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.

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.

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.

Enterococci from ready-to-eat food - horizontal gene transfer of antibiotic resistance genes and genotypic characterization by PCR melting profile.

BACKGROUND: The aim of this study was to evaluate the possibility of the horizontal transfer of genes encoding resistance to aminoglycosides (aac(6')-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(4')-Ia and ant(6')-Ia), tetracyclines (tetM, tetL, tetK, tetO and tetW), and macrolides (ermA, ermB, ermC, msrC, mefAB) in Enterococcus strains isolated from ready-to-eat dishes purchased in bars and restaurants in Olsztyn, Poland. RESULTS: It was found that 74% of tested strains were able to conjugal transfer at least one of the antibiotic resistance genes. Transfer of resistance to tetracyclines in strains was observed with a frequency ranging from 1.3 × 10-6 to 8.7 × 10-7 transconjugants/donor. The int gene and the tetM gene were transferred simultaneously, which indicated that a transposon of the Tn916/Tn1545 also participated in the conjugation process. The frequency of transferring genes of resistance to macrolides ranged from 3.2 × 10-6 to 2.4 × 10-8 transconjugants/donor. The ermB gene was transferred the most frequently. The frequency of acquisition of genes encoding aminoglycosides in strains isolated from food ranged from 1.7 × 10-6 to 3,2 × 10-8 transconjugants/donor. Transfer of the aac(6')-Ie-aph(2″) gene was the most frequent. In all reactions, the clonal character of transconjugants and recipients was confirmed by the polymerase chain reaction melting profile (PCR MP) method, which is an alternative to the pulsed field gel electrophoresis (PFGE) method. CONCLUSION: The findings of this study indicate that Enterococcus isolated from ready-to-eat food is able to horizontally transfer genes encoding various antibiotic resistance mechanisms. © 2018 Society of Chemical Industry.

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.

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.

Prevalence and Antibiotic Resistance of Bacillus sp. Isolated from Raw Milk.

Milk, due to its diversity in terms of its nutritional content, is an important element of the human diet, as well as a good medium for the development of bacteria. The genus Bacillus contains ubiquitous aerobic, rod-shaped, endospore-producing gram-positive bacteria. Representatives of the Bacillus cereus group and the Bacillus subtilis group contribute to shortening the shelf life of milk and dairy products by degrading milk components and its additives. They also produce a number of heat-stable toxins and can cause a number of ailments, mainly in the digestive system. The aim of this research was to identify Bacillus sp. strains isolated from raw milk and to determine their antibiotic resistance. Strains isolated from raw milk samples (n = 45) were identified by MALDI-TOF MS. Ninety strains of Bacillus sp. were identified, for which the antibiotic resistance phenotype was determined. A total of 90 strains of Bacillus were classified in five groups (the Bacillus cereus group (n = 35), B. licheniformis (n = 7), the B. subtilis group (n = 29), B. pumilus (n = 16), and Bacillus sp. (n = 3). All isolates were susceptible to chloramphenicol and meropenem. The antibiotic resistance profiles of the tested groups of Bacillus spp. differed from each other, which is of particular concern in relation to multidrug-resistant representatives of the B. cereus group resistant to cefotaxime (94.29%), ampicillin (88.57%), rifampicin (80%), and norfloxacin (65.71%). Our study provides data on the prevalence and antibiotic sensitivity of Bacillus sp. In raw milk, suggesting a potential risk to health and the dairy industry.

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.

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.

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.

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.

The Influence of Sous Vide Parameters on Nutritional Characteristics and Safety of Pikeperch Fillets.

The aim of the study was to investigate the influence of temperature and time combination on the quality of pikeperch fillets and to propose settings which allow high nutritional quality fish fillets to be obtained. The material for the study consisted of 24 farmed pikeperch (Sander lucioperca) fillets, which were evaluated raw and after sous vide (SV) cooking proceeded at 65 °C for 45 min (SV65), 75 °C for 20 min (SV75), and 90 °C for 10 min (SV90). The chemical composition was affected by SV procedure; SV90 was similar to raw samples in terms of moisture, protein, and fat content, whereas SV65 differed the most. Carnosine contents decreased in all SV samples compared with raw ones, and anserine only decreased in SV90. There were no differences in terms of fatty acid composition (% of total) between SV and raw samples. In SV75 and SV90 total viable counts, Enterobacteriaceae, Enterococcus sp., and Staphylococcus sp. were reduced below a detection level but not in SV65. These samples also showed a better sensory quality than SV65. Therefore, SV75 and SV90 might be recommended for pikeperch fillets preparation, taking into account safety and nutritional aspects.

First report of the presence of Vibrio vulnificus in the Gulf of Gdansk.

BACKGROUND: Vibrio infections are becoming more frequent in the Baltic Sea region, which is caused by an increase in the sea surface temperature. Climate change creates the conditions for the emergence of new environmental niches that are beneficial for Vibrio spp., especially in the summer months. Vibrio vulnificus, which causes wound infections and septicaemia, represents a particularly dangerous species of Vibrio spp. There are numerous publications on the prevalence of V. vulnificus in various regions of the Baltic Sea, but there is a lack of such data for the Polish coast. This prompted us to conduct a pilot study into the prevalence of the bacteria in the Gulf of Gdansk. The study aimed to detect Vibrio spp. in the coastal waters and the wet sand at the beaches and bathing areas in the Gulf of Gdansk. MATERIALS AND METHODS: During the period from June 16th to September 23rd 2020, 112 samples of seawater and 105 samples of wet sand were collected at 16 locations along the coast of the Gulf of Gdansk and Hel peninsula. Isolation of Vibrio spp. was conducted by filtering method and the isolated bacteria was cultured on CHROM agar Vibrio and TCBS agar. Final genus identification was performed by the MALDI TOF technique. RESULTS: In the present study, 10 isolates of Vibrio spp. were obtained from seawater and wet sand samples collected in the Gulf of Gdansk and Hel peninsula coast. Three of the isolates were identified as V. vulnificus; the presence of the species was confirmed in the seawater samples which had been collected in Hel (1 isolate), Jastarnia (1 isolate), and Chalupy (1 isolate). One strain of Vibrio alginolyticus was isolated from the seawater sample collected in Hel. Moreover, identification was incomplete for 6 of the isolated strains, these were identified as Vibrio cholerae/mimicus These strains were collected in Jastarnia (1 isolate), Kuznica (1 isolate), Gdansk-Brzezno (1 isolate), Puck (2 isolates), Chalupy (1 isolate). CONCLUSIONS: Our preliminary research study confirmed the presence of potentially pathogenic V. vulnificus in the Gulf of Gdansk in the summer months. Therefore, further monitoring of the presence of Vibrio spp. in the Baltic coast area is necessary.

Virulence Characterization of Listeria monocytogenes, Listeria innocua, and Listeria welshimeri Isolated from Fish and Shrimp Using In Vivo Early Zebrafish Larvae Models and Molecular Study.

Listeriosis is one of the most notable foodborne diseases and is characterized by high rates of mortality. L. monocytogenes is the main cause of human listeriosis outbreaks, however, there are isolated cases of disease caused by other species of the genus Listeria. The aim of this study was to evaluate strains of L. monocytogenes (n = 7), L. innocua (n = 6), and L. welshimeri (n = 2) isolated from fish and shrimps for their virulence based on the presence of virulence genes and the in vivo Danio rerio (zebrafish) larvae models. A total of 15 strains were analyzed. The zebrafish larvae model showed that the larvae injected with L. monocytogenes strains were characterized by the lowest survival rate (46.5%), followed by L. innocua strains (64.2%) and L. welshimeri (83.0%) strains. Multiplex PCRs were used for detection of selected virulence genes (luxS, actA2, prfA, inlB, rrn, iap, sigB, plcB, actA, hlyA), the majority of which were present in L. monocytogenes. Only a few virulence-related genes were found in L. welshimeri, however, no correlation between the occurrence of these genes and larval survival was confirmed. This research highlights the importance of the potential impact that Listeria spp. strains isolated from fish and shrimps may have on consumers.