Possibility of transfer and activation of 'silent' tetracycline resistance genes among Enterococcus faecalis under high-pressure processing.

In this study, the tetracycline resistance of Enterococcus faecalis strains isolated from food was determined and molecular analyses of the resistance background were performed by determining the frequency of selected tetracycline resistance genes. In addition, the effect of high-pressure stress (400 and 500 MPa) on the expression of selected genes encoding tetracycline resistance was determined, as well as changes in the frequency of transfer of these genes in isolates showing sensitivity to tetracyclines. In our study, we observed an increase in the expression of genes encoding tetracyclines, especially the tet(L) gene, mainly under 400 MPa pressure. The study confirmed the possibility of transferring genes encoding tetracyclines such as tet(M), tet(L), tet(K), tet(W) and tet(O) by horizontal gene transfer in both control strains and exposed to high-pressure. Exposure of the strains to 400 MPa pressure had a greater effect on the possibility of gene transfer and expression than the application of a higher-pressure. To our knowledge, this study for the first time determined the effect of high-pressure stress on the expression of selected genes encoding tetracycline resistance, as well as the possibility and changes in the frequency of transfer of these genes in Enterococcus faecalis isolates showing sensitivity to tetracyclines and possessing silent genes. Due to the observed possibility of increased expression of some of the genes encoding tetracycline resistance and the possibility of their spread by horizontal gene transfer to other microorganisms in the food environment, under the influence of high-pressure processing in strains phenotypically susceptible to this antibiotic, it becomes necessary to monitor this ability in isolates derived from foods.

High-Pressure Processing-Impacts on the Virulence and Antibiotic Resistance of Listeria monocytogenes Isolated from Food and Food Processing Environments.

High-pressure processing (HPP) is one of the non-thermal methods of food preservation considered to be safe but may cause an increase/decrease in virulence potential and antibiotic resistance. The aim of the present study was to evaluate the survival of L. monocytogenes isolates after high-pressure processing (200 and 400 MPa for 5 min) and to determine changes in phenotypic and genotypic antibiotic resistance and virulence after this treatment. The 400 MPa treatment was shown to be effective in reducing pathogens to safe levels; however, the potential for cell recovery during storage was observed. In addition, studies on changes in virulence indicated possibilities related to a decrease in actA gene expression, overexpression of the hly and osfX gene, and an increase in biofilm-forming ability. The studies on changes in antibiotic resistance of isolates showed that all isolates showing initial susceptibility to lincomycin, fosfomycin, trimethoprim/sulfamethoxazole, and tetracycline became resistant to these antibiotics, which was associated with an increase in the values of minimum inhibitory concentrations. An increase in the expression of antibiotic resistance genes (mainly tetA_1, tetA_3, tetC) was also observed (mainly after the application of 200 MPa pressure), which was isolate dependent. However, it is noteworthy that the induced changes were permanent, i.e., they persisted even after the restoration of optimal environmental conditions. The results presented in our work indicate that the stress occurring during HPP can affect both phenotypic and genotypic changes in the virulence and antibiotic resistance potential of pathogens isolated from food and food processing environments. The potential associated with cell recovery and persistence of changes may influence the spread of virulent isolates of pathogens with increased antibiotic resistance in the food and food processing environment.

Impact of the Food-Related Stress Conditions on the Expression of Enterotoxin Genes among Staphylococcus aureus.

Staphylococcus aureus is one of the most important foodborne pathogens. S. aureus has the capability to produce a variety of toxins, including staphylococcal enterotoxins (SEs). The aim of this study was to evaluate the survival rate of S. aureus cells and analyze enterotoxins gene expression after exposure to osmotic stress and acidic/alkaline stress. To determine survival rates, the traditional plate counting method and flow cytometry were used. The expression levels of the enterotoxin genes were performed by quantitative reverse transcription PCR (RT-qPCR). Expression changes differed depending on the stressors chosen. The obtained results in this study showed the effect of critical food-related stress conditions on SE gene expression in S. aureus. The study showed different expression levels of the tested enterotoxins genes depending on the stress. The most tested enterotoxin genes (seg, sei, and selo) after exposure to pH = 4.5 stress have similar expression as in the optimal condition. After alkaline treatment (pH = 9.6), a similar expression gene value as for the optimal condition was observed. The analysis of gene expression in response to stress caused by NaCl, showed that the expression of selp decreased, whereas selu, selm, and selo genes increased. A significantly decreased expression of the sea gene was observed.

A Comprehensive Study on Antibiotic Resistance among Coagulase-Negative Staphylococci (CoNS) Strains Isolated from Ready-to-Eat Food Served in Bars and Restaurants.

The present study aimed to characterize and assess the diversity of CoNS strains as potential vectors for the spread of resistance to antimicrobial agents from RTE foods served in bars and restaurants. Eighty-five CoNS strains, obtained from 198 RTE food samples, were investigated. Sixty-seven CoNS isolates (78.8%) were resistant to at least one antibiotic tested, and 37 (43.5%) were multidrug resistant (MDR-CoNS). Moreover, CoNS strains contained genes conferring resistance to antibiotics critically important in medicine, i.e., ß-lactams [mecA (29.4%); blaZ (84.7%)], aminoglycosides [aac(6')-Ie-aph(2″)-Ia (45.9%); aph(2″)-Ic (3.5%)], macrolides, lincosamides and streptogramin B-MLSB [msrA/B (68.2%); ermB (40%) and mphC (4.7%)], tetracyclines [tetK (31.8%); tetM (16.5%) and/or tetL (2.35%)]. We also found the fusB/C/D genes responsible for the acquired low-level fusidic acid resistance (17.6%) and streptogramin resistance determinant vgaA in 30.6% of isolates. In three linezolid resistant strains (2 S. epidermidis and 1 S. warneri), mutation was detected, as demonstrated by L101V and V188I changes in the L3 protein amino acid sequences. The high frequency in RTE food of MDR-CoNS including methicillin-resistant (MR-CoNS) strains constitutes a direct risk to public health as they increase the gene pool from which pathogenic bacteria can pick up resistance traits.

A Comprehensive Virulence and Resistance Characteristics of Listeria monocytogenes Isolated from Fish and the Fish Industry Environment.

Listeria monocytogenes is an important pathogen, often associated with fish, that can adapt and survive in products and food processing plants, where it can persist for many years. It is a species characterized by diverse genotypic and phenotypic characteristics. Therefore, in this study, a total of 17 L. monocytogenes strains from fish and fish-processing environments in Poland were characterized for their relatedness, virulence profiles, and resistance genes. The Core Genome Multilocus Sequence Typing (cgMLST) analysis revealed that the most frequent serogroups were IIa and IIb; sequence types (ST) were ST6 and ST121; and clonal complexes (CC) were CC6 and CC121. Core genome multilocus sequence typing (cgMLST) analysis was applied to compare the present isolates with the publicly available genomes of L. monocytogenes strains recovered in Europe from humans with listeriosis. Despite differential genotypic subtypes, most strains had similar antimicrobial resistance profiles; however, some of genes were located on mobile genetic elements that could be transferred to commensal or pathogenic bacteria. The results of this study showed that molecular clones of tested strains were characteristic for L. monocytogenes isolated from similar sources. Nevertheless, it is worth emphasizing that they could present a major public health risk due to their close relation with strains isolated from human listeriosis.

Molecular Analysis of Pathogenicity, Adhesive Matrix Molecules (MSCRAMMs) and Biofilm Genes of Coagulase-Negative Staphylococci Isolated from Ready-to-Eat Food.

This paper provides a snapshot on the pathogenic traits within CoNS isolated from ready-to-eat (RTE) food. Eighty-five strains were subjected to biofilm and slime production, as well as biofilm-associated genes (icaA, icaD, icaB, icaC, eno, bap, bhp, aap, fbe, embP and atlE), the insertion sequence elements IS256 and IS257 and hemolytic genes. The results showed that the most prevalent determinants responsible for the primary adherence were eno (57.6%) and aap (56.5%) genes. The icaADBC operon was detected in 45.9% of the tested strains and was correlated to slime production. Moreover, most strains carrying the icaADBC operon simultaneously carried the IS257 insertion sequence element. Among the genes encoding for surface proteins involved in the adhesion to abiotic surfaces process, atlE was the most commonly (31.8%) followed by bap (4.7%) and bhp (1.2%). The MSCRAMMs, including fbe and embp were detected in the 11.8% and 28.2% of strains, respectively. A high occurrence of genes involved in the hemolytic toxin production were detected, such as hla_yiD (50.6%), hlb (48.2%), hld (41.2%) and hla_haem (34.1%). The results of the present study revealed an unexpected occurrence of the genes involved in biofilm production and the high hemolytic activity among the CoNS strains, isolated from RTE food, highlighting that this group seems to be acquiring pathogenic traits similar to those of S. aureus, suggesting the need to be included in the routine microbiological analyses of food.

High-pressure processing effect on conjugal antibiotic resistance genes transfer in vitro and in the food matrix among strains from starter cultures.

This study analyzed the effect of high-pressure processing (HPP) on the frequency of conjugal gene transfer of antibiotic resistance genes among strains obtained from starter cultures. Gene transfer ability was analyzed in vitro and in situ in the food matrix. It was found that the transfer of aminoglycoside resistance genes did not occur after high-pressure treatment, either in vitro or in situ. After exposure to HPP, the transfer frequencies of tetracycline, ampicillin and chloramphenicol resistance genes increased significantly compared to the control sample, both in vitro and in situ. The frequency of resistance genes transfer in the food matrix in the pressurized samples did not differ significantly from the in vitro transfer rate. Minimum Inhibitory Concentrations (MICs) for these antibiotics determined for transconjugants were lower or equal to MICs determined for the donors. No significant differences were observed between the MIC values determined for the transconjugants obtained in vitro and in situ. The results suggest that HPP may contribute to the spread of antibiotic resistance. This points to the need to verify starter cultures strains for their antibiotic resistance and pressurization parameters to avoid spreading antibiotic resistance genes.

Effect of high pressure processing on changes in antibiotic resistance genes expression among strains from commercial starter cultures.

This study analyzed the effect of high-pressure processing on the changes in resistance phenotype and expression of antibiotic resistance genes among strains from commercial starter cultures. After exposure to high pressure the expression of genes encoding resistance to aminoglycosides (aac(6')Ie-aph(2″)Ia and aph(3')-IIIa) decreased and the expression of genes encoding resistance to tetracyclines (tetM and tetW), ampicillin (blaZ) and chloramphenicol (cat) increased. Expression changes differed depending on the pressure variant chosen. The results obtained in the gene expression analysis correlated with the results of the phenotype patterns. To the best of the authors' knowledge, this is one of the first studies focused on changes in antibiotic resistance associated with a stress response among strains from commercial starter cultures. The results suggest that the food preservation techniques might affect the phenotype of antibiotic resistance among microorganisms that ultimately survive the process. This points to the need to verify strains used in the food industry for their antibiotic resistance as well as preservation parameters to prevent the further increase in antibiotic resistance in food borne strains.

Identification of the Enterotoxigenic Potential of Staphylococcus spp. from Raw Milk and Raw Milk Cheeses.

This study aimed to genotypic and phenotypic analyses of the enterotoxigenic potential of Staphylococcus spp. isolated from raw milk and raw milk cheeses. The presence of genes encoding staphylococcal enterotoxins (SEs), including the classical enterotoxins (sea-see), non-classical enterotoxins (seg-seu), exfoliative toxins (eta-etd) and toxic shock syndrome toxin-1 (tst-1) were investigated. Isolates positive for classical enterotoxin genes were then tested by SET-RPLA methods for toxin expression. Out of 75 Staphylococcus spp. (19 Staphylococcus aureus and 56 CoNS) isolates from raw milk (49/65.3%) and raw milk cheese samples (26/34.7%), the presence of enterotoxin genes was confirmed in 73 (97.3%) of them. Only one isolate from cheese sample (1.3%) was able to produce enterotoxin (SED). The presence of up to eight different genes encoding enterotoxins was determined simultaneously in the staphylococcal genome. The most common toxin gene combination was sek, eta present in fourteen isolates (18.7%). The tst-1 gene was present in each of the analyzed isolates from cheese samples (26/34.7%). Non-classical enterotoxins were much more frequently identified in the genome of staphylococcal isolates than classical SEs. The current research also showed that genes tagged in S. aureus were also identified in CoNS, and the total number of different genes detected in CoNS was seven times higher than in S. aureus. The obtained results indicate that, in many cases, the presence of a gene in Staphylococcus spp. is not synonymous with the ability of enterotoxins production. The differences in the number of isolates with genes encoding SEs and enterotoxin production may be mainly due to the limit of detection of the toxin production method used. This indicates the need to use high specificity and sensitivity methods for detecting enterotoxin in future studies.

Impact of High-Pressure Processing (HPP) on Listeria monocytogenes-An Overview of Challenges and Responses.

High-pressure processing (HPP) is currently one of the leading methods of non-thermal food preservation as an alternative to traditional methods based on thermal processing. The application of HPP involves the simultaneous action of a combination of several factors-pressure values (100-600 MPa), time of operation (a few-several minutes), and temperature of operation (room temperature or lower)-using a liquid medium responsible for pressure transfer. The combination of these three factors results in the inactivation of microorganisms, thus extending food shelf life and improving the food's microbiological safety. HPP can provide high value for the sensory and quality characteristics of products and reduce the population of pathogenic microorganisms such as L. monocytogenes to the required safety level. Nevertheless, the technology is not without impact on the cellular response of pathogens. L. monocytogenes cells surviving the HPP treatment may have multiple damages, which may impact the activation of mechanisms involved in the repair of cellular damage, increased virulence, or antibiotic resistance, as well as an increased expression of genes encoding pathogenicity and antibiotic resistance. This review has demonstrated that HPP is a technology that can reduce L. monocytogenes cells to below detection levels, thus indicating the potential to provide the desired level of safety. However, problems have been noted related to the possibilities of cell recovery during storage and changes in virulence and antibiotic resistance due to the activation of gene expression mechanisms, and the lack of a sufficient number of studies explaining these changes has been reported.

Antimicrobial Resistance and Virulence Characterization of Listeria monocytogenes Strains Isolated from Food and Food Processing Environments.

Listeria monocytogenes is a particularly foodborne pathogen associated with listeriosis, which can be disseminated in food and food processing environments. This study aimed to determine the serotypes and characteristics of virulence factors and antibiotic resistance among 40 L. monocytogenes strains isolated from food (n = 27) purchased in Olsztyn (Warmia and Mazury region, Poland) and food processing environments in Poland (n = 13). Isolates were assigned to serotypes 1/2a, 1/2c, 3a, and 3c using polymerase chain reaction (PCR). The results showed that serotype 1/2a (66.7%) was the most prevalent among strains from food, and serotype 1/2c (53.8%) among strains from the food processing environments. Five different virulence factors (hlyA, prfA, inlB, luxS, sigB) were detected in all isolates from the food processing environments using PCR. The hlyA (100.0%), prfA (100.0%), and inlB (96.3%) were the most prevalent in food strains. Seven (25.9%) of the strains of food and ten (76.9%) strains from the food processing environments showed the ability to form biofilm. The tested isolates were subjected to antibiotic susceptibility testing against 12 antibiotics used in the mitigation of listeriosis, using the disk diffusion method. The most frequent were intermediate resistance and resistance to clindamycin. Twelve (92.3%) strains from the food processing environments, and twenty-three (85.2%) from food were non-susceptible to clindamycin. Generally, antibacterial resistance determinants (Lde, aadB, aac(3)-IIa(aacC2)a, penA, mefA, lnuA, lnuB, sulI, sulII) were detected in sixteen (59.0%) strains from food and four (30.8%) from the food processing environments, by PCR. The most frequent were the mefA-lnuA (n = 7; 20.0%) and lnuA (n = 6; 17.1%) genotypes. From this research, we can conclude that virulent and antimicrobial-resistant strains of L. monocytogenes are present in food and the food processing environment in Poland, which may pose a potential health risk to consumers. Monitoring for the control of virulent and antimicrobial-resistant L. monocytogenes strains in the food system can contribute to effective planning and prevention of their spread.

Occurrence and Characteristics of Staphylococcus aureus Strains along the Production Chain of Raw Milk Cheeses in Poland.

Cheeses produced from unpasteurized milk by traditional production methods may contain many groups of microorganisms, including Staphylococcus aureus. The aim of this study was to determine the occurrence of S. aureus in the artisanal cheese production chain from unpasteurized milk. We investigated the prevalence of S. aureus strains isolated from various stages of artisanal cheese of unpasteurized milk production from farms in the northeastern and southern parts of Poland and characterized them. Characterization included antimicrobial susceptibility by microbroth dilution and biofilm formation by in vitro assay. Among all strains, the presence of enterotoxigenic genes and genes involved with biofilm formation and antibiotic resistance were screened by PCR-based methods. A total of 180 samples were examined. A high percentage of strains were resistant to penicillin (54/58.1%) and tobramycin (32/34.4%). Some tested isolates also showed resistance to the macrolide class of antibiotics: azithromycin, clarithromycin, and erythromycin at 17/18.3%, 15/16.1%, and 21/22.6%, respectively. Among tested isolates, we also found phenotypic resistance to oxacillin (9/9.7%) and cefoxitin (12/12.9%). The blaZ gene encoding penicillin resistance was the most common gene encoding antibiotic resistance among the tested strains. All isolates showing phenotypic resistance to cefoxitin possessed the mecA gene. The study also evaluated the prevalence of biofilm-associated genes, with eno the most frequently associated gene. Eighty-nine out of 93 S. aureus isolates (95.7%) possessed at least one enterotoxin-encoding gene. The results of this study showed that production of raw milk cheeses may be a source of antibiotic resistance and virulent S. aureus. Our results suggest that artisanal cheese producers should better control production hygiene.

High pressure processing, acidic and osmotic stress increased resistance to aminoglycosides and tetracyclines and the frequency of gene transfer among strains from commercial starter and protective cultures.

This study analyzed the effect of food-related stresses on the expression of antibiotic resistance of starter and protective strains and resistance gene transfer frequency. After exposure to high-pressure processing, acidic and osmotic stress, the expression of genes encoding resistance to aminoglycosides (aac(6')Ie-aph(2″)Ia and aph(3')-IIIa) and/or tetracyclines (tetM) increased. After cold stress, a decrease in the expression level of all tested genes was observed. The results obtained in the gene expression analysis correlated with the results of the phenotype patterns. After acidic and osmotic stresses, a significant increase in the frequency of each gene transfer was observed. To the best of the authors' knowledge, this is the first study focused on changes in antibiotic resistance associated with a stress response among starter and protective strains. The results suggest that the physicochemical factors prevailing during food production and storage may affect the phenotype of antibiotic resistance and the level of expression of antibiotic resistance genes among microorganisms. As a result, they can contribute to the spread of antibiotic resistance. This points to the need to verify strains used in the food industry for their antibiotic resistance to prevent them from becoming a reservoir for antibiotic resistance genes.

Short Communication: Low Prevalence of Clinically Important Antibiotic-Resistant Strains among Non-Pathogenic Genera of the Tribe Klebsielleae.

Hafnia sp. and Serratia sp. belong to the Tribe Klebsielleae; although they are not considered pathogenic bacteria, there are many documented cases of diseases caused by these microorganisms. The aim of this study was to determine the antibiotic resistance profiles of strains belonging to the genus Hafnia and Serratia isolated from fish and shrimps. Phenotypic antibiotic resistance was determined using the semi-automatic Vitek 2 system (bioMérieux, Marcy-l'Étoile, France), while the presence of the extended-spectrum beta-lactamase, AmpC beta-lactamases, Klebsiella pneumoniae carbapenemases and Metallo-ß-Lactamase producing strains were determined using the MIC Test Strip (Liofilchem, Roseto degli Abbruzzi, Italy). As a result of the conducted research, it was observed that a vast number of Hafnia sp. strains were resistant to cefalexin (84.61%), while Serratia sp. Strains to cefuroxime (79.41%) and nitrofurantoin (85.29%). In addition, it was observed that of all strains, only one had an ability to produce enzymes typical for ß-lactamase-producing Enterobacterales. Although the strains of Hafnia sp. and Serratia sp. isolated from fish and shrimp are not characterized by frequent resistance to antibiotics, taking into account the constantly growing number of antibiotic-resistant strains, this may be a problem in the future, mainly due to gene transfer through mobile genetic elements and the acquisition of resistance expressed phenotypically through contact with stress factors. Therefore, studies monitoring the antibiotic resistance profile of these species should be carried out on a regular basis.

Antibiotic Resistance Carriage Causes a Lower Survivability Due to Stress Associated with High-Pressure Treatment among Strains from Starter Cultures.

High-pressure processing is one of the most promising novel food preservation methods that is increasingly used in the food industry. Its biggest advantage is that it is a nonthermal method that ensures the microbiological safety of the product while maintaining other features, including nutritional value. If products made with starter cultures are subjected to high-pressure treatment, the process parameters should be selected so as not to eliminate all microorganisms in the product. The aim of the study was to investigate if carrying antibiotic resistance genes affects the survival of lactic acid bacteria (Lactococcus and the former Lactobacillus) strains during high-pressure treatment. Survival was assessed using the plate count method. It was shown that the strains carrying antibiotic resistance genes showed a lower survival to high pressure. This might be explained by the phenomenon of fitness cost, consisting in a reduced adaptation of antibiotic-resistant strains related to metabolic expenditure. The obtained results indicate the need for further research in this field and the need to select food processing parameters depending on the strains intentionally included in the food.

A Comparison of Methods for Identifying Enterobacterales Isolates from Fish and Prawns.

Enterobacterales is a prevalent order, which inhabits a variety of environments including food. Due to the high similarities between pathogenic and non-pathogenic species, their identification might be difficult and laborious, and therefore there is a need for rapid and precise identification. The aim of this study was to compare the effectiveness of the available methods of identifying order Enterobacterales strains isolated from fresh fish and shrimps (n = 62). The following methods were used in this study: biochemical, sequencing and identification using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, biochemical identification was performed with the use of the EnteroTest 24N set, while the identification using the MALDI-TOF MS technology was operated on VITEK® MS. Results were compared with identification made by 16S rRNA sequencing. The results of the study showed that conventional identification methods might provide a false result. Identification by VITEK® MS to the species level was correct at 70.97%, and the accuracy of EnteroTest 24N identification did not exceed 50.0%. The genus identification reached 90.32% for the MALDI-TOF technique, while for EnteroTest 24N it was nearly 70.0%. Due to errors in identification, especially of pathogenic organisms, the use of each of these methods should be confirmed by another method, preferably sequencing.

Linezolid-Resistant Enterococcus spp. Isolates from Foods of Animal Origin-The Genetic Basis of Acquired Resistance.

Enterococci are important opportunistic pathogens with the capacity to acquire and spread antibiotic resistance. At present, linezolid-resistant enterococci (LRE) pose a great challenge. Linezolid is considered as a last resort antibiotic in the treatment of enterococcal infections, so it is important to monitor the occurrence of LRE in various environments. The aim of this study was to define the genetic mechanisms of linezolid resistance in enterococci (E. faecalis, E. faecium, E. hirae, E. casseliflavus) isolated from foods of animal origin (n = 104). Linezolid resistance (LR) was shown by 26.9% of isolates. All of them displayed linezolid MICs of 8-32 µg/mL, and 96.4% of them were multidrug multidrug-resistant. The most common acquired linezolid resistance gene in LR isolates was poxtA (64%), followed by optrA (28%) and cfr (12%). According to the authors' knowledge, this research is the first to indicate the presence of the cfr gene among isolates from food. In 28.6% of the isolates, the point mutation G2576T in the V domain of the 23S rRNA was responsible for linezolid resistance. All isolates harbored the wild-type rplC, rplD and rplV genes. The obtained results indicate that linezolid resistance among enterococci in animal-derived food may result from various genetic mechanisms. The most worrying is that this resistance is encoded on mobile genetic elements, so there is a risk of its rapid transmission, even despite the lack of selective pressure resulting from the use of antibiotics.

Ceviche-Natural Preservative: Possibility of Microbiota Survival and Effect on L. monocytogenes.

Ceviche is a marinated raw fish dish ready for consumption; it is a part of the cuisine of various countries on the Pacific coast and its preparation may differ among them. Although the process uses the traditional method of food preservation by lowering the pH, the exposure time is very limited, so the aim of the study was to determine the viability of bacteria often isolated from fish after the process of preparing traditional ceviche. For this purpose, the traditional plate method and flow cytometry were used, and for pathogenic L. monocytogenes strains, the influence of stress during the preparation of the dish on the pathogenic potential was determined. The study showed that the highest percentage of viable cells was observed in the case of L. monocytogenes and remained at the level of 98.54%, slightly less for L. innocua, 96.93%. For the remaining species the reduction did not exceed 10%, for E. faecalis it was 92.76%, for S. liqefaciens 91.44%, H. alvei 93.68%. In addition, the study of the antibacterial properties of individual ingredients showed that habanero and coriander did not show any bactericidal effect, while for onions the amount of live cells was 99.11%, and for lime juice 97.26%, Additionally, the study of changes in virulence, antibiotic resistance and gene expression showed that the stress during the preparation of ceviche has different effects depending on the strain and may cause virulence potential increase, levofloxacin and daptomycin minimum inhibiotory concentration increase and some crucial virulence gene expression induction; therefore, it is important to take care of the quality of the products used to prepare the ceviche and accurate pretreatment.

Occurrence of antibiotic resistance among Enterobacterales isolated from raw and ready-to-eat food - phenotypic and genotypic characteristics.

The aim of this study was phenotypic and genotypic characterization of antibiotic-resistant food-borne Enterobacterales. The largest number of isolates was identified as Enterobacter cloacae (42.4%) followed by Escherichia coli (9.8%), Proteus mirabilis, Salmonella enterica, Proteus penneri, Citrobacter freundii (7.6% each), Citrobacter braakii (6.6%), Klebsiella pneumoniae and Klebsiella oxytoca (5.4% each). More than half of isolates (52.2%) were resistant to at least one antibiotic. The majority were resistant to amoxicillin-clavulanate (28.3%) and ampicillin (19.5%). ESBL(+) phenotype was showed by 26 isolates and AmpC(+) phenotype by 32 isolates. The blaCTX-M gene was carried by 53.8% of ESBL-positive isolates, gene from CIT family by 43.8% of AmpC-positive isolates. Our results suggest that more attention should be paid to antibiotic resistance of food-borne Enterobacterales. The presence of transmissible antibiotic resistance markers is an important criterion in the evaluation of food safety.

Effects of osmotic and high pressure stress on expression of virulence factors among Enterococcus spp. isolated from food of animal origin.

The aim of study was to determine the occurrence of virulence factors and virulence-related genes among enterococci isolated from food of animal origin and effects of osmotic and high pressure stress on expression of virulence-related genes. The number of 78 isolates were analyzed. None of them showed a strong ability to form biofilm, 38.5% (n = 30) had the slime production ability, 41% (n = 32) had gelatinase activity, γ -type hemolysis was observed in 55% of isolates, and α-type hemolysis in 45%. All of the isolates carried 1-13 virulence-related genes. The most common genes were gelE (85.9%), sprE (78.2%) and asa1 (75.6%). There were also observed changes in the expression of the gelE, esp, asa1 and cylL genes in response to various NaCl concentration and high pressure processing. Results obtained in this study indicate that enterococci isolated from food may act as reservoirs of virulence genes. The presence of virulence factors among enterococci, especially the ability to biofilm formation is important for food safety and the protection of public health. The results presented in our work demonstrate that stress that can occur during food preservation and food processing can induce the changes in the virulence-related genes expression.