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 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.

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.

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.

Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food.

Although coagulase-positive staphylococci are considered to be the main factor responsible for food poisoning, an increasing role for the coagulase-negative staphylococci in the production of enterotoxins has been observed in recent years. This study was conducted to assess the occurrence of genes responsible for the production of staphylococcal enterotoxins (SE), enterotoxin-like toxins (SEI) and toxic shock syndrome toxin-1 (TSST-1) in coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food from bars and restaurants. One hundred and eighteen CoNS strains were tested using polymerase chain reaction (PCR) to five superantigenic toxin genes, including five different types of classical enterotoxins (sea, seb, sec, sed and see) and the toxic shock syndrome toxin-1 (tsst-1) as well as to supertoxin-like genes. PCR-positive isolates were then tested using immunoenzymatic methods (SET-RPLA, Vidas SET 2) for toxin expression. Out of 118 CoNS strains, the presence of staphylococcal enterotoxins was confirmed in 72% of them. The most frequently found enterotoxin-like genotype was ser, selu. Two of the tested strains had up to ten different enterotoxin genes in the genome at the same time. Although no production of enterotoxins was detected in the CoNS, which means that their possible role in the epidemiology of food-borne diseases is minimal, the data demonstrated that the toxigenic capacity of the CoNS should not be ignored, and that this group of microorganisms should be continuously monitored in food.