Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages − Rakyat Biologi

The current technologies employed to inactivate bacterial pathogens in foods are not always foolproof and, therefore, new approaches for improving food safety are necessary. Bacteriophages provide an attractive alternative since phages are ubiquitous in different environments, unable to infect human cells and, consequently, they have great potential for use as biocontrol agents in foods (Hudson, Billington, Carey-Smith, & Greening, 2005). The exploitation of bacteriophages has already become an interesting tool to fight the emergence of antibiotic-resistant bacteria (Kutter & Sulakvelidze, 2005). Staphylococcus aureus phages have been used in the treatment of staphylococcal infections in humans and animals (O’Flaherty et al., 2005; Wills, Kerrigan, & Soothill, 2005). In addition, phage components such as endolysins have also been tested for their anti-S. aureus activities (Donovan, Lardeo, & Foster-Frey, 2006; O’Flaherty, Coffey, Meaney, Fitzgerald, & Ross, 2005a). The role of bacteriophages in S. aureus biofilm differentiation and maturation has also been studied (Resch, Fehrenbacher, Eisele, Schaller, & Go, 2005) as well as the application of endolysins to inhibit biofilm formation (Sass & Bierbaum, 2007). The complete genomes and proteomes of 27 S. aureus bacteriophages have recently been obtained (Kwan, Liu, DuBow, Gros, & Pelletier, 2005) and will likely assist in the identification of other proteins involved in host growth inhibition (Liu et al., 2004).

S. aureus is one of the pathogenic bacteria considered as a major threat to food safety (de Buyser, Dufour, Maire, & Lafarge, 2001), and was responsible for the 1–9% outbreaks associated with milk and dairy products consumption during the period 1993–1998 in Europe (Tirado & Schmidt, 2000). In Spain, S. aureus was the causative agent in 13.9% and 11.1% of the foodborne outbreaks associated with cheeses and milk, respectively (Anonymous, 2003).

The manufacture of cheese from raw milk, particularly in cases of slow or insufficient acidification of curd, has led to staphylococcal outbreaks associated with this product (Le Loir, Baron, & Gautier, 2003). S. aureus may also contaminate heat-treated milk or curd if the hygienic conditions are inadequate. Therefore, S. aureus may be found in cheeses made either from raw or pasteurized milk (Coveney, Fitzgerald, & Daly, 1994). Furthermore, an initial population of 10³ cfu mL⁻¹ of S. aureus in milk may be sufficient for the production of enterotoxin A in cheese at detectable levels (Meyrand et al., 1998). Thus, the risk of enterotoxin production in cheese and the subsequent human intoxication indicates a need for new procedures to control S. aureus in curd and cheese. In this regard, we propose the use of phages in curd bearing in mind that limited data have been published on the effect of phages on S. aureus survival in milk (Gill, Sabour, Leslie, & Griffiths, 2006; O’Flaherty, Coffey, Meaney, Fitzgerald, & Ross, 2005b).

We have isolated from milk samples two phages, ΦH5 and ΦA72, that were able to infect several S. aureus also isolated from milk. This phage cocktail hampered the development of S. aureus in ultra-high-temperature (UHT) and pasteurized milk (García, P., unpublished data). However, a complete clearance of the pathogen was not achieved and S. aureus-resistant variants were easily generated. This prompted us to select lytic phages from their temperate counterparts, ΦH5 and ΦA72. The bactericidal effect of the cocktail of lytic phages on S. aureus during the manufacture of acid and enzymatic curd was investigated.

We have evaluated the suitability of S. aureus lytic phages for the biocontrol of this foodborne pathogen in some dairy products. S. aureus is one of the most frequent agents of bovine mastitis that contribute to milk contamination. Of particular relevance to the food processing industry is the ability of some strains to produce heat stable enterotoxins that cause staphylococcal food poisoning (Dinges, Orwin, & Schlievert, 2000). Therefore, new approaches to fight against this pathogen are necessary. Bacteriophages possess attributes that appear to be attractive to inhibit foodborne pathogens and spoilage organisms (Greer, 2005). They are antibacterial agents since they kill their host bacteria at the end of the lytic cycle. Indeed, phage therapy has been used successfully (Kutter & Sulakvelidze, 2005; Sulakvelidze, Alavidze, & Morris, 2001).

All attempts to isolate lytic phages from dairy environment were unsuccessful. Thus, we obtained two S. aureus lytic phages, Φ88 and Φ35, by DNA random deletion of their temperate counterparts, ΦH5 and ΦA72, isolated from raw milk. A mixture of both lytic variants infected six bovine strains out of a panel of 13 and all milk isolated strains available in our laboratory collection. These phages proved to be very efficient in the inhibition of the pathogen in UHT milk and in both acid and enzymatically produced curds. We have taken into account that the use of virulent phages holds several advantages in relation to temperate variants for use in phage-based biocontrol approach in food safety. First of all, the frequency of development of BIM, which could compromise the efficacy of a phage treatment, is often associated with point mutations in genes encoding receptor molecules on the bacterial cell surface (Forde & Fitzgerald, 1999), and commonly these mutants revert to phage sensitivity rapidly (O’Flynn, Ross, Fitzgerald, & Coffey, 2004). However, in temperate bacteriophages, higher BIM frequencies are found due to the acquisition of a lysogenic state that renders the cells resistant to infection. Consequently, our lytic variants showed a lower rate of BIM, due to their inability to lysogenize. Such properties are crucial for preparing phage mixtures for the control of unwanted bacteria in food. On the other hand, temperate phages are one of the leading causes of dissemination of antibiotic resistance and virulence factors (e.g., enterotoxin production) (reviewed by Brussow, Canchaya, & Hardt, 2004) and their deliberated spread in nature should be avoided. Considering the temperate origin of our lytic-derived phages, confirmation of the lack of any virulence trait in their genome should be obtained. Absence of several enterotoxins (enterotoxin A, D, E, J and leukotoxin lukM-lukF-PV) has been preliminary confirmed by PCR (data not shown).

A mixture of the lytic phages (Φ88+Φ35) was able to withstand the stresses found in acid curd manufacturing processes. Even though the phage cocktail was partially inactivated by low pH, it was able to completely eradicate viable S. aureus cells in curd made of heavily contaminated milk. Obviously, the presence of host cells, in which the phage is able to replicate, was enough to counteract pH inactivation. Further work is needed to determine the minimum host density which ensures phage replication in these conditions. Preliminary results in pasteurized milk indicate that lower contamination levels (10² cfu mL⁻¹) are still enough and the pathogen can be eliminated (García, unpublished observation).

Previous reports have shown that milk proteins could inhibit phage adsorption to the cell surface (Gill et al., 2006). However, according to our results, the phages were very stable and active during enzymatic curd formation, implying that pH is the most crucial inactivation factor. The activity of these phages in milk, in contrast to the inactivity of the bacteriophage K in raw milk (Gill et al., 2006), could be related to their milk origin. It is known that phages rapidly evolved along with their host and their environment (Brussow et al., 2004).

The use of a mixture of phages to control undesirable bacteria in food has been reported in several food systems (Carlton, Noordman, Biswas, de Meester, & Loessner, 2005; Hudson et al., 2005; Modi, Hirvi, Hill, & Griffiths, 2001). Furthermore, the use of an anti-Listeria phage preparation has been recently approved by the FDA (FDA, 2006). Studies on the application of phages to animals reported no adverse or unexpected effects (Biswas et al., 2002; Bruttin & Brüssow, 2005; Cerveny, DePaola, Duckworth, & Gulig, 2002). In addition, their specificity and ubiquitous presence in nature makes a disturbance in the intestinal microbiota unlikely. Hence, the intake of pathogen-specific phages along with food may be harmless to humans. Similarly, the food microbiota, particularly relevant in the production of fermented products, would not be disturbed. Data obtained concerning the S. aureus phages Φ88 and Φ35 suggest that their use as an additive for biopreservation of dairy products would be efficient and safe provided that no virulence traits are encoded in their genome. Challenge studies are in progress to determine the most advantageous conditions (e.g., host density, temperature) in which these phages can efficiently inhibit S. aureus in milk and other dairy products. These studies are necessary to implement phage biocontrol in dairy processes.