Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages
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.
Post Comment
No comments