Harnessing Nature's Defence: The Antimicrobial Efficacy of Pasteurised Cattle Milk-Derived Extracellular Vesicles on Staphylococcus aureus ATCC 25923.

Increasing antimicrobial resistance (AMR) challenges conventional antibiotics, prompting the search for alternatives. Extracellular vesicles (EVs) from pasteurised cattle milk offer promise, due to their unique properties. This study investigates their efficacy against five pathogenic bacteria, including Staphylococcus aureus ATCC 25923, aiming to combat AMR and to develop new therapies. EVs were characterised and tested using various methods. Co-culture experiments with S. aureus showed significant growth inhibition, with colony-forming units decreasing from 2.4 × 105 CFU/mL (single dose) to 7.4 × 104 CFU/mL (triple doses) after 12 h. Milk EVs extended lag time (6 to 9 h) and increased generation time (2.8 to 4.8 h) dose-dependently, compared to controls. In conclusion, milk EVs exhibit dose-dependent inhibition against S. aureus, prolonging lag and generation times. Despite limitations, this suggests their potential in addressing AMR.

In Vitro Antibacterial and Antioxidative Activity and Polyphenolic Profile of the Extracts of Chokeberry, Blackcurrant, and Rowan Berries and Their Pomaces.

The chemical composition of berries and berry pomaces is diverse, containing polyphenolic components that may have both antibacterial and antioxidative properties. In the present study, in vitro antibacterial effect of the extracts of chokeberry, blackcurrant, and rowan berries and berry pomaces against L. monocytogenes, S. aureus, E. coli, and C. jejuni was studied. In addition, the polyphenolic profile and antioxidant activity of these extracts were investigated. The polyphenolic profiles in the aqueous and 30% ethanolic extracts were determined chromatographically by HPLC-MS, and the total polyphenol content was estimated spectrophotometrically by HPLC-DAD-UV. The minimal inhibition concentrations (MICs) of the extracts against tested bacteria were determined by the broth microdilution method. The content of total polyphenols was highest and good antioxidative properties of the extracts were determined for chokeberry and blackcurrant berries and their pomaces. The highest proportions of total quercetin derivatives and anthocyanins were found in the extracts of chokeberry berry/pomace and blackcurrant berry/pomace, respectively. The sensitivity of tested microbes to the extracts of berries and berry pomaces was as follows: S. aureus > L. monocytogenes > E. coli and C. jejuni. In vitro antibacterial activity of tested extracts depended on the extraction solvent, mainly for the ethanolic extracts. Findings suggest that chokeberry and blackcurrant berries and their pomaces can be used as a good source of polyphenols with antioxidative properties, and they also have antibacterial activity against some foodborne pathogenic bacteria. It is important that the valuable compounds are extracted from juice press residues before their disposal.

The Effect of Fruit and Berry Pomaces on the Growth Dynamics of Microorganisms and Sensory Properties of Marinated Rainbow Trout.

Plant pomaces in suitable forms (powders, extracts) can be used in foods of animal origin to increase the nutritional value and safety of these foods. In the present study, water extracts of apple, black currant, rhubarb and tomato pomaces were used in fish marinade solutions to evaluate their effect on the growth dynamics of microorganisms and the growth potential of Listeria monocytogenes by challenge testing. The results showed that mesophilic aerobic microorganisms, Pseudomonas spp., yeasts and moulds remained at acceptable levels throughout the predetermined storage period. The challenge test results showed that the overall growth potential of L. monocytogenes in all marinated rainbow trout samples remained at ≤0.5 log10 cfu/g during the study period, and none of the marinated fish samples supported the growth of L. monocytogenes. In addition, the effect of fruit and berry pomaces on the sensory properties of marinated rainbow trout samples was evaluated. The results revealed that it is possible to effectively use fruit and berry pomaces in marinated fish products, ensuring food safety, high microbiological quality, acceptable sensory characteristics and a sufficiently long shelf life of the products.

Antimicrobial Resistance of Campylobacter coli Isolated from Caecal Samples of Fattening Pigs at Slaughter.

Pigs are known as the main Campylobacter coli reservoirs. Campylobacteriosis, the most commonly reported gastrointestinal disease in humans, is mainly caused by the consumption of poultry meat, and little is known about the role of pork. Pigs are often associated with C. coli, including antimicrobial-resistant isolates. Therefore, the entire pork production chain must be considered as an important source of antimicrobial-resistant C. coli. This study aimed to determine the antimicrobial resistance of Campylobacter spp. isolated from caecal samples of fattening pigs at the Estonian slaughterhouse level over a five-year period. The proportion of Campylobacter-positive caecal samples was 52%. All Campylobacter isolates were identified as C. coli. A high proportion of the isolates were resistant to most of the studied antimicrobials. The resistance to streptomycin, tetracycline, ciprofloxacin and nalidixic acid was 74.8%, 54.4%, 34.4% and 31.9%, respectively. In addition, a high proportion (15.1%) of the isolates were multidrug-resistant and, in total, 93.3% were resistant to at least one antimicrobial.

Antibiotic Resistance in Campylobacter spp. Isolated from Broiler Chicken Meat and Human Patients in Estonia.

Poultry meat is considered the most important source of Campylobacter spp. Because of rising antimicrobial resistance in Campylobacter spp., this study investigated the antimicrobial resistance of Campylobacter isolates from fresh broiler chicken meat originating from the Baltic countries sold in Estonian retail settings. Additionally, human clinical isolates obtained from patients with Campylobacter enteritis in Estonia were analysed. The aim of this study was to investigate the susceptibility of Campylobacter spp. to nalidixic acid, ciprofloxacin, tetracycline, streptomycin, erythromycin and gentamicin. The broth microdilution method with the EUCAMP2 panel was used for MIC determination and antimicrobial mechanisms were analysed using WGS data. A total of 46 Campylobacter strains were analysed, of which 26 (42.6%) originated from Lithuanian, 16 (26.2%) from Latvian, and 4 (6.6%) from Estonian fresh broiler chicken meat. In addition, 15 (24.6%) Campylobacter strains of patients with campylobacteriosis were tested. The antimicrobial resistance patterns of Campylobacter spp. isolated from fresh broiler chicken meat samples of Estonian, Latvian and Lithuanian origin collected in Estonian retail, and from patients with Campylobacter enteric infections, were determined. A total of 46 (75%) of the isolates tested were C. jejuni and 15 (25%) were C. coli. Campylobacter resistance was highest to nalidixic acid (90.2% of strains) and ciprofloxacin (90.2%), followed by tetracycline (57.4%), streptomycin (42.6%) and erythromycin (6.6%). All strains were sensitive to gentamicin. Additionally, antimicrobial resistance genes and point mutations were detected in 27 C. jejuni and 8 C. coli isolates previously assigned as resistant with the phenotypic method. A high antibiotic resistance of Campylobacter spp. in Lithuanian- and Latvian-origin broiler chicken meat and Estonian clinical isolates was found. Similar antibiotic resistance patterns were found for broiler chicken meat and human Campylobacter isolates.

The prevalence, counts, and MLST genotypes of Campylobacter in poultry meat and genomic comparison with clinical isolates.

Since 2005 campylobacteriosis has been the most commonly reported gastrointestinal infection in humans in the European Union with more than 200,000 cases annually. Also Campylobacter is one of the most frequent cause of food-borne outbreaks with 319 outbreaks reported to EFSA, involving 1,254 cases of disease and 125 hospitalizations in EU in 2019. Importantly poultry meat is one of the most common source for the sporadic Campylobacter infections and for strong-evidence campylobacteriosis food-borne outbreaks in EU. In present study, 429 fresh broiler chicken meat samples of Estonian, Latvian, and Lithuanian origin were collected from Estonian retail level and analyzed on a monthly basis between September 2018 and October 2019. Campylobacter spp. were isolated in 141 (32.9%) of 429 broiler chicken meat samples. Altogether 3 (1.8%), 49 (36.8%), and 89 (66.9%) of Estonian, Latvian, and Lithuanian origin broiler chicken meat samples were positive for Campylobacter spp. Among Campylobacter-positive samples, 62 (14.5%) contained Campylobacter spp. below 100 CFU/g and in 28 (6.5%) samples the count of Campylobacter spp. exceeded 1,000 CFU/g. A high prevalence of Campylobacter spp. in fresh broiler chicken meat of Lithuanian and Latvian origin in Estonian retail was observed. Additionally, 22 different multilocus sequence types were identified among 55 genotyped isolates of broiler chicken meat and human origin, of which 45 were Campylobacter jejuni (C. jejuni) and 10 were Campylobacter coli (C. coli). The most prevalent multilocus sequence types among C. jejuni was ST2229 and among C. coli ST832, ST872. C. jejuni genotypes found in both broiler chicken meat and human origin samples were ST122, ST464, ST7355, and ST9882, which indicates that imported fresh broiler chicken meat is likely the cause of human campylobacteriosis in Estonia.

Prevalence and Serotype Diversity of Salmonella enterica in the Estonian Meat Production Chain in 2016-2020.

BACKGROUND: Salmonella enterica represents a considerable public concern worldwide, with farm animals often recognised as an important reservoir. This study gives an overview of the prevalence and serotype diversity of Salmonella over a 5-year period in the meat production chain in Estonia. Data on human salmonellosis over the same period are provided. METHODS: Salmonella surveillance data from 2016 to 2020 were analysed. RESULTS: The prevalence of Salmonella at the farm level was 27.7%, 3.3% and 0.1% for fattening pigs, cattle and poultry, respectively. S. Derby was the most prevalent serotype at the farm level for fattening pigs and S. Dublin for cattle. The top three serotypes isolated at the slaughterhouse and meat cutting levels were S. Derby, monophasic S. Typhimurium and S. Typhimurium with proportions of 64.7%, 9.4% and 7.0%, respectively. These serotypes were the top five most common Salmonella serotypes responsible for human infections in Estonia. S. Enteritidis is the main cause (46.9%) of human salmonellosis cases in Estonia, but in recent years, Enteritidis has not been detected at the slaughterhouse or meat cutting level. CONCLUSION: In recent years, monophasic S. Typhimurium has become epidemiologically more important in Estonia, with the second-highest cause in human cases and third-highest among the most prevalent serotypes of Salmonella enterica in the meat chain.

Retrospective Use of Whole-Genome Sequencing Expands the Multicountry Outbreak Cluster of Listeria monocytogenes ST1247.

Listeria monocytogenes sequence type 1247 clonal complex 8 caused a prolonged multicountry outbreak in five EU countries: Denmark, Estonia, Finland, France, and Sweden. A total of 22 disease cases were identified with onset of symptoms between July 2014 and February 2019. Five patients died due to, or with, the disease. The retrospective analysis of L. monocytogenes isolate VLTRLM2013 revealed the presence of an outbreak-related strain (cgMLST type L2-SL8-ST1247-CT4158) in ready-to-eat fish product more than a year prior to the first outbreak-related cases. Reference outbreak strain and VLTRLM2013 strain were compared using core genome and whole-genome multilocus sequence typing analyses. Genomic level differences of the persistent L. monocytogenes strains associated with a prolonged multicountry foodborne listeriosis outbreak are described. It was concluded that the persistent nature of the multicountry outbreak-related L. monocytogenes strain VLTRLM2013 together with stress island, virulence, and antibiotic resistance genes could potentially be the determining factors for the extensive and prolonged outbreak affecting five European Union countries. Our results support the systematic application of whole-genome sequencing in food and public health surveillance and further encourages its wide adoption.

Draft Genome Sequence of a Multicountry Outbreak-Related Listeria monocytogenes Sequence Type 1247 Strain, VLTRLM2013.

We sequenced the genome of a multicountry outbreak-related Listeria monocytogenes sequence type 1247 strain (VLTRLM2013) that was isolated from a vacuum-packaged sliced salted salmon product of an Estonian fish-processing company that was obtained from an Estonian retail outlet in 2013.

The source attribution analysis revealed the prevalent role of poultry over cattle and wild birds in human campylobacteriosis cases in the Baltic States.

The reservoir and source of human campylobacteriosis is primarily considered to be poultry, but also other such as ruminants, pets and environmental sources are related with infection burden. Multilocus sequence typing is often used for Campylobacter epidemiological studies to determine potential sources of human infections. The collection of 420 Campylobacter jejuni isolates with assigned MLST genotype from poultry (n = 139), cattle (n = 48) and wild birds (n = 101) were used in source attribution analysis. Asymmetric island model with accurate and congruent self-attribution results, was used to determine potential sources of human C. jejuni infections (n = 132) in Baltic States. Source attribution analysis revealed that poultry (88.3%) is the main source of C. jejuni human infections followed by cattle and wild bird with 9.4% and 2.3%, respectively. Our findings demonstrated that clinical cases of C. jejuni infections in Baltic countries are mainly linked to poultry, but also to cattle and wild bird sources.

Nitrite and nitrate content in meat products and estimated nitrite intake by the Estonian children.

Public interest in nitrate and nitrite content reduction in meat products has increased; therefore, the aim of the present study was to determine nitrate and nitrite levels in commercial meat products as the main source of added nitrites, and to estimate their dietary intake by children. The intake of nitrite from processed meat products and drinking water by Estonian children was estimated. Daily intake estimations were based on the food consumption data of the National Institute for Health Development. In addition, nitrite/nitrate concentrations of meat and processed meat products were measured using a liquid chromatographic method with UV detection. Mean nitrite intakes among 1087 studied children were 0.015 and 0.016 mg kg-1 b.w. day-1, respectively, among children aged 12-35 months and 3-10 years. Acceptable daily intake (ADI) of 0.07 mg nitrite kg-1 b.w. day-1 was exceeded in 3.1% of children, being more prevalent in the younger age group. Considering the consumption of processed meat and drinking water, mean nitrite intakes in the younger and the older age groups were, respectively, 21.9% and 22.9% of the ADI. Study results indicated that over a period of 10 years, children's dietary nitrite intake from processed meat products has declined, which is probably caused by changes in food preferences and decreased usage of nitrite in cured meat products by meat industries.

Composition and Antibacterial Effect of Mint Flavorings in Candies and Food Supplements.

Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n = 45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcusson's type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01 - 0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (-)-carvone, as most active against both bacteria, had the lowest MIC90 values. (+)-Menthyl acetate, (-)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus. Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.

Antimicrobial and Antioxidative Effects of Plant Powders in Raw and Cooked Minced Pork.

It is a challenge for scientists to find new plant-based food constituents simultaneously possessing antimicrobial and antioxidative properties to prolong the shelf life of meat products. In this study, various plant powders and their blends were added to minced pork to carry out a complex study of their effect on sensory characteristics, microbial growth, and lipid oxidation of the meat in raw and cooked forms during storage. Microbiological shelf life parameters were evaluated by determining the total counts of microorganisms, yeasts, and molds. The growth potential of Listeria monocytogenes was estimated by challenge testing. The impact on lipid oxidation processes was assessed using thiobarbituric acid reactive substances (TBARS) and high performance liquid chromatography (HPLC) methods. The results showed that the blend of rhubarb petioles and tomato powder added a pleasant color and a combined taste to the product, similar to the taste of salt. In raw samples, considerable microbial growth inhibition was achieved with rhubarb petioles, tomato, and their mixture. Nine treatments of cooked samples had a stronger inhibitory effect on microbial growth compared to control treatments. Among all plant powders, tomato was the most effective inhibitor of yeast and mold growth. However, the challenge test revealed that L. monocytogenes growth in cooked samples was not inhibited during shelf life. In raw samples, rhubarb roots combined with blackcurrant or chokeberry berries effectively inhibited lipid oxidation, and in cooked samples, rhubarb petioles showed a similar effect. In conclusion, it was found that powdered plant materials are well suited for use as antimicrobial and antioxidative agents in minced meat products.

Prevalence and Numbers of Listeria monocytogenes in Various Ready-to-Eat Foods over a 5-Year Period in Estonia.

HIGHLIGHTS: This study is a significant contribution to L. monocytogenes risk analysis. L. monocytogenes was found in 3.6% of 30,016 analyzed RTE food samples. A food safety criterion of 100 CFU/g was exceeded for 0.3% of the RTE food samples. Salted and cold-smoked fish products were found to be potentially high-risk foods.

Whole-genome multilocus sequence typing of closely related broiler chicken meat origin Campylobacter jejuni ST-5 isolates.

Broiler chicken meat is considered as the main source of foodborne Campylobacter infection. Campylobacter jejuni multilocus sequence type ST-5 has been previously identified as predominant among Latvian and Lithuanian broiler chicken meat products and is often isolated from Lithuanian patients with Campylobacter enteric infection. The market share of Lithuanian and Latvian origin fresh broiler chicken meat sold at Estonian retail is estimated close to 50%. In this study, we used the whole-genome sequences of Latvian and Lithuanian origin C. jejuni ST-5 broiler chicken meat isolates obtained from Estonian retail. Applied genome-by-genome whole-genome MLST revealed high similarity of investigated isolates with 1589 shared alleles, of which 218 loci were polymorphic. Latvian and Lithuanian isolates had 199 to 205 intergroup differences in shared alleles, whereas intragroup isolates differed by 27 to 31 alleles. Allele differences divided the isolates into 2 distinct groups based on the country of origin, which was supported by sequence-based phylogenetic tree. The resulting geographic separation of the isolates found in this study should be carefully interpreted taking into account the small sample size.

Antibacterial and antioxidative properties of different parts of garden rhubarb, blackcurrant, chokeberry and blue honeysuckle.

BACKGROUND: It is important to find plant materials that can inhibit the growth of Listeria monocytogenes and other food-spoiling bacteria both in vitro and in situ. The aim of the study was to compare antibacterial and antioxidative activity of selected plant-ethanol infusions: leaves and berries of blackcurrant (Ribes nigrum L.), berries of chokeberry (Aronia melanocarpa (Michx.) Elliott) and blue honeysuckle (Lonicera caerulea L. var. edulis); petioles and dark and light roots of garden rhubarb (Rheum rhaponticum L.) for potential use in food matrices as antibacterial and antioxidative additives. RESULTS: The strongest bacterial growth inhibition was observed in 96% ethanol infusions of the dark roots of rhubarbs. In 96% ethanol, nine out of ten studied plant infusions had antibacterial effect against L. monocytogenes, but in 20% ethanol only the infusions of dark rhubarb roots had a similar effect. Chokeberry and other berries had the highest antioxidative activity, both in 20% and 96% ethanol infusions. CONCLUSION: The combination of dark rhubarb roots or petioles and berries of black chokeberry, blackcurrant or some other anthocyanin-rich berries would have potential as both antibacterial and antioxidative additives in food. © 2018 Society of Chemical Industry.

Antimicrobial resistance and multilocus sequence types of Campylobacter jejuni isolated from Baltic broiler chicken meat and Estonian human patients.

Genotypes of Campylobacter jejuni in Baltic fresh broiler chicken meat and in Estonian human origin samples were determined by using MLST. We observed associations between clonal complexes, sequence types, and antibiotic resistance of C. jejuni isolates. The detection of high proportions of fluoroquinolone resistance among Latvian (100.0%) and Lithuanian (84.6%) origin broiler chicken meat C. jejuni isolates, found in this study, suggests the wide use of these antibiotics in poultry in these countries. MLST of 48 Baltic origin C. jejuni isolates revealed a total of 22 sequence types belonging to 13 clonal complexes. From the 13 assigned CCs and 22 STs, the most prevalent were ST-353 CC and ST-5 consisting of human, Latvian and Lithuanian chicken meat isolates. Among human and chicken meat isolates the resistance rate was 63.6 and 62.2%, respectively. ST-353 isolated from humans were more often resistant to nalidixic acid and ciprofloxacin than the other STs. Strain types ST-5, ST-50, ST-353, ST-354, and ST-429 isolated from poultry were more often resistant to both nalidixic acid and ciprofloxacin than the other STs. Furthermore, ST-353 and ST-354 isolated from poultry were more often resistant against tetracycline than the other STs. Fluoroquinolone resistance was significantly (P < 0.01) associated with ST-353 CC and ST-5 in comparison with all other CCs, STs. We found that three Estonian broiler isolates belonged to the novel ST-8188.The study indicates the potential public health concern posed by the very high fluoroquinolne resistance in Campylobacter jejuni found in Latvian and Lithuanian poultry meat.

Acrylamide in commercial foods and intake by infants in Estonia.

Acrylamide is formed when certain foods with low moisture are prepared at above 120 ºC, especially those foods containing asparagine and reducing sugars such as glucose and fructose. Acrylamide is a probable carcinogen, and from animal evidence the margins of exposure indicate a concern for neoplastic effects. On a body weight basis infants´ acrylamide intakes are often higher than those of adults. The aim of the study was to determine acrylamide levels in different commercially-available foods and to assess dietary acrylamide intakes by infants. The acrylamide content in samples ranged widely, from

Changes in Polyphenols Contents and Antioxidant Capacities of Organically and Conventionally Cultivated Tomato (Solanum lycopersicum L.) Fruits during Ripening.

Polyphenols of fruits and vegetables form an important part of human dietary compounds. Relatively little is known about accumulation of phenolics during fruits ripening process. The goal of this work was to study the changes in antioxidant activity and in content of 30 polyphenols during ripening of tomato fruits. Five organically and conventionally grown tomato cultivars were investigated at three different ripening stages. Phenolic compounds were extracted with methanol and extracts were analyzed by HPLC-DAD-MS/MS. During ripening, four different changing patterns were observed: (1) high level in green fruits with minimal changes; (2) continuous increase with maximum level in red-ripe fruits; (3) decrease; (4) increase and achieving maximum level at half-ripe stage. Similar change patterns were found for organic and conventional fruits. The accumulation patterns of phenolic compounds were similar in standard-type tomatoes but differed in several cases in cherry-type cultivar. Although contents of some polyphenols decreased during ripening, total phenolics and free radical scavenging activity increased in all studied cultivars and in case of both cultivation modes. The changes in content of phenolic compounds during ripening were greatly influenced by cultivars, but cultivation mode had only minor impact on dynamics in polyphenols contents in tomato fruits.

Phenogenotypic Characterization of Escherichia coli O157:H7 Strains Isolated from Cattle at Slaughter.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 was isolated from 30 (4%) of 744 cattle hide swab samples collected at Estonian slaughterhouses within a 3-year monitoring program of zoonotic pathogens. The isolates were characterized by determining the presence of STEC main virulence factors, the antimicrobial resistance profiles, and the genetic relatedness by pulsed-field gel electrophoresis (PFGE). Thirteen strains carried the stx2 gene alone and 17 both the stx1 and stx2 genes. The most frequently detected stx subtype was stx2c, occurring alone (n = 12) or in combination with subtype stx1a (n = 13). All isolates harbored the intimin-coding eae gene and produced enterohemolysin. Twelve isolates (40%) showed resistance to at least one of the 14 antimicrobials and the isolates were predominantly resistant to streptomycin, sulfamethoxazole, and ampicillin. No extended-spectrum beta-lactamase-producing isolates were detected. PFGE characterization of the isolates showed an overall similarity higher than 75%, and four clusters based on 100% similarity were revealed.