by ceftriaxone with 60.4%, and tetracycline (37.8%). However only three C. jejuni strains were resistant to erythromycin (0.9%) and all tested thermophilic Campylobacter strains were sensitive to gentamicin. Most of the examined C. jejuni isolates (80.6%) showed resistance to at least one of three profiles: CIP+AXO (28.1%), TET+CIP+AXO (26.7%) and CIP (25.8%). Statistically significant differences in resistance to tetracycline were found between C. jejuni strains obtained from cattle (85.4%) and broiler products (64.3%) (P < 0.05). The majority (87.1%) of the tested strains from wild birds were resistant to ciprofloxacin (P < 0.05). The results showed that strains of novel ST’s showed significantly lower resistance to ceftriaxone (P < 0.05). The ST-21 (CC21) (78.8%) was identified with significantly higher multidrug resistance relatively to other tested ST’s in this study. Our results emphasize the high antimicrobial resistance of phylogenetically diverse C. jejuni strains isolated from different sources including specific genotypes of wild bird’s strains in Lithuania. The results support the opinion that not only broiler products but cattle and wild birds may be a reservoir of resistant C. jejuni and stipulate a risk of spread or resistant bacteria. There is increasing need for broad surveillance and control measures to track changes and pathways of antimicrobial resistance of C. jejuni in epidemiologically distinct populations

MLST genotypes of Campylobacter jejuni isolated from broiler products, dairy cattle and human campylobacteriosis cases in Lithuania

Background Campylobacter (C.) jejuni is the leading cause of human campylobacteriosis worldwide. We performed a molecular epidemiological study to investigate the genetic relationship among C. jejuni strains isolated from human diarrhoeal patients, broiler products and dairy cattle in Lithuania. Methods The C. jejuni isolates from human clinical cases, dairy cattle and broiler products were genotyped using multilocus sequence typing (MLST). Allele numbers for each housekeeping gene, sequence type (ST), and clonal complex (CC) were assigned by submitting the DNA sequences to the C. jejuni MLST database (http://pubmlst.org/campylobacter). Based on the obtained sequence data of the housekeeping genes a phylogenetic analysis of the strains was performed and a minimum spanning tree (MST) was calculated. Results Among the 262 C. jejuni strains (consisting of 43 strains isolated from dairy cattle, 102 strains isolated from broiler products and 117 clinical human C. jejuni strains), 82 different MLST sequence types and 22 clonal complexes were identified. Clonal complexes CC21 and CC353 predominated among the C. jejuni strains. On ST-level, five sequence types (ST-5, ST-21, ST-50, ST-464 and ST-6410) were dominating and these five STs accounted for 35.9% (n = 94) of our isolates. In addition, 51 (19.5%) C. jejuni strains representing 27 (32.9%) STs were reported for the first time in the PubMLST database (http://pubmlst.org/campylobacter). The highest Czekanowski index or proportional similarity index (PSI) was calculated for C. jejuni strains isolated from human campylobacteriosis cases and broiler products (PSI = 0.32) suggesting a strong link between broiler strains and human cases. The PSI of dairy cattle and human samples was lower (PSI = 0.11), suggesting a weaker link between bovine strains and human cases. The calculated Simpson’s index of all C. jejuni isolates showed a high genetic diversity (D = 0.96). Conclusion Our results suggest that broiler products are the most important source of human campylobacteriosis in Lithuania. The study provides information on MLST type distribution and genetic relatedness of C. jejuni strains from humans, broiler products and dairy cattle in Lithuania for the first time, enabling a better understanding of the transmission pathways of C. jejuni in this country

Prevalence, antimicrobial susceptibility and virulence gene profiles of Arcobacter species isolated from human stool samples, foods of animal origin, ready-to-eat salad mixes and environmental water

Background Members of the genus Arcobacter are considered as emerging zoonotic food and waterborne pathogens that cause gastroenteritis and bacteremia in humans. However, the potential risk that Arcobacter species pose to public health remains unassessed in various countries, including Baltic states. Therefore, the aim of this study was to determine the prevalence, antimicrobial susceptibility and presence of putative virulence genes of Arcobacter isolates recovered from humans, food products and environmental water in Lithuania. Results A total of 1862 samples were collected and examined from 2018 to 2020 in the city of Kaunas. Overall, 11.2% (n = 208) of the samples were positive for the presence of Arcobacter spp. The highest prevalence was detected in chicken meat (36%), followed by environmental water (28.1%), raw cow milk (25%), ready-to-eat salad mixes (7.1%) and human stool (1.7%). A. butzleri was the most frequently isolated species (n = 192; 92.3%), followed by A. cryaerophilus (n = 16; 7.7%). Arcobacter spp. antimicrobial susceptibility testing revealed unimodally distributed aggregated minimal inhibitory concentrations (MICs) for gentamicin, tetracycline, ciprofloxacin, ampicillin and erythromycin. However, a bimodal distribution for azithromycin was found with 96.2% of determined MICs above the epidemiological cut-off value (ECOFF) defined for Campylobacter jejuni (0.25 µg/ml). Majority of the Arcobacter isolates (n = 187; 89.9%) showed high susceptibility to ciprofloxacin with MICs below or equal to the ECOFF value of 0.5 µg/ml. The putative virulence genes cadF (100%), ciaB (100%), cj1349 (99%), tlyA (99%), mviN (97.9%) and pldA (95.8%) were the predominant genes detected among A. butzleri isolates. In contrast, the mviN and ciaB genes were present in all, whereas cj1349 (12.5%), tlyA (25%) and hecA (12.5%) were only detected in few A. cryaerophilus isolates. Conclusions Our results demonstrate that food products and environmental water in Lithuania are frequently contaminated with Arcobacter spp. that carry multiple putative virulence genes. Furthermore, A. butzleri were isolated from 1.7% of inpatients. Fluoroquinolones and aminoglycosides were found to be more effective against Arcobacter in comparison to other antimicrobial agents. However, further studies are needed to determine the pathogenic mechanisms and factors that facilitate the spread of Arcobacter infections

Helminths of red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania

Red foxes and raccoon dogs are hosts for a wide range of parasites including important zoonotic helminths. The raccoon dog has recently invaded into Europe from the east. The contribution of this exotic species to the epidemiology of parasitic diseases, particularly parasitic zoonoses is unknown. The helminth fauna and the abundance of helminth infections were determined in 310 carcasses of hunted red foxes and 99 of raccoon dogs from Lithuania. Both species were highly infected with Alaria alata (94·8% and 96·5% respectively) and Trichinella spp. (46·6% and 29·3%). High and significantly different prevalences in foxes and raccoon dogs were found for Eucoleus aerophilus (97·1% and 30·2% respectively), Crenosoma vulpis (53·8% and 15·1%), Capillaria plica (93·3% and 11·3%), C. putorii (29·4% and 51·5%), Toxocara canis (40·5% and 17·6%) and Uncinaria stenocephala (76·9% and 98·8%). The prevalences of the rodent-transmitted cestodes Echinococcus multilocularis, Taenia polyacantha, T. crassiceps and Mesocestoides spp. were significantly higher in foxes than in raccoon dogs. The abundances of E. multilocularis, Mesocestoides, Taenia, C. plica and E. aerophilus were higher in foxes than those in raccoon dogs. A. alata, U. stenocephala, C. putorii and Echinostomatidae had higher abundances in raccoon dogs. The difference in prevalence and abundance of helminths in both animals may reflect differences in host ecology and susceptibility. The data are consistent with red foxes playing a more important role than raccoon dogs in the transmission of E. multilocularis in Lithuani

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

Application of Edible Coating Based on Liquid Acid Whey Protein Concentrate with Indigenous Lactobacillus helveticus for Acid-Curd Cheese Quality Improvement

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Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance

The demand for healthy foods without artificial food additives is constantly increasing. Hence, natural food preservation methods using bioprotective cultures could be an alternative to chemical preservatives. Thus, the main purpose of this work was to screen the indigenous lactobacilli isolated from fermented cow milk for their safety and antifungal activity to select the safe strain with the strongest fungicidal properties for the development of bioprotective acid whey protein concentrate (AWPC) based fermentates and their coatings intended for fresh cheese quality maintenance. Therefore, 12 lactobacilli strains were isolated and identified from raw fermented cow milk as protective cultures. The safety of the stains was determined by applying antibiotic susceptibility, haemolytic and enzymatic evaluation. Only one strain, Lacticaseibacillus paracasei A11, met all safety requirements and demonstrated a broad spectrum of antifungal activity in vitro. The strain was cultivated in AWPC for 48 h and grew well (biomass yield 8 log10 cfu mL−1). L. paracasei A11 AWPC fermentate was used as a vehicle for protective culture in the development of pectin-AWPC-based edible coating. Both the fermentate and coating were tested for their antimicrobial properties on fresh acid-curd cheese. Coating with L. paracasei A11 strain reduced yeast and mould counts by 1.0–1.5 log10 cfu mL−1 (p ≤ 0.001) during cheese storage (14 days), simultaneously preserving its flavour and prolonging the shelf life for six days.Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality MaintenancepublishedVersio