Safety assessment of coagulase-negative staphylococci used in food production

Coagulase-negative staphylococci (CNS) are used in starter cultures for the production of fermented meat products due to their involvement in the development of desired red color, characteristic flavor as well as ensuring stability. But also other CNS species like S. condimenti, S. piscifermentans, S. equorum and S. succinus have a potential for future use in starter cultures. The safety of fermented food products is principally proven by long-term experience as traditional methods are considered safe based on their long “history of safe use”. However, for the last mentioned species long-term experience concerning sanitary harmlessness exists only with limitations.\nTo get an insight in safety relevant properties of food associated CNS in Chapter III-V strains of the species S. carnosus, S. condimenti and S. piscifermentans (S. carnosus-group) as well as S. equorum, S. succinus and S. xylosus (S. xylosus-group) were phenotypically and partly genotypically investigated. Based on these insights in Chapter VI a DNA microarray was developed for rapid and simultaneous detection of various safety relevant properties in CNS with future use in the food production. To increase the application potential of this microarray, additionally technological relevant properties were considered in the array design. Subsequently, the designed microarray was used for the genotypic investigation of phenotypically characterized CNS concerning the presence of safety relevant properties.\nIn Chapter III, antibiotic resistances of 330 CNS belonging to S. carnosus- and S. xylosus-group isolated from food and starter cultures were examined. Resistances to 21 antibiotics were phenotypically determined and resistance genes blaZ, lnuA and tetK were detected in strains showing phenotypic resistances to ß-lactam antibiotics, lincomycin and tetracycline. Antibiotic resistance profiles in strains of the species S. equorum, S. succinus and S. piscifermentans are described and due to the high number of investigated strains an insight regarding the occurrence of antibiotic resistances in food associated CNS is given.\nIn Chapter IV toxin production of food associated CNS belonging to S. carnosus- and S. xylosus-group was investigated. First, 330 strains isolated from food, starter cultures and clinical isolates have been analyzed to hemolytic activity on human and sheep blood agar plates. Secondly, the ability of 35 selected strains to produce staphylococcal enterotoxins, toxic shock syndrome toxin 1 and exfoliative toxin A has been examined by immunoblot analysis. The chapter demonstrates that CNS strains present in high numbers in fermented food cannot necessarily be regarded as safe. Thus, strains used in the production of fermented food should be analyzed with respect of their toxigenic potential to avoid negative effects on human health.\nChapter V is dealing with the formation of binding proteins to extracellular matrix proteins (ECM) and the production of biogenic amines (BA) by 32 CNS of S. carnosus- and S. xylosus-group. Binding capacity of CNS to the ECM fibronectin and fibrinogen was investigated by detection of fluorescent labeled cells which were added to microtiter plates coated with ECM. The formation of six important BA was examined by HPLC using growing and resting cells. By the results of this chapter the ability of food associated CNS to develop undesired properties like the formation of binding proteins to ECM and BA was demonstrated. Thus, further research is needed concerning potential risks and the importance on human health if strains with these properties are used in the production of fermented food.\nIn Chapter VI, the design of a polynucleotide based DNA microarray as screening tool to detect genes of potential health concern and technological relevance in food associated CNS is described. The array considered 220 genes encoding for antibiotic resistances, hemolysins, toxins, amino acid decarboxylases (involved in the formation of BA), binding proteins to ECM, lipases, proteases, stress response factors, and nitrate dissimilation. Hybridization experiments were performed using genomic DNA isolated of 32 in Chapter III-V phenotypically characterized CNS allowing the detection of e.g. antibiotic resistance genes blaZ, lnuA, and tetK. Genes coding for decarboxylases as well as fibronectin and fibrinogen binding proteins were rarely correlated with the phenotype. Toxin genes could not be detected, whereas technological relevant genes like genes coding for proteases, lipases, catalase, superoxide dismutase or genes involved in dissimilatory nitrate reduction resulted in hybridization signals. \nThe present thesis provides data concerning safety relevant properties in food associated CNS which are important for accurate safety assessment. Comparison of the results of Chapter III-V with them of Chapter VI showed that antibiotic resistances, formation of toxins and binding proteins to ECM are more present in strains of S. xylosus- than in S. carnosus-group. In context with safety assessment of food associated CNS, the designed microarray can be used as screening tool for the detection of safety relevant combined with technologically important properties (nitrate dissimilation, control of oxidative damage by catalase, flavor formation by proteases and lipases). Summarizing, the array is able to make a contribution in enhancing the selection criteria of CNS used as starter organisms in respect to food safety as well as technologically relevant properties.