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Title: Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses
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Authors: Edita Bueno, Pilar García, Beatriz Martínez and A. Rodríguez*
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Addresses: Instituto de Productos Lácteos de Asturias (IPLA-CSIC). Department of
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Technology and Biotechnology of Dairy Products. 33300- Villaviciosa, Asturias, Spain.
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*
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Dr. Ana Rodriguez
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IPLA-CSIC
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33300-Villaviciosa, Asturias
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Spain.
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e-mail: anarguez@ipla.csic.es
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Phone: +34 985 89 21 31 (Ext. 24)
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Fax: +34 985 89 22 33
Corresponding author:
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Abstract
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Bacteriophages are regarded as natural antibacterial agents in food since they are able to
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specifically infect and lyse food-borne pathogenic bacteria without disturbing the
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indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages
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(vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the
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dairy environment, were evaluated for their potential as biocontrol agents against this
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pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was
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contaminated with S. aureus Sa9 (about 106 CFU/mL) and a cocktail of the two lytic
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phages (about 106 PFU/mL) was also added. For control purposes, cheeses were
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manufactured without addition of phages. In both types of cheeses, the presence of
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phages resulted in a notorious decrease of S. aureus viable counts during curdling. In
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test fresh cheeses, a reduction of 3.83 log CFU/g of S. aureus occurred in 3 h compared
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with control cheese, and viable counts were under the detection limits after 6 h. The
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staphylococcal strain was undetected in both test and control cheeses at the end of the
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curdling process (24 h) and, of note, no re-growth occurred during cold storage. In hard
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cheeses, the presence of phages resulted in a continuous reduction of staphylococcal
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counts. In curd, viable counts of S. aureus were reduced by 4.64 log CFU/g compared
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with the control cheeses. At the end of ripening, 1.24 log CFU/g of the staphylococcal
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strain was still detected in test cheeses whereas 6.73 log CFU/g was present in control
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cheeses. Starter strains were not affected by the presence of phages in the cheese
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making processes and cheeses maintained their expected physico-chemical properties.
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Keywords
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Lytic phage, biocontrol, cheese, Staphylococcus aureus
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1. Introduction
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The current consumer demand for nutritious food products, containing minimal
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amounts of chemically synthesized additives, with acceptable shelf-life and high
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organoleptical quality has driven research into alternative food preservation methods to
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fight against pathogenic and spoilage microorganisms in food. Namely, biopreservation
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aims at enhancing food safety by using natural microbiota and/or their metabolites with
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antimicrobial properties. Bacteriocins produced by lactic acid bacteria and
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bacteriophages (phages) are good examples of biopreservation agents (García et al.,
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2010; Galvez et al., 2010).
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The use of virulent phages that infect and lyse bacterial pathogen and spoilage
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bacteria in food is quite recent and this strategy has been explored along the food chain
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from the production of primary commodities to shelf life extension of manufactured
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food products (García et al., 2010; Mahony et al., 2011). A remarkable advantage of the
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application of phages as food biopreservatives is their specificity towards their hosts,
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meaning that undesired target bacteria are infected, and consequently killed, without
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disturbing the endogenous microbiota (i.e., starter cultures used in fermented foods).
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Besides, no adverse effects on the human commensal microbiota have been reported
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after oral administration of bacteriophages in rats and humans (Carlton et al., 2005;
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Bruttin and Brüssow, 2005).
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The potential of phages as biocontrol agents in food is supported by several
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studies that indicate an efficient reduction of pathogen levels in meat (Atterbury et al.,
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2003; Whichard et al., 2003), fresh-cut produce (Leverentz et al., 2001, 2003), dairy
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products (Modi et al., 2001, García et al., 2007, Guenther and Loessner, 2011) or
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reconstituted infant formula (Kin et al., 2007). Phages were also effective in controlling
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the growth of beer spoilage bacteria (Deasy et al., 2011). Some phage preparations are
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already commercially available. This is the case of ListexTM P100 that has been
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recognized as safe by the Food and Drug Administration (FDA) and has been also
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approved by the US Department of Agriculture (USDA) as an antimicrobial processing
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aid to combat Listeria monocytogenes in foods (www.micreosfoodsafety.com).
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Nevertheless, phages intended as biocontrol agents in food must be evaluated carefully
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to reduce the risk of spreading virulent factors among the bacterial population (García et
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al., 2008; Cheng and Novick, 2009). Accordingly, the use of obligately lytic instead of
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temperate phages is encouraged because the latter are the leading cause of dissemination
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of virulent factors (Brüssow et al., 2004).
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The food industry is mostly concerned with food-borne pathogens such as L.
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monocytogenes, Salmonella, Campylobacter jejuni, Staphylococcus aureus and
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enteropathogenic Escherichia coli. In particular, S. aureus has been responsible for
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outbreaks associated with milk and dairy products, particularly those strains able to
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produce heat stable enterotoxins (de Buyser et al., 2001; Tirado and Schmidt, 2000, Le
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Loir et al., 2003; Little et al., 2008). Of note, the largest proportion of verified outbreaks
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caused by staphylococcal toxins (21.6 %) was attributed to cheese in a recent report
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(EFSA and ECDC, 2011). Bovine mastitis is an important source of milk contamination
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by S. aureus as this pathogen is one of the most prevalent agents of intramammary
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infections in dairy ruminants (Katsuda et al., 2005). Postpasteurization contamination of
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milk and dairy products by food handlers has also been reported (Waldvogel, 2001).
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The European regulation has set the upper limit for coagulase positive staphylococci at
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105 CFU/g in cheese. Above this limit, enterotoxin determination must be conducted in
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the cheese batch (Commission Regulation [EC] Nº 2073/2005). Therefore, it is very
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important to control S. aureus growth throughout the cheese-making process (Delbes et
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al., 2006; Meyrand et al., 1999).
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Bacteriophages vB_SauS-phi-IPLA35 (in short, phiIPLA35) and vB_SauS-phi-
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IPLA88 (in short, phiIPLA88) belong to the Siphoviridae family and are lytic
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derivatives of temperate phages ΦA72 and ΦH5, previously isolated from the dairy
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environment. These bacteriophages were able to inhibit S. aureus grown in milk and
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curd manufacturing processes (García et al., 2007; 2009). Regarding this, the aim of this
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study was to evaluate the suitability of these lytic S. aureus phages as biocontrol agents
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against this pathogen in both fresh and hard-type cheeses.
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2. Materials and methods
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2.1. Bacterial strains and growth conditions
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S. aureus Sa9, isolated from a mastitic bovine milk sample, was grown in 2xYT
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broth (Sambrook et al., 1989) at 37ºC for 18 h. Baird-Parker Agar supplemented with
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egg yolk-tellurite (Scharlau Chemie, S.A. Barcelona, Spain) was used for differential
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counting. The strains Lactococcus lactis subsp. lactis IPLA 542, L. lactis subsp. lactis
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biovar. diacetylactis IPLA 838, and Leuconostoc citreum IPLA 979, grown in
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commercial skimmed UHT milk, were mixed in the ratio 56: 19: 25 (%, v/v) and used
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as starter cultures (1% v/v) in cheesemaking trials, as previously described (Cárcoba et
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al. 2000).
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2.2. Bacteriophages
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Bacteriophages phi-IPLA35 and phi-IPLA88 were routinely propagated on S.
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aureus Sa9 as previously described (García et al., 2007). Concentrated phage
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preparations were obtained by ultracentrifugation (100,000 × g for 90 min) of culture
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supernatants followed by CsCl gradient centrifugation (Sambrook et al., 1989). Double-
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plaque assays were performed to determine the phage titer by using 100 µL of a S.
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aureus Sa9 overnight culture and 100 µL of the appropriate phage dilution.
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2.3. Bacterial-phage challenge test during cheese manufacture
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Three independent trials of fresh and hard-type cheeses were manufactured in
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the Pilot Plant at the Instituto de Productos Lácteos de Asturias (IPLA-CSIC) following
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traditional manufacturing methods. For each fresh-type cheese (acid-coagulated cheese)
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trial, pasteurized whole bovine milk (72 °C, 15 s) was cooled to 25 °C and placed into
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two 12 L vats (control and test) and supplemented with 0.02% CaCl2. Each vat was
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inoculated at 1% (v/v) (about 107 CFU/mL) with an overnight culture of the mixed
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starter culture described above and contaminated with S. aureus Sa9 (about 1.7  106
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CFU/mL). A final concentration of about 106 PFU/mL of a cocktail of phages phi-
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IPLA35 and phi-IPLA88 (1:1) was also added to the test vat as biocontrol agent. A
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small amount of calf rennet (0.025 g/liter; activity 1:10,000, Laboratorios Arroyo,
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Santander, Spain) was added 2 h after the starter addition to increase the firmness of the
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coagulum. At the end of the coagulation process (24 h), the acid curd was placed in a
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linen-covered perforated container and kept overnight at room temperature for
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completed draining. Drained curd was salted with dry salt (1.5% w/w) and packaged in
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100 ml sterile containers and stored at 4ºC for up to 14 days.
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For each hard-type cheese trial, pasteurized whole bovine milk was cooled to
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32ºC and placed into two 12 L vats (control and test). CaCl2 supplementation and starter
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culture addition were performed as indicated for fresh cheeses. Both cheese vats were
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contaminated with S. aureus Sa9 (about 8 × 105 CFU/mL). The phage mixture (5 × 106
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PFU/mL) was also added to the test vat. Calf rennet (0.3 g/L, activity 1:10,000,
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Laboratorios Arroyo), was added 60 min after inoculation, and milk coagulation was
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performed along 45 min at 32ºC. Curd was cut into cubes of ca. 5 mm and stirred into
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the whey for 90 min. Whey was drained off, the curd filled into cylindrical molds and
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pressed for 1.25 h at 1.5 kg/cm2. Cheese pieces (about 300 g each) were placed in
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saturated brine for 30 min. Ripening took place at 11ºC and 80% relative humidity for
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30 days.
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2.4. Microbiological analyses
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Samples of milk (10 ml), curd (10 g), and cheese (10 g) were aseptically taken in
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duplicate at different times. Curd and cheese samples were homogenized in 90 ml of a
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prewarmed sterile 2% sodium citrate solution in a Stomacher Lab-Blender (Seward
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Medical, London, UK). Decimal dilutions of milk and homogenates were made in
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quarter-strength Ringer solution (Oxoid, Basingstoke, Hampshire, UK) and plated in
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triplicate in the appropriate medium. MRS agar was used for lactic acid bacteria
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counting, and Baird-Parker Agar supplemented with egg yolk-tellurite for S. aureus
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counting. Phage titere was obtained by plaque assays on 2xYT medium.
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2.5 Physico-chemical analyses of cheeses
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pH was measured with a MicropH 2001 pHmeter (Crison, Barcelona, Spain).
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Dry matter, fat and protein content were determined according to the International Dairy
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Federation (IDF, 1982, 1991, 1993). A 926 Chloride Analyser (Corning Medical and
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Scientific, England) was used to determine the NaCl content.
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2.6. Statistical analysis
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Statistical analysis was performed using the SPSS-PC +11.0 software (SPSS,
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Chicago, IL, USA). Data related to gross composition (dry matter, protein, fat and
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sodium chloride content) and S. aureus counts were subjected to ANOVA. Tests were
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performed within each sampling time using type of cheese as a factor with two
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categories: cheese manufactured in the presence and in the absence of phages.
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3. Results and Discussion
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3.1. Effect of phage cocktail on S. aureus Sa9 growth in fresh-type cheese
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The ability of the phage cocktail to control the development of S. aureus Sa9
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was investigated during manufacturing and cold storage of fresh cheese. Figure 1 shows
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the evolution of the lactic acid microbiota and staphylococcal counts throughout
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curdling and storage at 4ºC in the presence and in the absence of phages. Similar trend
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was shown by the starter strains whose level increased about 2.5 log units during the
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coagulation process (24 h) and decreased during the storage period in both control and
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test cheeses (P>0.05). Moreover, pH similarly dropped from 6.6 in milk to 4.24-4.25 at
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the end of the coagulation process in both cheese batches and a slight increase was
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observed during storage. As expected, because of their high specificity, the presence of
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phages did not compromise starter performance and cheese making proceeded
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undisturbed. In fact, the physico-chemical parameters of test and control cheeses were
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very similar showing no significant differences (P>0.05). Mean values of dry matter
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(20.06  0.42 % [w/w]), protein content (43.91  0.32 % of dry matter), fat content
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(45.91  1.82 % of dry matter) and NaCl (1.5  0.15 g /100 g cheese) in both cheeses
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during cold storage were within the standards of fresh-type (acid coagulated) cheeses
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(Schulz-Collins and Senge, 2004).
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The presence of phages resulted in a quick and significant reduction of the
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staphylococcal population compared with control cheeses (P<0.05). Viable counts
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dropped from 6.25 log CFU/g in milk to 3.38 log units after 3 h and were under the
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detection limits (<10 CFU/g) in 6 h (Fig. 1B). By contrast, in control cheeses, S. aureus
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was able to multiply reaching levels of 7.20 log CFU/g at 6 h (Fig. 1A). In both cases,
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staphylococcal counts were undetectable at 24 h. This is most likely due to the
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restrictive environment for staphylococcal survival created by the low pH (4.24) after
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curdling. It is worth mentioning that S. aureus re-growth was not observed in either
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cheese batch during cold storage for 14 d.
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The negative impact of the low pH on S. aureus survival has been previously
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reported (Lanciotti et al., 2001; Lindqvist et al., 2002; Alomar et al., 2008). Of note,
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lactic acid bacteria can contribute to S. aureus inhibition not only by decreasing pH but
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also by producing bacteriocins (Rilla et al. 2004) or H2O2 (Ito et al. 2003). In any case,
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according to our results, phages contributed considerably to accelerate S. aureus
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inactivation lowering the risk of enterotoxin accumulation by effectively inhibiting S.
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aureus multiplication long before pH values were low enough to be safe.
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Remarkably, our results also showed that phage replication could take place
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within the cheese matrix under these specific conditions. Indeed, an increase of the
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phage titre was observed in the first 3 h and then remained stable until 6 h (Fig. 1B).
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However, phage titre decreased thereafter probably due to partial inactivation of the
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phages caused by low pH (García et al., 2007).
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3.2. Effect of phage cocktail on S. aureus Sa9 growth in hard-type cheeses
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The lactic acid microbiota showed a similar evolution in control and phage-
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treated cheeses throughout manufacturing and ripening (P>0.05) (Figure 2). The mean
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counts in milk (6.64 log CFU/mL) increased about 2 log units during the coagulation
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process. This was due to the growth of the starter strains, as reflected by the decrease in
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pH from 6.64 in milk to 6.05-5.98 in curd, and to the concentration of cells in the curd
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after whey drainage. Further multiplication of bacteria occurred until 3-day control
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cheeses in which the viable counts reached 9.5 log CFU/g and remained unchanged
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until the end of ripening. Regarding the gross composition parameters, both cheeses
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showed a similar trend. However, significant differences were observed in dry matter
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content of 15-day old cheeses (P<0.05) and in salt content of 15-day and 30-day old
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cheeses (P<0.05) (Table 1).
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A very strong bactericidal effect of the lytic phages on the S. aureus Sa9 strain
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was observed during cheese manufacturing (Fig. 2). The presence of phages resulted in
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a fast and continuous decrease of staphylococcal viable counts dropping from the initial
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milk contamination level (5.9 log CFU/mL) to 1.24 log CFU/g at the end of the ripening
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period. This contamination level is low enough to avoid the production of sanitary risk
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levels of enterotoxins (Belay & Rasooly, 2002), one of relevant virulent factors
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associated to S. aureus and produced by about 25% of staphylococcal isolates from
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foods (Dinges et al., 2000). Enterotoxin in soft-cheese made with milk highly
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contaminated with S. aureus (7.5 log CFU/g) and treated with 500 MPa has even been
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detected after 30 days of ripening in spite of a viable count reduction of 4.7 log CFU/g
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was observed (López Pedemonte et al., 2006). Thus, a very quick reduction of the initial
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staphylococcal contamination levels is essential to avoid enterotoxin production.
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In the absence of phages, by contrast, an increase of 2.27 log CFU/g of
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staphylococcal population occurred during the coagulation process as a consequence of
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growth and cell concentration. The highest level of the strain was detected in one-day
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old cheese (8.17 log CFU/g). A slight and continuous decline further occurred until 6.73
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log CFU/g at 30 days of ripening is reached, this level being 5.5 log CFU/g higher than
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that detected in phage-treated cheeses.
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In parallel with S. aureus Sa9 initial decrease, a slight increase of phage titer
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occurred that remained constant until the end of ripening. The stability of phage titer
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was previously observed in enzymatic curds (García et al., 2007). In spite of the
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presence of a stable phage titre throughout ripening, a complete elimination of S. aureus
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was not achieved. This supports previous observations indicating that successful phage
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infection and subsequent killing of the host cells are strongly dependent on the ratio
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phages/bacteria (Cairns et al., 2009). Thus, the phage concentration to be used must be
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specifically optimized for each food system to ensure the contact of the passively
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diffusing phage particles with their host cells (Hagens and Offerhaus, 2008). Regarding
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this, a minimum phage concentration (about 2 × 108 PFU/mL) has been reported for
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inactivating S. aureus in milk (Obeso et al., 2010). Similar phage concentration has
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required for inactivation close to 100% of Salmonella in LB broth (Bigwoog et al.,
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2009). High phage concentration (over 3 × 108 PFU/cm2) was also used to control the
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surface contamination of soft cheeses (Camembert and Limburger-type) by Listeria
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monocytogenes. The higher the phage dose, the higher the effectiveness of virulent
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phage A511 on both cheeses (Guenther and Loessner, 2011).
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Physical and chemical factors associated with the food matrix could also
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compromise the successful use of phages as biocontrol agents (Guenther et al., 2009). It
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has been reported that whey milk proteins inhibited phage K binding to S. aureus (Gill
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et al., 2006). On the other hand, a reduced firmness and higher moisture of coagulum
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facilitated phage activity against Salmonella in Cheddar cheese made with pasteurized
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milk compared to that of raw milk (Modi et al., 2001). In our case, the quick increase of
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the dry matter (and the concomitant moisture decrease) that we observed in hard-type
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cheese (Table 1) could have hampered phages to encounter the bacterial cell host and
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thus, it could contribute to the presence of viable staphylococcal cells at the end of
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ripening.
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In conclusion, phages have been shown to be effective on reducing S. aureus
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counts in both fresh and hard-type cheeses without compromising the performance of
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the starter culture or their physico-chemical properties. In fresh cheeses, both low pH
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and phages contributed to clearance of the pathogen. In hard cheeses, the pathogen was
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not totally eliminated but its numbers were kept far below the limits for toxin
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production in the presence of phages.
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Acknowledgments
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This work was supported by grants AGL2009-13144-C02-01 (Ministry of Science and
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Innovation, Spain), and IB08-052 and COF07-006 (Science, Technology and
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Innovation Programme, Principado de Asturias, Spain)
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18
441
Table 1
442
One-way ANOVA of different gross composition parameters performed within each
443
ripening time to compare control and test (phages added) hard-type cheeses
Cheese
Dry mattera
Proteinb
Fatb
NaClc
5
Control
Test
61.041.85
61.080.97
37.380.43
37.240.57
53.600.91
52.391.36
1.710.20
1.690.04
8
Control
Test
63.272.77
61.241.07
38.311.59
38.181.81
51.430.97
53.370.44
3.080.17
3.100.18
15
Control
Test
68.720.93
63.891.60*
37.041.53
39.134.93
51.430.97
53.632.16
3.710.14
3.270.22*
30
Control
Test
69.204.55
70.244.97
36.982.25
39.624.94
52.631.64
54.166.25
3.940.04
3.590.20*
Ripening
time (days)
444
445
446
447
448
449
Data reported are means  standard deviations of three independent trials
a
Dry matter (%, w/w)
b
Data expressed as percentage of dry matter
c
Salt content expressed as g NaCl/ 100 g cheese
*P<0.05
19
450
Figures captions
451
452
Fig. 1. Effect of phage cocktail (phi-IPLA35 and phi-IPLA88) on growth of S. aureus
453
Sa9 in fresh-type cheese throughout manufacturing and cold storage. (A) Control cheese
454
(no phage cocktail added) (B) Test cheese (phage cocktail added). Symbols: (♦ lactic
455
acid microbiota, starter culture); (■ S. aureus Sa9); (∆ pH); (× phages). Data reported
456
are means ± standard deviations of three independent trials. One-way ANOVA was
457
performed within each sampling time to compare S. aureus Sa9 counts in control and
458
test cheeses. **P<0.01; ***P<0.001.
459
460
Fig. 2. Effect of phage cocktail (phi-IPLA35 and phi-IPLA88) on growth of S. aureus
461
Sa9 in hard-type cheese throughout ripening. (A) Control cheese (no phage cocktail
462
added) (B) Test cheese (phage cocktail added). Symbols: (♦ lactic acid microbiota,
463
starter culture); (■ S. aureus Sa9); (∆ pH); (× phages). Data reported are means ±
464
standard deviations of three independent trials. One-way ANOVA was performed
465
within each sampling time to compare S. aureus Sa9 counts in control and test cheeses.
466
*P<0.05; **P<0.01; ***P<0.001.
467
20
Bacterial counts
(log CFU/g)
10
4
8
8
7
6
6
5
4
2
3
0
2
Bacterial counts (log CFU/g
Phage titre (log PFU/g)
468
Fig. 1
469
470
21
10
4
8
8
7
6
6
5
4
2
3
0
2
Bacterial counts (log CFU/g)
9
7
5
3
1
7
6
5
4
3
Bacterial counts (log CFU/g)
Phage titre (log PFU/g)
471
Fig. 2
8
2
472
22
9
8
7
5
3
1
7
6
5
4
3
2