Yersinia enterocolitica is one of the priority biological hazards in pork inspection. Persistence of the pathogen, including strains resistant to antimicrobials, should be evaluated in pigs from ...different housing systems for risk ranking of farms. In this 2019 study, tonsils were collected from 234 pigs, of which 69 (29.5%) were fattened on 3 big integrated farms, 130 (55.5%) on 10 medium-sized farms, and 35 (15%) on 13 small family farms. In addition, 92 pork cuts and minced meat samples from the same farms were tested for the presence of Y. enterocolitica using the culture method. Phenotypic and genetic characteristics of the isolates were compared with previously collected isolates from 2014. The overall prevalence of Y. enterocolitica in pig tonsils was 43% 95% CI 36.7−49.7. In pigs from big integrated, medium-sized, and small family farms, the prevalence was 29%, 52%, and 40%, respectively. All retail samples of portioned and minced pork tested negative for pathogenic Y. enterocolitica, likely due to high hygienic standards in slaughterhouses/cutting meat or low sensitivity of culture methods in these matrices. The highest recovery rate of the pathogen from tonsils was found when alkali-treated PSB and CIN agar were combined. The biosecurity category of integrated and medium farms did not affect the differences in prevalence of Y. enterocolitica (p > 0.05), in contrast to family farms. Pathogenic ail-positive Y. enterocolitica biotype 4 serotype O:3 persisted in the tonsils of pigs regardless of the type of farm, slaughterhouse, and year of isolation 2014 and 2019. PFGE typing revealed the high genetic concordance (80.6 to 100%) of all the Y. enterocolitica 4/O:3 isolates. A statistically significant higher prevalence of multidrug-resistant Y. enterocolitica 4/O:3 isolates was detected in the tonsils of pigs from big integrated farms compared to the other farm types (p < 0.05), with predominant and increasing resistance to nalidixic acid, chloramphenicol, and streptomycin. This study demonstrated multidrug resistance of the pathogen in pigs likely due to more antimicrobial pressure on big farms, with intriguing resistance to some clinically relevant antimicrobials used in the treatment of yersiniosis in humans.
This study aimed to implement a microencapsulated form of selected autochthonous lactic-acid bacteria (LAB) isolated from the cheese-production chain and natural rennet obtained from suckling lambs ...in the traditional production of hard sheep cheese, “Paški sir”, from the island of Pag, Croatia. Two different formulations of microparticles were prepared: (i) microparticles containing the strain of both Lactiplantibacillus plantarum and Lactococcus lactis (S2) and (ii) microparticles containing both strains and natural rennet (S3). These formulations were used in the production of Paški sir cheese simultaneously with standard production using non-encapsulated commercial starter cultures and commercial rennet (S1). The number of Lc. lactis isolates decreased at day 30 and were not isolated during the remaining ripening process, whereas the number of L. plantarum remained stable throughout the ripening process. The level of LAB and the release of the rennet from microsphere formulations at the end allowed for the production of cheese with the same characteristics as the commercial product, indicating no negative interactions of natural rennet, bacterial culture, and chemical components of microparticles. To our knowledge, this is the first report of a microencapsulated L. plantarum (isolated from the abomasum of lambs) coupled with natural lamb’s rennet used in the production of hard sheep cheese. This pilot study showed the great potential for maintaining authenticity in cheese production by combining traditional and sustainable innovative technologies.
Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) ...collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.
Wastewaters from a treatment plant discharging into a canal harboring fish may present sources of microbiological hazard for wild fish. Such fish, inhabiting microbiologically polluted bodies of ...water, can be contaminated by human pathogens and, if used for human consumption, may pose a risk to public health. Hence, in this work the aim was to identify tested strains from tissues of wild fish living in the receiving water bodies, captured from locations up to 12 km from the point of discharge of treated water of town Virovitica in order to assess the bacterial threat of the WWTP on fish and potentially on public health. A rather rich diversity of bacterial genera was isolated from gill tissues and internal organs. The most frequent isolate was
which has gained public health recognition as an opportunistic pathogen.
, an indicator bacterium for aquatic contamination, was retrieved from all investigated tissues. Opportunistic human pathogens as well as some zoonotic agents were also retrieved from fish tissues (
,
spp.,
,
,
,
and others). Public health hazard is particularly pronounced regarding local recreational fishermen who fish out, handle and consume fish from respective waters.
The aim of this research is to use isolated and characterized autochthonous functional starter cultures from traditional Croatian dry sausages and to evaluate their capacity for industrial production ...of five sausages (Cajna sausage, Zimska sausage, Becka sausage, Srijemska sausage and Slavonski kulen). These defined autochthonous functional starter cultures (combination of Lactobacillus and Staphylococcus strains) were used to produce five different industrial sausages which were compared by a panel. The viability of introduced autochthonous Lactobacillus and Staphylococcus strains and their effect on the final product characteristics, namely microbiological, physicochemical and sensory properties were monitored. The obtained results indicate that autochthonous starter cultures survived industrial production of sausages and can be used for production of sausages under controlled conditions. Autochthonous starter cultures obtained better results in the organoleptic evaluation, microbial safety and prolonged shelf-life in comparison with commercial starter cultures.
The aim of the present study was to investigate the diversity, distribution of virulence factor genes and antimicrobial resistance phenotypes of Vibrio Harveyi clade species from two distinct and ...spatially distant near-shore bivalve farming environments located in protected marine reserves of the eastern Adriatic. Tissues of farmed Mediterranean mussel Mytilus galloprovincialis and European flat oyster Ostrea edulis, seawater and sediment samples were collected during six bi-monthly sampling events conducted over one year from farming locations in Lim Bay and Mali Ston Bay, Croatia. Eight distinctive Harveyi subclades, namely, V. harveyi, V. alginolyticus, V. diabolicus, V. jasicida, V. parahaemolyticus, V. rotiferianus, V. campbellii and V. communis, were detected by Phylogenetic Multilocus Sequence Analysis (MLSA) based on rpoD, toxR and rctB concatenated genes sequences. Genomic fingerprinting of MLSA-classified samples revealed substantial diversity of environmental isolates. The virulence-related genes considered typical for the Harveyi clade and their homologues were represented in all subclades with varying frequencies. Of twelve antibiotics commonly used in human and veterinary medicine, the most frequent resistance phenotypes were resistance to ampicillin (90%), erythromycin (39%), imipenem (29%) and streptomycin (22%). >60% of isolates were resistant to at least two classes of antibiotics. Seasonal or site-specific differences were found in the proportion of isolates carrying virulence genes and displaying specific antibiotic resistance phenotype.
This study is the first to provide a broader perspective on the properties of Vibrio Harveyi clade species associated with ecosystems under presumably low anthropogenic pressures, such as marine protected areas. New data on virulence and antibiotic resistance are presented of Harveyi clade species coexisting in different environmental compartments, including those that have been largely overlooked or understudied in the context of bivalve aquaculture. Taken together, these results highlight the importance of monitoring the presence of potentially pathogenic strains in the natural Vibrio population of bivalve farming areas to reduce the risk to seafood consumers.
•Eight Harveyi subclades identified in the eastern Adriatic bivalve aquaculture.•Isolates of all Harveyi subclades displayed substantial genetic heterogeneity.•Typical V. harveyi virulence factors spread across all Harveyi clade species.•>60% of isolates displayed resistance to at least two antibiotic classes.•Monitoring of Vibrio spp. in the Adriatic bivalve aquaculture strongly recommended.
Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an ...esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.Iron plays vital roles in important biological processes in fish, but can be toxic in high concentrations. The information on metalloproteins that participate in maintenance of Fe homeostasis in an esocid fish, the northern pike, as an important freshwater bioindicator species, are rather scarce. The aim of this study was to identify main cytosolic constituents that sequester Fe in the northern pike liver. The method applied consisted of two-dimensional HPLC separation of Fe-binding biomolecules, based on anion-exchange followed by size-exclusion fractionation. Apparent molecular masses of two main Fe-metalloproteins isolated by this procedure were ~360 kDa and ~50 kDa, with the former having more acidic pI, and indicated presence of ferritin and hemoglobin, respectively. MALDI-TOF-MS provided confirmation of ferritin subunit with a m/z peak at 20.65 kDa, and hemoglobin with spectra containing main m/z peak at 16.1 kDa, and smaller peaks at 32.1, 48.2, and 7.95 kDa (single-charged Hb-monomer, dimer, and trimer, and double-charged monomer, respectively). LC-MS/MS with subsequent MASCOT database search confirmed the presence of Hb-β subunits and pointed to close relation between esocid and salmonid fishes. Further efforts should be directed towards optimization of the conditions for metalloprotein analysis by mass spectrometry, to extend the knowledge on intracellular metal-handling mechanisms.
It was assumed that gas-phase ligation of metal monocations by flavonoids might provide some insight on the intrinsic antioxidant activity of the latter. Thus, the ligation of Fe+ and Cu+ ions by ...apigenin (1), luteolin (2), kaempferol (3), quercetin (4), myricetin (5), and naringenin (6) was investigated in the gas phase in a Fourier transform mass spectrometer (FTMS). Both of the metal ions, which were produced by laser desorption ionization (LDI), bind consecutively to two neutral flavonoid molecules either with or without the simultaneous loss of some part (H, CO, H2O) of the latter. The flavonoids are present in the instrument at steady concentrations. The formation of flavonoid positive ions by charge exchange is also a common observation but is accompanied, in some cases, by a loss of H, CO, or H2O fragments. The reaction paths and observed fragmentations are presented. The results are supported by DFT B3LYP calculations that indicate a preference for metal ion attack at C-ring and not at the B-ring site considered to be mainly responsible for flavonoid antioxidant activity. Keywords: Flavonoids; antioxidants and antiradical activity; DFT calculation; FTICR MS; ligation and fragmentation products with Fe+ and Cu+
is considered one of the most common bacterial causative agents of reproductive tract disorders in poultry. In this study, phylogenetic analysis of partial
sequences and biotyping using MALDI-TOF MS ...was done in order to investigate the genetic diversity of
isolates from thirteen farms with different biosecurity measures and management practices. Sampling was done as a part of regular monitoring, except for Farms 9-13 that were included in the study to represent extensive production system with lowest biosecurity levels. Pharyngeal and cloacal swabs were taken from live animals, while swabs from trachea, liver, peritoneum and oviduct were taken during necropsies. After cultivation and identification, strains from each farm were randomly selected for sequencing and biotyping. Both results showed high level of heterogeneity among the isolates originating from farms with low biosecurity levels, unlike isolates from farms with higher biosecurity levels and proper management that were more closely related and clustered together. Such correlation was statistically significant. Low biosecurity levels enable horizontal transmission of the pathogens, as well as gene transfer. The results confirm the importance of adequate biosecurity measures and management on poultry farms as they greatly affect the genetic diversity of the pathogens. Therefore, implementation of basic biosecurity measures could help control the heterogeneity of
strains, which would alleviate control of the infection prevalence on farms through immunoprophylaxis and consequently improve poultry production. Also, the genetic diversity of
on poultry farms could be a good bioindicator of management practice and biosecurity measures used.
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