Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses

https://doi.org/10.1016/j.ijfoodmicro.2012.06.012Get rights and content

Abstract

Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 106 CFU/mL) and a cocktail of the two lytic phages (about 106 PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83 log CFU/g of S. aureus occurred in 3 h compared with control cheese, and viable counts were under the detection limits after 6 h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24 h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64 log CFU/g compared with the control cheeses. At the end of ripening, 1.24 log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73 log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties.

Highlights

► A cocktail of two staphylococcal lytic phages reduced S. aureus counts in both fresh and hard-type cheeses. ► In fresh cheese, S. aureus counts were not detected after 6 h of coagulation process. ► In hard cheese, S. aureus counts were below the limits for toxin production throughout ripening. ► Starter performance was not affected by the presence of phages.

Introduction

The current consumer demand for nutritious food products, containing minimal amounts of chemically synthesized additives, with acceptable shelf-life and high organoleptical quality has driven research into alternative food preservation methods to fight against pathogenic and spoilage microorganisms in food. Namely, biopreservation aims at enhancing food safety by using natural microbiota and/or their metabolites with antimicrobial properties. Bacteriocins produced by lactic acid bacteria and bacteriophages (phages) are good examples of biopreservation agents García et al., 2010, Gálvez et al., 2010.

The use of virulent phages that infect and lyse bacterial pathogen and spoilage bacteria in food is quite recent and this strategy has been explored along the food chain from the production of primary commodities to shelf life extension of manufactured food products García et al., 2010, Mahony et al., 2011. A remarkable advantage of the application of phages as food biopreservatives is their specificity towards their hosts, meaning that undesired target bacteria are infected, and consequently killed, without disturbing the endogenous microbiota (i.e., starter cultures used in fermented foods). Besides, no adverse effects on the human commensal microbiota have been reported after oral administration of bacteriophages in rats and humans Carlton et al., 2005, Bruttin and Brussow, 2005.

The potential of phages as biocontrol agents in food is supported by several studies that indicate an efficient reduction of pathogen levels in meat Atterbury et al., 2003, Whichard et al., 2003, fresh-cut produce Leverentz et al., 2001, Leverentz et al., 2003, dairy products Modi et al., 2001, García et al., 2007, Guenther and Loessner, 2011 or reconstituted infant formula (Kim et al., 2007). Phages were also effective in controlling the growth of beer spoilage bacteria (Deasy et al., 2011). Some phage preparations are already commercially available. This is the case of ListexTM P100 that has been recognized as safe by the Food and Drug Administration (FDA) and has been also approved by the US Department of Agriculture (USDA) as an antimicrobial processing aid to combat Listeria monocytogenes in foods (www.micreosfoodsafety.com). Nevertheless, phages intended as biocontrol agents in food must be evaluated carefully to reduce the risk of spreading virulent factors among the bacterial population García et al., 2008, Cheng and Novick, 2009. Accordingly, the use of obligately lytic instead of temperate phages is encouraged because the latter are the leading cause of dissemination of virulent factors (Brüssow et al., 2004).

The food industry is mostly concerned with food-borne pathogens such as L. monocytogenes, Salmonella, Campylobacter jejuni, Staphylococcus aureus and enteropathogenic Escherichia coli. In particular, S. aureus has been responsible for outbreaks associated with milk and dairy products, particularly those strains able to produce heat stable enterotoxins De Buyser et al., 2001, Tirado and Schmidt, 2000, Le Loir et al., 2003, Little et al., 2008. Of note, the largest proportion of verified outbreaks caused by staphylococcal toxins (21.6%) was attributed to cheese in a recent report (EFSA and ECDC, 2011). Bovine mastitis is an important source of milk contamination by S. aureus as this pathogen is one of the most prevalent agents of intramammary infections in dairy ruminants (Katsuda et al., 2005). Postpasteurization contamination of milk and dairy products by food handlers has also been reported (Waldvogel, 2001). The European regulation has set the upper limit for coagulase positive staphylococci at 105 CFU/g in cheese. Above this limit, enterotoxin determination must be conducted in the cheese batch (Commission Regulation [EC] No., 2073/2005). Therefore, it is very important to control S. aureus growth throughout the cheese-making process Delbes et al., 2006, Meyrand et al., 1999.

Bacteriophages vB_SauS-phi-IPLA35 (in short, phiIPLA35) and vB_SauS-phi-IPLA88 (in short, phiIPLA88) belong to the Siphoviridae family and are lytic derivatives of temperate phages ΦA72 and ΦH5, previously isolated from the dairy environment. These bacteriophages were able to inhibit S. aureus grown in milk and curd manufacturing processes García et al., 2007, García et al., 2009. Regarding this, the aim of this study was to evaluate the suitability of these lytic S. aureus phages as biocontrol agents against this pathogen in both fresh and hard-type cheeses.

Section snippets

Bacterial strains and growth conditions

S. aureus Sa9, isolated from a mastitic bovine milk sample, was grown in 2xYT broth (Sambrook et al., 1989) at 37 °C for 18 h. Baird–Parker agar supplemented with egg yolk-tellurite (Scharlau Chemie, S.A. Barcelona, Spain) was used for differential counting. The strains Lactococcus lactis subsp. lactis IPLA 542, L. lactis subsp. lactis biovar. diacetylactis IPLA 838, and Leuconostoc citreum IPLA 979, grown in commercial skimmed UHT milk, were mixed in the ratio 56:19:25 (%, v/v) and used as

Effect of phage cocktail on S. aureus Sa9 growth in fresh-type cheese

The ability of the phage cocktail to control the development of S. aureus Sa9 was investigated during manufacturing and cold storage of fresh cheese. Fig. 1 shows the evolution of the lactic acid microbiota and staphylococcal counts throughout curdling and storage at 4 °C in the presence and in the absence of phages. Similar trend was shown by the starter strains whose level increased about 2.5 log units during the coagulation process (24 h) and decreased during the storage period in both control

Acknowledgments

This work was supported by grants AGL2009-13144-C02-01 (Ministry of Science and Innovation, Spain), and IB08-052 and COF07-006 (Science, Technology and Innovation Programme, Principado de Asturias, Spain).

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