Using Essential Oils to Reduce Yersinia enterocolitica in Minced Meat and in Biofilms.

Yersiniosis, one of the leading foodborne infections in the European Union, is caused by Yersinia enterocolitica. In this study, the antibacterial and antibiofilm effects of cinnamon (Cinnamomum zeylanicum Nees), clove (Syzygium aromaticum L.), oregano (Origanum vulgare L.), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.), and winter savory (Satureja montana L.) essential oils were investigated against Y. enterocolitica strains belonging to the bioserotype 4/O:3. Cinnamon essential oil showed the highest antibacterial activity, with an MIC value 0.09 µL/mL, followed by oregano and thyme essential oils, with MIC values from 0.09 to 0.18 µL/mL, and from 0.18 to 0.23 µL/mL, respectively. Thyme essential oil at 0.23 µL/g (MIC) and at 0.46 µL/g (2MIC) significantly (p < 0.05) reduced the number of Y. enterocolitica by 0.38 log CFU/g and 0.64 log CFU/g, respectively, in minced pork meat during storage at 4 °C for 4 days. The Y. enterocolitica strains formed biofilms at 15 °C and 37 °C in tryptic soy broth and Luria-Bertani broth, while no biofilms were obtained at 5 °C, and in meat broth nutrient media. Applying the minimum bactericidal concentrations of cinnamon, clove, oregano, rosemary, thyme, and winter savory essential oils on preformed biofilms led to significant reductions being observed in the range from 45.34% to 78.89%. A scanning electron microscopy assay showed the devastating impact of oregano and thyme essential oils on the morphology of Y. enterocolitica bacterial cells. In conclusion, the results of this study show that essential oils possess high anti-Yersinia and antibiofilm effects.

Multilocus sequence analysis (MLSA) of a Nocardia cyriacigeorgica strain causing severe bovine mastitis in Bosnia and Herzegovina.

Nocardia cyriacigeorgica is a well-known agent of human nocardiosis and is considered an emerging pathogen, however, its identification to the species level is complex for many clinical laboratories. Available data on the clinical significance of N. cyriacigeorgica in veterinary medicine are sparse and mainly concern isolated reports of pyogranulomatous lesions in domestic animals. We report a case of severe bovine mastitis caused by N. cyriacigeorgica that did not respond to conventional antimicrobial therapy in a small holding in Bosnia and Herzegovina. After isolation of the pathogen, further identification by routine microbiological methods was not possible. Susceptibility to antimicrobials was tested using the disc diffusion method according to published recommendations. The sample was also tested by MALDI-ToF MS with inconclusive results. In addition, 16S rRNA sequence analysis, verified by multilocus sequence analysis (MLSA) using the gyrB, 16S rRNA, secA1, and hsp65 sequences, confirmed the species N. cyriacigeorgica. To our knowledge, this is the first report of isolation of N. cyriacigeorgica from a clinical case of bovine mastitis in a European dairy farm and the first MLSA method approach to distinguish a Nocardia spp. strain isolated from animals.

Novel extraction of polyphenols from sour cherry pomace using natural deep eutectic solvents - Ultrafast microwave-assisted NADES preparation and extraction.

In this work, new approaches for the green extraction of polyphenols from sour cherry pomace were explored. Three Natural Deep Eutectic Solvents (NADES) systems based on choline chloride (ChCl) as a hydrogen bond acceptor (HBA) and malic acid, urea, and fructose (MalA, Ur, and Fru) as hydrogen bond donors (HBD) were used. NADES systems were prepared by heating and stirring (H&S), ultrasound (US), and microwave (MW) methods. It was found that MW-assisted preparation was the fastest requiring less than 30 s. Polyphenol extraction from cherry pomace was performed also by three mentioned methods, and compared with conventional methods. MW extraction was the most rapid with less than 5 min necessary for the extract preparation. All three NADES systems were highly efficient for anthocyanin extraction, but the most efficient was ChCl:MalA system. Extract based on ChCl:MalA system was for 62.33% more efficient for anthocyanin extraction comparing with the conventional solvent.

Medicinal Plants Extracts Impact on Oxidative Stress in Mice Brain Under the Physiological Conditions: the Effects of Corn Silk, Parsley, and Bearberry.

This study was performed to examine the effects of medicinal plant extracts of corn silk (Stigma maydis), parsley leaf (Petroselini folium), and bearberry leaf (Uvae ursi folium) on antioxidant status of the brain of experimental animals (mice) under the physiological conditions. Biological properties of these plants are insufficiently investigated and the aim was to explore their possible antioxidant effects that can alleviate oxidative damage of the brain tissue. Corn silk extract showed positive effect on activities of antioxidant enzymes in mice brain tissue. Parsley extract induced the increase in glutathione content and decrease of lipid peroxidation. Bearberry leaf extract induced catalase activity and decrease of hydroxyl radical content, while malonyldialdehide accumulation was maintained at the control level. Results obtained in this study support the use of corn silk, parsley and bearberry leaves as natural antioxidant sources in the prevention and treatment of brain tissue damages and different diseases caused by oxidative stress.

The evaluation of phenolic content, in vitro antioxidant and antibacterial activity of Mentha piperita extracts obtained by natural deep eutectic solvents.

The focus of this study was to evaluate whether six choline chloride-based natural deep eutectic solvents (NADES) could serve as solvents for the extraction of bioactives from the leaves of Mentha piperita. NADES extracted significantly higher amounts of phenols from peppermint than 70% ethanol and may be useful in the extraction of targeted major compounds from peppermint, like rosmarinic acid, at a similar level as 70% ethanol. The microdilution method for in vitro antibacterial activity showed that all NADES exhibit bacterial growth inhibition at a lower concentration than 70% ethanol, especially NADESs containing organic acids. The majority of NADES extracts neutralize DPPH radical at a lower concentration than conventional solvent and showed similar ability to reduce Fe3+ to Fe2+ ions in FRAP assay. NADES can be useful in the isolation of phenolic compounds from plant sources and should be considered as novel, sustainable, and low-cost solvents with a variety of applications.

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis.

The aim of the present study was to determine the bioactive compounds in four essential oils (EO's) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO's and EOC's (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO's and EOC's in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO's and EOC's (the essential oil components, carvacrol and thymol) on eradication of preformed 48 h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.

Molecular characterization of Listeria monocytogenes isolates from a small-scale meat processor in Montenegro, 2011-2014.

The presence of Listeria monocytogenes was evaluated in a small-scale meat processing facility in Montenegro during 2011-2014. L. monocytogenes isolates from traditional meat products and environmental swabs were subjected to a) molecular characterization b) serotyping by both multiplex PCR and next generation sequencing (NGS) c) potential antimicrobial resistance (AMR) was assessed by extraction of specific genes from NGS data and d) screening for the presence of some disinfectant resistance markers. Overall, traditional meat products were contaminated, most likely from incoming raw materials, with 4 major specific STs of L. monocytogenes (ST515, ST8, ST21, ST121) representing 4 clonal complexes (CC1, CC8, CC21, CC121) identified during the four-year period. These strains belonged to serogroup IIa which predominated, followed by IVb (ST515, CC1). The strains from environmental swabs belonged, exclusively, to ST21 and were isolated from cutting board and floor swabs in 2011. Furthermore, we found Tn6188, a novel transposon conferring tolerance to BC, to be specific to sequence type ST121. In addition, antimicrobial resistance genes mprF and fosX were present in clonal complexes CC21 and CC121, while complexes CC8 and CC1 exclusively harbored the mprF antimicrobial resistance gene.

Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province, Serbia.

The aim of the study was to characterize multidrug-resistant (MDR) Escherichia coli isolates collected in Serbia from bovine clinical mastitis cases and diseased pigs, mainly with molecular methods. A total of 48 E. coli isolates was collected during the years 2013-2014, of which 22 were MDR and were included in further analysis. Phylogenetic typing showed that 17 isolates belonged to group A, while two isolates were classified in group B1 and a single one in group D. All isolates showed unique macrorestriction patterns. Phenotypic susceptibility testing revealed resistances of the isolates against up to 13 antimicrobial agents, including resistance to fluoroquinolones. A wide variety of resistance genes was detected by PCR amplification and sequencing of amplicons. Sequence analysis of the quinolone resistance determining regions of topoisomerase genes revealed mutations in gyrA, parC, and/or parE. Plasmid-mediated quinolone resistance genes were detected in two porcine (aac-6'-Ib-cr and qnrS, respectively) isolates and a single bovine (aac-6'-Ib-cr) isolate. Resistance genes were found to be located on conjugative plasmids in 16 cases, many of which conferred a multidrug resistance phenotype. In conclusion, the plentitude of resistance genes located on conjugative plasmids and integrons in E. coli from cows and pigs in Vojvodina, Serbia, pose a high risk for horizontal gene transfer in bacteria from livestock husbandry.

Tracking of Listeria monocytogenes in meat establishment using Whole Genome Sequencing as a food safety management tool: A proof of concept.

Repeated Listeria outbreaks particularly associated with Ready-To-Eat (RTE) delicatessen meat products have been reported annually at global level. The most frequent scenario that led to foodborne outbreaks was the post-thermal treatment cross-contamination of deli meat products during slicing and modified atmosphere packaging (MAP). The precondition for such cross contamination is the previous introduction of Listeria into meat processing facilities and subsequent colonization of the production environment, associated with formation of biofilms resilient to common sanitation procedures regularly applied in meat establishments. The use of Whole Genome Sequencing (WGS) can facilitate the understanding of contamination and colonization routes of pathogens within the food production environment and enable efficient pathogen tracking among different departments. This study aimed to: a) provide a proof of concept on practical use of WGS in a meat establishment to define the entry routes and spread pattern of L. monocytogenes, and b) to consider the regular use of WGS in meat processing establishments as a strong support of food safety management system. The results revealed that Listeria spp. was present in slaughter line, chilling chambers, deboning, slicing, MAP, as well as in corridors and dispatch (53 positive samples, out of 240). Eight L. monocytogenes isolates (out of 53) were identified from the slaughterhouse, chilling chambers, deboning, MAP and dispatch. L. monocytogenes isolates were of three different serotypes (1/2a, 1/2c, 4b) and correspondingly of three MLST sequence types. Overall, two pairs of L. monocytogenes isolates were genetically identical, i.e. two serotype 4b isolates (ST1), isolated from water drain at dispatch unit and two isolates obtained from slaughterhouse (floorwall junction at the carcass wash point) and MAP (water drain). These findings indicated that L. monocytogenes isolates identified in meat processing units (MAP, chilling chamber and dispatch) originated from the slaughter line. Further, all eight L. monocytogenes isolates were confirmed to be biofilm producers on glass and stainless steel surfaces. The identification of the main entry routes of L. monocytogenes into meat establishments and tracking the routes for spread of the pathogen are of essential importance to define appropriate risk mitigation strategies for L. monocytogenes in meat production environment. The routine use of WGS for bacterial characterization, as a strong support of food safety management system in meat establishments, will require the cost-effective approach. It may encompass in-house sequencing when sequencing equipment is used for multiple applications (e.g. WGS of pathogens, starter cultures and spoilage organisms).

Clonal persistence of Salmonella enterica serovars Montevideo, Tennessee, and Infantis in feed factories.

INTRODUCTION: Novel molecular techniques applied in biotechnology research have provided sound evidence on clonal persistence of distinct serovars of Salmonella in feed factory environments, over long periods of time (months, even years), which can be responsible for repeated in-house contamination of final products. In this study, we examined the possibility of clonal persistence of isolates of three Salmonella serovars that have been repeatedly identified in animal feed samples from three feed factories throughout a two-year period. METHODOLOGY: The isolates Salmonella enterica serovars Tennessee (n = 7), Montevideo (n = 8), and Infantis (n = 4) were tested for genetic diversity using pulsed-field gel electrophoresis (PFGE) and multicellular behavior patterns by applying the Congo red agar test. RESULTS: SpeI and XbaI macro-restriction profiles indicated that isolates S. Montevideo and S. Infantis were identical, whereas isolates of S. Tennessee demonstrated greater genetic diversity, although the genetic differences did not exceed 10%. All Salmonella serovars demonstrated the ability to produce predominant matrix compounds essential for biofilm formation, curli fimbriae and cellulose. CONCLUSIONS: The identification of identical clones of S. Montevideo and S. Infantis, as well as the minor genetic diversity of S. Tennessee, which have been repeatedly isolated from animal feed in three production plants throughout a two-year period, indirectly suggests the possibility of their persistence in feed factory environments. Their ability to express the key biofilm matrix components further supports this hypothesis.

Results of bacteriological and cytological examinations of the endometrium of subfertile mares in stud farms in Serbia.

Uterine microbiology, antimicrobial susceptibility and endometrial cytology were investigated in a total of 51 mares with fertility problems from 16 different stud farms in Serbia. Uterine cultures were performed after collection with a double guarded uterine swab, and endometrial cytology was evaluated after collection of endometrial cells with a special device (cytology brush). In 21 of 51 mares, at least one bacterial species was isolated from the uterus; the most frequent were Streptococcus equi subsp. zooepidemicus (13 isolates) and E. coli (four isolates). All isolates of Streptococcus equi subsp. zooepidemicus were susceptible to penicillin. Results from endometrial cytology were inconsistent; in 17 animals with positive bacteriological culture, cytology was not altered. It can be concluded that in Serbia, as in many other contries, Streptococcus equi subsp. zooepidemicus is the main cause for equine endometritis. It can be easily diagnosed by uterine culture but endometrial cytology does not always prove the existence of an endometrial infection with this agent.