Assessment of control strategies against Staphylococcus aureus biofilms potentially present in the fishery industry

Resumo

Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing a long-term persistence of this pathogen in food-related environments. Novel trends in food production (e.g. minimal processing, mass production, globalization) have additionally introduced new scenarios that can enhance the presence and subsequent growth of S. aureus. Moreover, an increased number of antibiotic-resistant S. aureus has been detected in non-clinical ambits, including the food industry, which can lead to the transmission of resistances to the human microbiome through the ingested food and causing infections hard to be treated. Therefore, this work was aimed to improve the control of S. aureus in the food industry (particularly, in fisheries) through the identification of the most risky scenarios of contamination and the evaluation of promising disinfection strategies against this pathogen. The first objective was thus the assessment of the incidence of S. aureus in different commercialized fishery products. A total of 298 fishery products of different origin and type of processing were sampled. Isolates were identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA sequencing), and characterized by RAPD-PCR with three primers (AP-7, ERIC-2 and S). In addition, the enterotoxin-producing ability (i.e. presence of se genes) and the resistance to several antibiotics were also evaluated for all isolates. S. aureus was detected in a significant proportion of products (~ 25%), being the highest incidence in fresh (43%) and frozen products (30%). In addition, a significant proportion of smoked fish, surimis, fish roes and other ready-to-eat products did not comply with legal limits in force. Isolates displayed 33 fingerprint patterns, and each one was attributed to a single bacterial clone. Cluster analysis based on similarity values between RAPD fingerprints did not find relationship between any RAPD pattern and any product category. Most isolates (88%) were found to be sea positive. Putative enterotoxigenic strains counts reached high risk levels in 17 products. No relationship was found between the presence of se genes and RAPD patterns. All isolates were resistant to penicillin, chloramphenicol and ciprofloxacin, and most to tetracycline (82.4%), but MRSA were not detected. In fact, none strain carried mecA gen, which is related to the resistance to beta-lactam antibiotics such as methicillin. Subsequently, the prevalence in fishery-processing facilities of putative enterotoxigenic S. aureus strains was examined by studying their biofilm-forming ability and disinfectant resistance. All strains were described as S. aureus able to produce exopolysaccharides (positive phenotype in red Congo agar and icaA- and icaD-carriers), but none carried bap gene (expression of biofilm accessory protein). Most strains showed a biofilm-forming ability higher than S. aureus ATCC 6538 -reference strain in bactericidal standard tests- on common food-contact surface materials (stainless steel, polystyrene) and under different environmental conditions (temperature, nutrient content, osmolarity) potentially present in processing plants. In general, initial adhesion of S. aureus was increased by the presence of high ionic strength conditions, whereas biofilm formation was significantly promoted by the presence of glucose (e.g., additive in surimis and smoked fish), but moderately by sodium chloride or magnesium (e.g., wastes of seawater and seafood). Nevertheless, transcriptional analysis of genes related with biofilm formation (icaA, sarA, rbf and ?B) showed a high variability between strains in the response to these environmental conditions. Moreover, it seems that food-processing could have produced a selective pressure and strains with a high biofilm-forming ability were more likely to be found in highly handled and processed products. Biofilms formed by all strains showed a marked resistance to disinfectants applied commonly in the food industry (benzalkonium chloride or BAC, peracetic acid or PAA, sodium hypochlorite or NaClO), being higher than ATCC 6538 in most cases. As expected, the resistance of S. aureus biofilms was significantly higher than that of planktonic cells in all cases. But no correlation was found between the resistance of biofilms to BAC, PAA and NaClO and that of planktonic cells, so no extrapolation seems thus feasible. However, most standard bactericidal tests used in the European Union are based in suspension cultures, and only EN 13697 is biofilm-based, but it does not seem to truly simulate environmental conditions found in the food industry. The antimicrobial resistance increased as biofilm aged. Biofilm formation also seemed to attenuate the effect of low temperatures on BAC resistance. PAA was found to be most effective against both biofilms and planktonic cells, followed by NaClO and BAC. But the resistance of strains did not follow the same order for each biocide, which shows the present limitation of using a few type strains (and only one S. aureus) in standard tests in order to ensure a proper application of disinfectants. Consequently, doses recommended by manufacturers for BAC, PAA and NaClO to disinfect food-contact surfaces were lower than data obtained in this study, so they are not able to guarantee biofilm removal. Microorganisms could therefore be exposed to sub-lethal doses of disinfectants and this could generate the emergence of antimicrobial resistance. For this reason, this work was then focused in the study of the efficacy and applicability of innovative and more environmental-friendly disinfection strategies to control S. aureus biofilms on fishery-processing plants. Particularly, it was investigated the bactericidal activity of electrolyzed water (EW) and a range of essential oils (EOs), as well as combined treatments with BAC or PAA. The four S. aureus strains with the highest potential prevalence in food-processing plants and with a high incidence in fishery products (St.1.01, St.1.04, St.1.07 and St.1.08) were evaluated. In the case of EW, its efficacy against biofilms was hardly any affected by variations in the pH of production. Neutral EW (NEW) was therefore used in subsequent studies as it has a higher potential for long-term application than acidic EW (due to a lower corrosiveness and toxicity) and due to the higher yield rate of the production unit at neutral pH. The application of NEW caused a high reduction in the number of viable biofilm cells initially. However, a high available chlorine concentration (800 mg/L ACC) was needed to achieve logarithmic reductions (LR) demanded by the European quantitative surface test of bactericidal activity (? 4 log CFU/cm2 after 5 min). A double sequential application of NEW at much lower concentrations for 5 min each allowed LR ? 4 log CFU/cm2 to be reached in most of the experimental range. Sequential applications of NEW and either BAC or PAA showed a similar effect, with PAA-NEW being most effective. The combination of NEW with other antimicrobial treatments can thus be an effective alternative to disinfection protocols traditionally used in the food industry. Otherwise, the effectiveness of nineteen EOs (anise, carrot, citronella, coriander, cumin, Eucalyptus globulus, Eucalyptus radiata, fennel, geranium, ginger, hyssop, lemongrass, marjoram, palmarosa, patchouli, sage, tea-tree, thyme and vetiver) was assessed against planktonic cells of S. aureus St.1.01. Planktonic cells showed a wide variability in resistance to EOs, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight EOs most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All EOs reduced significantly the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli oils were the most effective, but high concentrations were needed to achieve LR over 4 log CFU/cm2 after 30 min exposure. The use of sub-lethal doses of thyme oil prevented biofilm formation and enhanced the efficiency of thyme oil and BAC against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, EO-based treatments should be based on the rotation and combination of different EOs or with other biocides to prevent the emergence of antimicrobial-resistant strains. Combined thyme oil-based treatments can be an effective, environmentally-friendly, safe-to-use and relatively inexpensive alternative to control the formation of S. aureus biofilms on food-processing facilities