Interactions between Staphylococcus aureus and lactic acid bacteria: An old story with new perspectives

International Journal of Food Microbiology 131 (2009) 30–39

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Interactions between Staphylococcus aureus and lactic acid bacteria: An old story with new perspectives C. Charlier, M. Cretenet, S. Even, Y. Le Loir ⁎ UMR1253 STLO INRA Agrocampus Rennes, 65, rue de Saint Brieuc CS84215 35042 Rennes, France

A R T I C L E

I N F O

Keywords: Lactic acid bacteria Staphylococcus aureus Bacterial interactions Ecosystems

A B S T R A C T Staphylococcus aureus is a Gram positive opportunistic pathogen and a major concern for both animal and human health worldwide. In some contexts where Lactic Acid Bacteria (LAB) are the normal dominant microbiota, such as in fermented food or in the vaginal ecosystem, S. aureus sometimes colonises, persists, expresses virulence factors and produces food poisoning or urogenital infections, respectively. Studies on the interactions between LAB and S. aureus began a few decades ago and were pursued to shed light on the inhibitory capabilities that LAB might have on S. aureus growth and/or enterotoxin production in fermented foodstuffs. These early studies had the aim of developing methods to prevent staphylococcal food poisoning, thus improving food safety. More recently, the concept of vaginal probiotic LAB has emerged as a promising way to prevent urogenital infections, S. aureus being one of the potential pathogens targeted. This review provides an up-to-date look at the current hypotheses of the mechanisms involved in the inhibition of S. aureus by LAB in both the vaginal ecosystem and in fermented food ecosystems. We also emphasise that post-genomic approaches can now be envisioned in order to study these diverse and complex interactions at the molecular level. Further works in this field will open up new avenues for methods of biocontrol by LAB and/or for biotechnological uses of LAB-compounds to fight against the long-standing, yet incumbent menace of staphylococcal infection. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Staphylococcus aureus is an opportunistic pathogenic Gram positive bacterium and the causative agent of a wide panel of infections ranging from superficial lesions to life-threatening septicaemia. The natural ecological niches of this species are the nasal cavity and the skin of warm-blooded animals (Kluytmans and Wertheim, 2005). Because of its importance and high prevalence in nosocomial infections, S. aureus is one of the most studied Gram positive pathogen. The effects that antibiotic compounds and physicochemical parameters such as pH, temperature, H2O2 or salt concentration, amongst others, have on S. aureus physiology, as well as host– pathogen interactions and immune response to staphylococcal infections are well-documented. The interactions between S. aureus cells have been well studied because it has been found that they tightly regulate and determine key steps in the infectious process (Novick, 2003). In contrast, little is actually known about the interactions between S. aureus cells and those belonging to different species, especially within contexts where S. aureus must colonise and persist in ecological niches occupied by other microorganisms. There is currently an increasing interest in bacterial interactions and in the ⁎ Corresponding author. Tel.: +33 223485904; fax: +33 223485350. E-mail address: [email protected] (Y. Le Loir). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.06.032

understanding of the mechanisms by which the inhibition of pathogens by other bacterial species may occur. These studies have been notably aimed at providing a basis for the developments of methods envisaging new means of controlling the emergence and spread of antibiotic resistant S. aureus strains. From a food safety and human health point of view, cheese and vaginal ecosystems are two ecosystems wherein lactic acid bacteria (LAB) are the dominant microbiota and where S. aureus is prone to posing major public health problems (vaginosis, toxic shock syndrome and food poisoning). LAB compose a heterogeneous bacterial group comprising non-sporulating, Gram positive cocci and bacilli, whose principal common characteristic is the ability to ferment sugars predominantly into lactic acid which in turn leads to an acidification of the environment down to a pH of 3.5. These bacteria have a facultative anaerobic metabolism and do not produce catalase. LAB occupy various ecological niches. Some species, such as Lb. plantarum, are reportedly adaptable to several different environments and are associated to numerous ecosystems varying from plant surfaces to the human digestive tract (Kleerebezem et al., 2003). Many species of lactobacilli have been isolated from the vaginal or the digestive microbiota. Other species, such as L. lactis, are less ubiquitous and some strains seem to have evolved towards the adaptation to a particular niche, such as certain plant species or milk (van Hylckama Vlieg et al., 2006).

C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39 Table 1 Examples of food preservation involving LAB. Food/products Milk products Butter and sour cream Yoghurts Churn buttermilk Fresh cheese Curded cheese

Kefir Kummis Taette

Ingredients

LAB

Cow milk

L. lactis ssp. cremoris Ln. mesenteroides Lb. delbrueckii ssp. bulgaricus S. thermophilus, Lb. delbrueckii ssp. bulgaricus

Cow milk Cow milk Cow, ewe or goat milk Cow, ewe or goat milk

L. lactis, Ln. mesenteroides, Lb. rhamnosus L. lactis ssp., Lactis L. lactis ssp. cremoris, S. thermophilus, Lb. delbrueckii ssp., Lactis Lb. delbrueckii ssp. helveticus Cow, mare or goat milk Lb. lactis, Lb. bulgaricus Mare milk Lb. bulgaricus, Lb. leichmanii Cow milk S. thermophilus

Meat and fish products Dry sausage Pork, beef Semi-dry sausage Beef Burong dalag Fish, rice Izushi Fish, rice, vegetable

Lb. plantarum, Lb. brevis, Pediococcus sp. Lb. plantarum, Lb. brevis, Pediococcus sp. Pediococcus sp. Ln. mesenteroides, Lb. plantarum, Lactobacilli

Vegetal products Silages «kenkey» Ogi Olives

Corn Corn Corn Green olives

Pickles Sauerkraut Soya sauce Wine Sake

Cucumber Cabbage Soybean Grapes Rice

Lb. plantarum Lactobacilli Lb. plantarum, L. lactis Ln. mesenteroides, Lb. plantarum, Pediococcus sp., Ln. mesenteroides Lb. plantarum, Pediococcus sp. Ln. mesenteroides, Lb. plantarum Lb. delbruckii Ln. oenos Lb. sakei, Lb. homohioshi, Ln. mesenteroides

Wheat flour

Lb. sanfranciscensis

Wheat flour

Ln. mesenteroides

Rice and bean flour

Ln. mesenteroides

Breads Sanfrancisco sourdough Sour pumpernickel Idli

Lactic fermentation is one of the oldest forms of preparation and preservation of foods. LAB are essential to the fabrication of fermented products such as cheese, yogurt, fermented milk and butter (Stiles and Holzapfel, 1997). Some species are also involved in the production of wine (malolactic fermentation), sourdough breads, curing, sauerkraut as well as vegetable brining and silage (Table 1). LAB also contribute to the development of the organoleptic characteristics (flavour, aroma, texture) of the final products. Their ability to promote food preservation is linked to the fact that they cause a decrease in pH, as a consequence of lactic acid production, and additionally, the production of a number of antimicrobial agents (such as bacteriocins and non-proteic organic compounds). A combination of these factors limits the proliferation of undesirable microorganisms (spoilage- or pathogenic- microorganisms). LAB therefore undoubtedly play a role in promoting food safety. LAB have a GRAS (Generally Regarded As Safe) status. Infections by LAB are likely the result of opportunistic infections that rely on host factors rather than on intrinsic pathogenicity. Only rare cases of clinical infections involving LAB have been reported in humans (Aguirre and Collins, 1993) and few case reports have described the isolation of LAB species in patients with endocarditis, local infections or septicaemia (Ishibashi and Yamazaki, 2001). One example of such negative and severe impact was recently reported: the administration of a combination of probiotic strains to patients suffering from severe acute pancreatitis resulted an increased risk of mortality (Besselink et al., 2008). Of note, this negative impact occurred in an abnormal context, i.e. pancreatitis. The only one active and deleterious role attributed to LAB as a pathogen has been its identification as a participant in the

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development of dental caries due to the formation of biofilms that acidify the dental environment (Chestnutt et al., 1994). Beyond their involvement in industrial fermentations, LAB make part of natural ecosystems such as the vagina or the gastro-intestinal tract of humans and other animals. In these environments, LAB also play a role in the inhibition of pathogenic microorganisms. Some species of LAB reportedly have a beneficial role on health and wellbeing of the host. The definition of this so-called probiotic effect has evolved during past years. A bacterial strain was initially considered probiotic when its ingestion had a beneficial effect on health beyond its intrinsic nutritive value (Guarner and Schaafsma, 1998). The current definition of a probiotic is a “live micro-organism which when administered in adequate amounts confers a health benefit on the host” (FAO/WHO report, October, 2001), which does not imply that microorganisms have to be orally ingested. Probiotics are generally isolated from the intestinal microbiota of a host. The most studied species of probiotics are lactobacilli and bifidobacteria (Lb. casei, Lb. rhamnosus, Lb. acidophilus, Bf. bifidum) and many studies report their use for the treatment of diarrhoea, as a prophylactic agent against cancer, and both as a prophylactic and therapeutic agent against inflammatory disease of the digestive tract and against hypercholesterolaemia (de Roos and Katan, 2000; Steidler, 2003). In the last ten years, the concept of using LAB as a vaginal probiotic emerged from our advances in the understanding of the vaginal ecosystem and the use of vaginal LAB species to prevent vaginosis and urogenital diseases is now considered (Reid and Bruce, 2003). 2. S. aureus and lactobacilli of the vaginal microbiota S. aureus is part of the opportunist pathogenic bacteria that can be isolated from the vaginal microbiota of several hosts. It is involved in toxic shock syndrome (which is linked to the production of TSST-1, a potent superantigenic toxin) and in post-partum metritis in cows (Otero et al., 2006). Lactobacilli belong to the dominant microbiota of the normal and balanced vaginal ecosystem in several species of warm-blooded animals. Among the lactobacilli species found in the vaginal microbiota, Lb. gasseri, Lb. vaginalis, Lb. delbrueckii spp. lactis are the most frequently isolated in healthy women (Reid and Bruce, 2003; Aslim and Kilic, 2006), however, several other species have been identified (Witkin et al., 2007). In cattle, lactobacilli species such as Lb. fermentum, Lb. gasseri and Lb. rhamnosus are isolated from the vaginal microbiota of cows (Otero et al., 2000, 2006). Other LAB species have also been isolated from the vaginal microbiota of other warm-blooded animals such as hen or mares and their use as probiotics has been envisaged (Table 2). The equilibrium of the vaginal ecosystem is one of the main elements of natural defence against urogenital infections (RedondoLopez et al., 1990; Hillier et al., 1993). This antagonistic potential against pathogenic bacteria like S. aureus is nevertheless poorly understood. Some hypotheses have been proposed however, there has been some difficulty in demonstrating them.

Table 2 Lactic acid bacteria species isolated from vagina or cloaca of warm-blooded animals and humans. Host

LAB species

Women Lb. gasseri, Lb. iners, Lb. vaginalis, Lb. delbrueckii spp. lactis, Lb. jensenii, Lb. cellobiosus, Lb. curvatus, Lb. acidophilus, Lb. cripatus, Lb. plantarum, Lb. salivarius, Lb. brevis, Lb. oris Cow Lb. fermentum, Lb. gasseri Lb. rhamnosus Mare Lb. pantheris, Lb. mucosae, Lb. equi, Enterococcus faecalis, E. faecium Hen Lb. acidophilus, Lb. reuteri, Lb. salivarius

References (Aslim and Kilic, 2006; Witkin et al., 2007)

(Otero et al., 2000, 2006) (Fraga et al., 2008) (Van et al., 2007)

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C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39

Table 3 Environmental conditions allowing Staphylococcus aureus growth. Condition

Optimum

Range

Water activity pH Temperature

N 0.99 6–7 37

% NaCl Oxygen

0–4 Aerobiosis

0.83–N0.99 4.6–10 7–47. 8 0–20 Aerobiosis–anaerobiosis

The role that LAB play in inhibiting pathogens could be linked to their ability to acidify the vaginal tract. However, there are controversies as to this being the sole reason by which pathogens are inhibited. The vaginal pH varies from 6.6 (+/−0.3) to 4.2 (+/−0.2) between day 2 and day 14 of the menstrual cycle (Wagner and Ottesen, 1982). When close to 4–4.5, pH probably contributes to S. aureus inhibition as it is close to or even below the minimum pH allowing S. aureus growth (Table 3). The production of lactic acid by LAB can contribute to this acidification, nevertheless, this low pH is also maintained by the secretion of organic acids by the vaginal epithelial cells themselves (Boskey et al., 1999; Pybus and Onderdonk, 1999). Equilibrium of the vaginal microbiota is also attained by the production of bacteriocins by certain species which compose it (Kaewsrichan et al., 2006). However, the secretion of these substances by lactobacilli isolated from the vagina has not yet been characterised in situ. In screening studies, up to 80% of vaginal lactobacilli are found to produce bacteriocin or bacteriocin-like substances (Aroutcheva et al., 2001). However, only a few strains producing bacteriocin (or bacteriocin-like substance) active against S. aureus are described among those vaginal lactobacilli. Only 1 out of 9 vaginal lactobacilli strains isolated from 30 healthy women presented an anti-S. aureus bacteriocin-like activity (Aroutcheva et al., 2001; Voravuthikunchai et al., 2006). The scarcity of data may be due to the fact that bacteriocin activity screenings are primarily carried out on typical causative agent of bacterial vaginosis such as Gardnerella vaginalis but rarely include S. aureus strains in the species tested (Falagas et al., 2007). By contrast, production of bacteriocin with an anti-S. aureus activity is much better documented among LAB isolated from fermented food (see hereafter). The production of hydrogen peroxide (H2O2) is a widely accepted hypothesis to explain the inhibitory activity of vaginal LAB. Lactobacilli strains producing H2O2 are in fact widely represented within the vaginal microbiota in healthy women (Klebanoff et al., 1991) and are also the principal constituent of the vaginal ecosystem in cows (Otero et al., 2000). The inhibitory effect of H2O2 produced by lactobacilli has been demonstrated in vitro on S. aureus (Klebanoff et al., 1991; Ocana et al., 1999a,b; Otero et al., 2006). However, the inhibitory potential of lactobacilli on S. aureus has been observed during mixed cultures in laboratory media under aeration. In vivo, conditions upon which lactobacilli generate H2O2 in an environment poor in oxygen such as the vaginal tract, remain to be elucidated. It is thought that the inhibitory action of H2O2 could also result from the action of peroxidases conjugated to halogenic compounds (chloride or bromide) generating reactive oxygen species that are highly toxic for certain pathogens (Klebanoff et al., 1991; Bauer, 2001). The capacity that lactobacilli have to adhere and compete for adhesion sites in the vaginal epithelium can also be involved in the impairment of colonisation by a pathogen. The renewal of the superficial epithelium of the vagina, as well as cyclic changes in pH and secretion can affect the equilibrium of the vaginal microbiota. Studies have demonstrated the capacity of lactobacilli to adhere to the vaginal epithelium (Reid et al., 2003) and to interfere with pathogen colonisation (viral, bacterial or fungal) of the urogenital system (Reid and Bruce, 2003). Competition for adhesion sites may play a role in the positive effects displayed by certain lactobacilli strains isolated from healthy women against urogenital pathogens such as S. aureus as demonstrated in vitro on vaginal epithelial cells (Zarate and Nader-

Macias, 2006) and in a mouse model (Zarate et al., 2007). It was also recently shown that S. aureus adheres to human intestinal mucus and can be displaced by certain LAB (Vesterlund et al., 2006). The production of biosurfactants as a hypothetical mechanism for inhibition of pathogens has not yet been investigated. Biosurfactants produced by bacteria can be of different classes (glycolipids, lipopeptides, lipopolysaccharides, phospholipids) and their major physiologic role is to facilitate the acquisition of insoluble substrates, however, they can also present an antimicrobial action or be involved in the capacity of adhering to certain surfaces. It has been showed that the production of “surlactin” by certain strains of Lb. acidophilus can interfere in the adhesion of several microorganisms on silicon plates, amongst which is S. epidermidis (Velraeds et al., 1998). It has now been established that maintaining an equilibrated vaginal microbiota can prevent many urogenital infections. Beginning a few years ago, the concept of “vaginal probiotics” has emerged, and there is an increasing number of studies on the selection and use of lactobacilli to restore a healthy vaginal microbiota in women or other animals via vaginal or oral administration. 3. LAB antagonism on S. aureus in the food context S. aureus can grow in a wide range of environmental conditions (Table 3) and is a frequent contaminant of food. Contamination by S. aureus can come from raw material (e.g. mastitic milk) from the processing plant environment (e.g. biofilm on surfaces of processing plant) or from human activity (e.g. healthy carriage, sneeze, whitlow…) during food preparation and manipulation. Cooked meals and fermented milk products are the most frequent types of food involved in S. aureus food poisoning (Le Loir et al., 2003). Certain S. aureus strains can produce enterotoxins whose ingestion causes staphylococcal food poisoning. The frequency of S. aureus contamination and the impact of staphylococcal food poisoning on public health has justified an early and strong interest in combating this situation by the scientific community and agrofood industries. Conditions affecting growth of S. aureus and toxinogenesis in various food contexts have been investigated since the 1960s. Because of the early interest in inhibitory potential of LAB on S. aureus and because food fermentations can be readily reproduced and controlled in laboratory conditions, many hypotheses of the mechanisms involved in the inhibition phenomenon (acidification, bacteriocin production, H2O2 production) have been tested and reported in the scientific literature. We shall focus here on the particular case of fermented foods (especially milk

Fig. 1. pH of S. aureus colonisation site in humans and in fermented foodstuffs. ⁎: Indicates ecosystems where LAB are the dominant flora. After (Weinrick et al., 2004; completed by the authors).

C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39

and meat products). The impact of the LAB and LAB-related stresses, such as acidification and nutritional-related phenomena, on S. aureus growth will be discussed in the following paragraphs. 3.1. Acidification and organic acid production S. aureus is reportedly able to grow in pH values ranging from 4.6 to 10 with an optimal growth at pH value close to neutrality (Fig. 1; Table 3). The deleterious effects of pH variations can be amplified when these variations are combined with other factors. For example, S. aureus strains are more sensitive to acidification when salt concentration is high (resulting in lower water activity) (Table 4), although S. aureus is reportedly halotolerant (Iandolo et al., 1964). Similarly, Barber and Deibel have shown that oxygen tension affects the resistance of S. aureus to acidification in a fermented sausage model (Barber and Deibel, 1972). Acids do not have the same inhibition capacity and for a given pH value, the impact on S. aureus physiology will vary with the nature of the acid causing acidification. In food fermentations, lactic acid and sometimes acetic acid are predominantly produced. Acidification of a laboratory medium or of milk by addition of lactic acid down to pH 4.5–4.4, completely inhibits growth of S. aureus (Tatini et al., 1971; Haines and Harmon, 1973a; Charlier et al., 2008). By acidifying milk down to 4.6 with lactic acid, Minor and Marth (1970) showed a 99% reduction of biomass yield compared to a control culture without acid. The same inhibition effect was observed for pH 5 using acetic acid, for pH 4.5 using citric acid, for pH 4.1 using phosphoric acid and for pH 4 using hydrochloric acid. Inhibition by organic acids is usually achieved by the non-dissociated form of the acid (Minor and Marth, 1970), which is able to diffuse across the cytoplasmic membrane. Since the pH is higher in the cytoplasm, the acid dissociates and releases a proton. Thus, acetic acid and propionic acid, which have pKa values of 4.8 and 4.9, respectively, are more inhibitory than lactic acid whose pKa is 3.9. Acidic stress was studied in several bacterial species, notably in LAB. Generally, the drop in intracellular pH alters membrane structure and leads to a decrease in the activity of several enzymes that are pHsensitive. Bacterial growth is then strongly altered because most of the energy available in the cell is used to de-acidify the cytoplasm by generating a proton gradient across the cytoplasmic membrane (Cotter and Hill, 2003). Recently, S. aureus gene profile expression was investigated in a condition of mild acidification (Weinrick et al., 2004). Results revealed that the expression of several genes was affected by a variation in pH from 7.5 to 5.5. This set of genes was designated MAS, for “Mild Acidification Stimulon”. Modulation of the transcription of 400 MAS genes represents the adaptation of S. aureus to mild acidification. Among the MAS genes, one can find genes involved in maintaining the

Table 4 Influence of temperature, pH and NaCl concentration on lag phase and growth rate (µ) of S. aureus MF31 TSB medium (Trypto caseine Soja Broth).

Temperature (°C)

pHc

15.5 27.0 37.0 45.0 5.0 6.0 7.5 8.0

0.5%

NaCl

4%

NaCl

8%

NaCl

Tl (h)a

µ (h− 1)

Tl (h)a

µ (h− 1)

Tl (h)a

µ (h− 1)

11 3 1 1 6 3 1 2

0.4 1.6 3.1 1.5 1.6 2.2 2.9 2.6

22 5 3 4 6 5 4 4

0.6 1.8 3.5 1.3 2.0 2.4 3.9 3.1

45 9 5 –b 17 6 6 9

0.3 1.1 2.4 –b 0.8 1.5 1.7 0.9

After Iandolo (Iandolo et al., 1964). a Lag phase. b No growth. c Incubation at 37 °C.

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intracellular pH, such as the urease operon (ure), genes encoding intracellular chaperones (clpB) and many genes involved in metabolism and transport of amino acids and carbohydrates. This moderate stress also affects the expression of virulence factors (surface and secreted proteins). Interestingly, MAS genes are also involved in S. aureus response to other environmental stresses, notably osmotic stress (opuC and kdp) or stationary phase (sigB) (Weinrick et al., 2004). Medium acidification resulting from lactic fermentation by LAB seems to be one of the main factors involved in the inhibition of S. aureus growth. The impact of acidification on S. aureus growth was taken as a physico-chemical parameter in several studies (see above). Some studies showed a direct correlation between acidification of the medium and the level of S. aureus inhibition (impact on growth rate or survival) during mixed cultures with LAB (Kao and Frazier 1966; Barber and Deibel, 1972; Metaxopoulos et al., 1981). These studies led to the generally accepted idea that the more acidifying a starter is, the more inhibitory it will be. Among studies in which inhibition was derived from acidification, several consequences were evaluated, such as whether the impact was bacteriostatic or bacteriolytic, or whether the rate of acidification played a role in the inhibition. Several examples illustrate these different assessments. In a rich culture medium (TSB), some LAB strains (E. faecium and L. mesenteroides) can have a bactericidal effect on S. aureus. When the pH of the culture medium is controlled (TSB at pH 6.3), the bactericidal effect is lost, however, significant inhibition of S. aureus persists (Kao and Frazier,1966). Similarly, mixed cultures of S. aureus and commercial streptococci used for cheddar production have been carried out in milk maintained at pH 6.6. Results showed that in these conditions (constant pH), S. aureus is still sensitive to the action of streptococci and its population reaches only 2.104 cfu mL− 1 after 6 h of cultivation at 32 °C (Gilliland and Speck, 1974). Thus, pH can affect S. aureus survival (or cultivability) and be confounded with an apparent bacteriolytic effect. However, this inhibitory effect can be partially or totally abolished if culture conditions are changed. Daly et al. (1972) observed an inhibitory effect of L. lactis ssp. diacetylactis on S. aureus growth during mixed cultures in a meat matrix (ham). Nevertheless, the inhibition disappeared during cultures in laboratory media at a constant pH (pH 6.8) (Daly et al., 1972). The acidification rate seems also to be an important parameter. Several studies on cheese production models showed that S. aureus was able to grow in the presence of the starters used during the first phase of production. However, the pH reached after the initial hours of production was determinant for the evolution of the staphylococcal population. These observations were made on non-cooked semi-hard cheese as a matrix (Saint-Nectaire or Salers) where an increase in the population of S. aureus was observed during the first 6 h of production independent of the initial level of contamination. In this cheese technology, the low acidification rate achieved during the initial hours may be permissive for staphylococcal growth. The staphylococcal population after 6 h of production would change depending on the pH achieved at this point in time, the higher the pH, the lower the rate of decrease of the staphylococcal population (Delbes et al., 2006). Similar results were obtained in the production of other types of cheese such as Tomme de Savoie or Cantal (Lamprell, 2003). Studies on soft cheese technology (like Camembert) showed a staphylococcal growth (~ 3 log10 increase) during the first phase of production (~ 22 h) from inoculation to salting (Meyrand et al., 1998). One part of the biomass increase (1 to 1.5 log10, expressed in cfu g− 1) can be attributed to curd draining and to retention/concentration of staphylococci during stripping. After the growth period of S. aureus, the authors observed a stabilisation of the population. In this study, the experimental data obtained did not demonstrate any impact of pH on the staphylococcal population after the first growth phase. Recently, Charlier et al. (2008) showed that a significant inhibition of S. aureus growth was observed in milk medium both when the pH

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C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39

was regulated or not, suggesting that acidification is not involved in the early inhibition of S. aureus growth by L. lactis in milk. In fermented meat products, dominant LAB species are lactobacilli belonging to Lactobacillus sakei and Lactobacillus curvatus species. Other lactobacilli such as Lb. plantarum, Lb. casei, Lb. brevis or Lb. alimentarius can be isolated as well (Rantsiou and Cocolin, 2008). Although coagulase negative staphylococci like S. xylosus or S. carnosus are often found (or used as starter) in fermented meat products, lactobacilli are the bacterial group reportedly involved in the acidification. The role of low pH and acidification rate in meat products is also well-documented. The addition of starters including lactobacilli in dry sausages increases the rate of acidification compared to the one observed with only natural contaminating lactobacilli, and, as a consequence leads to a reduced S. aureus contamination (Barber and Deibel, 1972; Niskanen and Nurmi, 1976; Metaxopoulos et al., 1981; Sameshima et al., 1998; Kaban and Kaya, 2006). However, sometimes it does not completely prevent from transient staphylococcal growth as reported by Niskanen and Nurmi (1976) and Metaxopoulos et al. (1981). On the contrary, Kaban and Kaya (2006) observed a bactericidal effect of two different starter cultures (including strains of Pediococcus acidilactici, Lb. curvatus, Staphylococcus xylosus, Lb. sakei and Staphylococcus carnosus) on S. aureus growth in Turkish dry fermented sausages, even without any transient S. aureus growth. Likewise, Sameshima et al. (1998) observed a bacteriostatic effect of the addition of a strongly acidifying commercial strain of L. sakei as well as of two intestinal lactobacilli on S. aureus growth in sausages. Altogether, these data suggest a major role of the rate of acidification in addition to the final pH reached. Moreover, other parameters may interfere with acidification as illustrated by Barber and Deibel (1972) who reported S. aureus growth inhibition in fermented sausage by biological acidification with a high inoculum of Pediococcus cerevisiae, yet with variable efficiency depending on oxygen tension. On the whole, these data show that acidification resulting from LAB activity can have different impacts on S. aureus growth depending on the medium used (laboratory media, meat or milk) and on which food technology is considered. In addition to the effect of pH, these results show the importance of analysing the kinetics of inhibition. A biphasic effect (tolerance or even stimulation of growth and then stabilisation or inhibition) has been observed in laboratory media (Kao and Frazier, 1966) but also during the cheese-making process (Meyrand et al., 1998; Delbes et al., 2006). If the long term effect of acidification on S. aureus growth is the only parameter taken into account, there is a risk of ignoring a possible stimulation observed in the early steps of the process and then conclude that an inhibitory effect took place, even though the staphylococcal population might have reached a sufficient density to produce one or several enterotoxins.

mixed culture in laboratory media (Haines and Harmon, 1973b). On the other hand, Gilliland and Speck (1974) showed that the inhibition of S. aureus in milk during mixed cultures with lactococci and streptococci strains was independent of H2O2 production . More recently, Ito et al. (2003) screened LAB from various food samples and evaluated their hydrogen peroxide production. However, when they further investigated the antimicrobial activity of the cell-free filtrate corresponding to the L. lactis strain producing the highest amount of H2O2, they only observed a weak effect on S. aureus. The actual role of hydrogen peroxide production by LAB in the inhibition of S. aureus remains difficult to demonstrate and thus is still controversial. 3.3. Production of bacteriocins The antagonistic potential of LAB may also involve the production of bacteriocins which are active against S. aureus (Cotter et al., 2005). Bacteriocins are ribosomally-synthesized peptides or proteins secreted by certain strains of bacteria. Many LAB produce bacteriocins with rather broad spectra of inhibition and several LAB bacteriocins offer potential applications for food preservation (Galvez et al., 2007). They generally have an inhibitory or even a bacteriolytic effect on target species. Many studies have focused on the search, identification and characterisation of bacteriocins produced by LAB, notably by LAB isolated from foodstuffs (e.g. fermented milk or meat products) in order to combine technological and preservative capacities (Sobrino et al., 1991; Vignolo et al., 1993; Sudirman et al., 1993; Mataragas et al., 2002). Bacteriocin-producing strains are indeed of considerable interest for the preservation of fermented products which may be achieved by inhibiting the growth of pathogens such as Listeria monocytogenes and/or S. aureus. These two bacterial species are usually used as targets to screen for the production of antimicrobial agents such as bacteriocins by LAB. Nisin (a lantibiotic, produced by certain strains of L. lactis) and pediocin (a non-lantibiotic, produced by certain strains of Pediococcus acidilactici) are among the most well

Table 5 Example of LAB bacteriocins and action spectrum. BL (bacteriocins)

Inhibition spectrum/activity on S. aureus (+/−)

Lactococcus sp. Nisin Lacticin 3147 Lacticin 481 Lactococcin A, B et M

Broad spectrum (+) Broad spectrum (+) Mean spectrum (nd) Narrow spectrum (−)

Lactobacillus sp. Lactocin 27 Sakacin A Sakacin B Plantaricin C Curvaticin 13

Narrow spectrum (nd) Narrow spectrum (nd) Narrow spectrum (nd) Broad spectrum (nd) Broad spectrum (nd)

Pediococcus sp. Pediocin A Pediocin AcH (PA-1)

Broad spectrum (+) Broad spectrum (+)

Leuconostoc sp. Leucocin A-UAL187

Broad spectrum (nd)

Enterococcus sp. Enterocin A Enterocin P

Narrow spectrum (−) Broad spectrum (+)

Carnobacterium sp. Carnocin H Piscicolin 126 Divercin V41

Broad spectrum (+). Broad spectrum (−) Broad spectrum (nd)

3.2. Production of hydrogen peroxide (H2O2) by LAB The production of hydrogen peroxide (H2O2) by LAB, particularly by lactobacilli, is also antagonistic to S. aureus. The production of H2O2 by LAB and its potential role in S. aureus inhibition is well-documented especially in lactobacilli isolated from the vaginal ecosystem (see Section 2). Unlikely, very few and old studies are found in the literature regarding the role of H2O2 producing isolates in food context (Dahiya and Speck, 1968; Haines and Harmon, 1973b; Gilliland and Speck, 1974). Some lactobacilli strains are able to inhibit the growth of S. aureus by producing H2O2 at a concentration of 0.18 mmol L− 1. H2O2 has a bacteriostatic effect at these concentrations and is bactericidal for concentrations up to 0.6 to 1.0 mmol L− 1. The addition of catalase to the medium where a L. lactis or Pediococcus cerevisiae (renamed Pediococcus acidilactici) strain had been grown partially halted the inhibition of S. aureus. The authors concluded that hydrogen peroxide was involved in the capacity of these strains to inhibit S. aureus in

+, S. aureus inhibition. −, no inhibition. nd, undetermined activity on S. aureus or not mentioned in the studies characterising these bacteriocins.

C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39 Table 6 Examples of bacteriocins produced by Staphylococcus aureus. Bacteriocins

Spectrum

References

Bac1829 (S. aureus KSI1829) BacR1 (S. aureus UT0007) Staphylococcin 462 Staphylococcin 162 Staphylococcin C55 (S. aureus C55) Bac188 (S. aureus AB188) Aureocin A53 (S. aureus A53) Aureocin A70 (S. aureus A70)

Broad spectrum Broad spectrum n.d. Broad spectrum Narrow spectrum Broad spectrum n.d. Broad spectrum

(Crupper and Iandolo, 1996) (Crupper et al., 1997) (Hale and Hinsdill, 1975) (Hale and Hinsdill, 1973) (Navaratna et al., 1998, 1999) (Saeed et al., 2004) (Netz et al., 2002) (Netz et al., 2001)

characterised bacteriocins and the most frequently used in fermented products. Nisin is currently used commercially as a food preservative in around 50 countries (Delves-Broughton et al., 1996). The production by LAB strains of bacteriocins which are active against S. aureus (Table 5) and other uropathogenic bacteria are also screened for in order to develop vaginal probiotics (Voravuthikunchai et al., 2006). By contrast, the ability of S. aureus to produce bacteriocins is poorly studied, however, this ability would provide an advantage over other organisms competing for the same ecological niche and thus explain the ability that certain strains of S. aureus have to colonise some of the ecological niches in which LAB are prevalent. The first S. aureus bacteriocins were described long time ago (Jetten and Vogels, 1973) (Table 6). The genes involved in the production of most staphylococcal bacteriocins are located on mobile genetic elements such as prophages (Navaratna et al., 1999) or plasmids (Netz et al., 2001, 2002) and can thus be passed from one S. aureus strain to another or even to another species. Epidermin-like compounds and genes involved the biosynthesis of epidermidin, an bacteriocin produced by S. epidermidis, were indeed found in certain S. aureus strains (Sahl 1994; Ben Zakour et al., in press). The production of such staphylococcal bacteriocins which are active against vaginal lactobacilli could provide certain S. aureus strains a selective advantage (Scott et al., 1992). 3.4. Nutritional competition The nutritional demands of S. aureus and LAB have long been studied when each is cultivated in a pure culture, thanks to the development of chemically defined media. These studies defined the amounts of vitamins, amino acids, sugars, minerals, metals, and other

35

nutritional components required in media to ensure bacterial growth (Snell, 1945; Banville, 1964; Miller and Fung, 1973; Courcol et al., 1997; Chervaux et al., 2000; Letort and Juillard, 2001). S. aureus is a chemo-organotrophic species and thus requires a complex organic source of energy. Substrates used by S. aureus can be sugars such as fructose, glucose, galactose, mannose, ribose, maltose, sucrose, trehalose, alcohols (mannitol), organic acids (acetate), and in some conditions amino acids (glutamine, arginine). The development of in silico metabolic networks has permitted virtual assays which suggest that S. aureus N315 might be able to synthesize almost any amino acids since all the genes involved in amino acid biosynthesis pathways are present in N315 genome. Depending on the in silico model, S. aureus will either have auxotrophy for only one amino acid (Becker and Palsson, 2005), or no auxotrophy at all (Heinemann et al., 2005). In vitro studies have revealed that these models were not validated, since the N315 strain indeed required six amino acids to be able to grow (Kuroda et al., 2001). The most commonly used method to identify essential nutrients is the progressive simplification of a chemically defined medium by a step-by-step omission of one or several amino acids. Table 7 summarizes the results obtained for about 80 S. aureus strains isolated from various origins. Altogether, these results show that four amino acids are frequently or almost always required for the growth of S. aureus: arginine, cysteine, proline and valine. In contrast, eight other amino acids are rarely required (alanine, aspartate, histidine, isoleucine, lysine, serine, threonine and tryptophan). The nutritional requirement also depends on growth conditions. In aerobiosis or anaerobiosis, S. aureus is able to metabolise different sources of carbon, such as glucose, lactose, maltose and or mannitol. Genome sequence analyses have revealed the presence of lactose phosphotransferase systems (Kuroda et al., 2001). This system enables S. aureus growth in milk (Simoni and Roseman, 1973). Moreover, Sharer et al. (2003) showed that a lactose-specific permease (EII) may facilitate the growth of S. aureus in a lactose-rich environment and is thus likely to be involved in the invasion of the mammary gland during mastitis. The nutritional aspects of mixed cultures have, however, been addressed only by a small number of studies. Some studies have shown that a co-culture with LAB reduced S. aureus growth because of nutritional competition. Iandolo et al. (1965) showed that nutriment depletion in a laboratory culture medium (and notably depletion of nicotinamide) was involved in the inhibition of S. aureus by L. lactis

Table 7 Essential amino acids for Staphylococcus aureus growth. Number of strains

50

18

7

5

1 (S. aureus N315)

1 (S. aureus S6)

Strains ORIGIN

Human (skin)

Not given

Cow mastitis

Not given

Human (pharynx)

Frozen shrimps

Reference

(Emmett and Kloos, 1975)

(Taylor and Holland, 1989)

(Lincoln et al., 995)

(Onoue and Mori, 1997)

(Kuroda et al., 2001)

(Mah et al., 1967)

(Miller and Fung, 1973)

Ala Arg Asp Cys Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

+/− + − + − +/− +/− − + − +/− +/− + − +/− +/− +/− +

− + + + + + +/− − − +/− − − + +/− − − +/− +/−

− + − + +/− + +/− +/− + +/− +/− +/− + − − +/− +/− +

− + − + − +/− − − +/− − +/− +/− +/− − − − +/− +

+ + − − − + − + − − − − + − − − − +

− + − + − − − − − − − − + − − − − +

− + − + + − − − − − − + − − − − − −

36

C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39

ssp. diacetylactis. However, the availability of a given compound is closely related to the pH of the culture medium. For example, at pH 5.5, the availability of nicotinamide is sufficient to allow S. aureus growth, however, the amount of nicotinamide needed increases directly proportionally with pH. The biological activity of nicotinamide is indeed higher for low pH values (Iandolo et al., 1965). Moreover L. diacetylactis may be capable of rapidly metabolising vitamins thus lowering the quantity available for S. aureus. Haines and Harmon (1973a,b) also reported that competition for vital nutritive elements, such as biotin and niacin, is involved in the mechanism of inhibition of S. aureus in mixed cultures with L. lactis at 30 °C in laboratory media (Haines and Harmon, 1973a). It is worth noting that some studies were carried out in mixed cultures using laboratory media whereas others were performed on food matrices, which are much more complex than the former. For example, an inhibitory effect of L. lactis on S. aureus growth can be observed in a milk medium and yet be totally absent in a rich medium in similar conditions (regulated pH) demonstrating that the inhibitory effect is also dependent on the composition of the culture medium (Charlier et al., 2008). LAB antagonism in milk or meat likely involves phenomena linked to nutritional factors including factors such as the production of inhibitory metabolites (e.g. release of bioactive peptides from proteolysis), rather than to a sensu stricto nutritional competition. The example of S. aureus 8325-4, which is prototrophic for phenylalanine, illustrates this complex relationship between nutrients and physiology. Inactivation of pheP, the gene encoding a permease for phenylalanine, leads to a growth impairment of the mutant in pig serum in anaerobe conditions (Horsburgh et al., 2004). This suggests that, in some environmental conditions, the biosynthetic capabilities of the strain are probably too weak to provide enough phenylalanine to the cell. This thus creates an apparent auxotrophy for this amino acid. Some other authors have pointed out that environmental factors (including the presence of LAB) may affect S. aureus growth through complex nutritional phenomena (Troller and Frazier, 1963b; Haines and Harmon, 1973a). Nevertheless, evaluating the impact of these nutritional factors appears to be a daunting task to be pursued in further investigations. 3.5. Influence of the LAB/S. aureus ratio The ratio between the inoculum of (or level of milk contamination by) S. aureus and that of LAB at the beginning of the culture determines the efficiency of the inhibition. This was demonstrated by several studies carried out in laboratory media, in milk, or in a cheese or meat matrix. Therefore, in rich media (APT), when the S. aureus population is larger than that of L. lactis (Ratio 10/1, i.e. 105 cfu mL− 1 for S. aureus vs 104 for L. lactis), the size of the S. aureus population in a mixed culture can reach the same as that which is observed in a control culture (i.e. ~1010 cfu mL− 1, a level at which enterotoxin production occurs). However, for (S. aureus:L. lactis) ratios of 1/1 and 1/10, the maximal population reached by S. aureus is greatly affected and drops down to 106 and 105 cfu mL− 1, respectively (Haines and Harmon, 1973b). The prevalence of the level of inoculum seems to be independent of the media and even from, in some way, the competitor species, since similar results were indeed obtained on semi-synthetic media during mixed culture with an E. coli strain (Troller and Frazier, 1963b). Several studies also showed that the ratio of S. aureus/LAB was a determining parameter for the efficiency of inhibition, in situ, in cheese or meat matrix models. In non-cooked pressed cheese (SaintNectaire or Salers type) made with raw milk, the population of coagulase-positive staphylococci (CPS, where S. aureus is predominant) at 24 h was determined by the amount of initial contamination. However, the population of CPS increased in any of these cheese technologies during the first 6 h of production. Thus, the population after 24 h also depended on the pH reached after the first 6 h of

production (Meyrand et al., 1998; Delbes et al., 2006). Comparable results were obtained in the manufacturing of other non-cooked pressed cheeses (Cantal and Tomme de Savoie) (Lamprell, 2003). In a soft cheese model (Camembert type) produced from goat raw milk, the inoculum of S. aureus (from 102 to 106 cfu mL− 1) partly determines the population level it reaches by the time of the salting step (after ~ 22 h of mixed culture). However, it is worth noting that the population of S. aureus increased during the formation of curd, independent of the initial inoculum, and was stable after salting and decreased during curing (Meyrand et al., 1998). Similar results were obtained for Manchego type cheese (goat and cow milk, Spain) (Gomez-Lucia et al., 1992). In a meat matrix, Metaxopoulos et al. (1981) tested different inocula of S. aureus (104 and 105 cells/g) with lactobacilli (0, 105 and 106 cells/g) during sausage fermentation (Metaxopoulos et al., 1981). They noticed that the ratio of LAB:S. aureus was directly proportional to the inhibition of S. aureus. Additionally, the temperature of incubation had an impact on the amount of inhibition. In this case, as in milk, the inhibition was stronger at temperatures lower than 30 °C (Troller and Frazier, 1963a; Kao and Frazier, 1966; Haines and Harmon, 1973b). 3.6. Inter- and intra-species variability of the inhibition capacity Few studies have addressed the variability of the inhibitory capability of different LAB species. Haines and Harmon (1973a,b) tested 8 species belonging to the Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus genera. They demonstrated that in mixed cultures on laboratory media with S. aureus and several strains belonging to different species, L. lactis, S. thermophilus and P. cerevisiae (renamed Pediococcus acidilactici) displayed the most marked effect on the development of S. aureus (Haines and Harmon, 1973b). Only two studies have measured the variability of the inhibitory capacity of LAB within a certain species. Haines and Harmon (1973a,b) used a panel of 5 L. lactis strains to test the potential of L. lactis in inhibiting S. aureus during mixed cultures in laboratory media and concluded that its capacity to antagonise the latter was homogeneous in this species. More recently, Charlier et al. (2008) screened a panel of 75 L. lactis strains for their potential to inhibit S. aureus growth in milk and demonstrated that the inhibition potential of the strains was not homogeneous within L. lactis species. Most of the lactococcal strains (93%) indeed displayed a strong inhibitory effect whereas a few strains (7%) were poor inhibitors. This high prevalence of the inhibition potential within L. lactis may explain why previous studies, carried out on small numbers of strains, concluded that the prevalence of the inhibition potential was homogeneous among LAB species. Thus, in a food- as well as in a vaginal-context, the inhibitory potential of LAB species may be strain dependent. Conversely, few published studies have discussed the variability of S. aureus susceptibility to inhibition by LAB within the species. The sensitivity of S. aureus to inhibition by LAB is reportedly homogeneous however, inhibition tests were carried out on only two (Kao and Frazier, 1966) and fifteen (McCoy and Faber, 1966) strains. Yet, S. aureus is reportedly a phenotypically and genotypically variable species with a great capacity for adaptation (Clements and Foster, 1999; Fitzgerald et al., 2003). 4. Conclusions and perspectives The inhibitory potential of LAB on S. aureus growth results from many different factors described in this review. These factors compose a complex and intricate network and it is difficult to evaluate the prevalence of one inhibition factor over another. One other important message emerging from these studies is that the inhibition of S. aureus growth (or expression of the LAB inhibitory potential) is dependent on the environment (e.g. effect vs no effect depending on which medium is used and on physico-chemical parameters). Many hypotheses

C. Charlier et al. / International Journal of Food Microbiology 131 (2009) 30–39

resulting from in vivo observations are difficult to reproduce in vitro, and vice-versa. Classical microbiological approaches are useful for the high throughput screening of strain libraries and can shed light on important, yet poorly understood information such as what is the extent of the variability of the inhibitory potential of LAB strains and of susceptibility of S. aureus strains to their inhibition, within each species. Similarly, classical microbiological approaches may help taking into account S. aureus–LAB interactions in terms of genetic exchange (e.g. horizontal gene transfer of antibiotic resistance genes). The reducing capacities of LAB may also affect conditions for the growth of S. aureus during co-culture and may indirectly result in variation of the inhibition capacities within a species (Brasca et al., 2007). These aspects have been, as of yet, poorly addressed in the literature available. As well as LAB being able to inhibit the growth of S. aureus, it may be the case that the expression of virulence of an S. aureus strain may also be inhibited, even if inhibition of growth is not achieved. Whenever S. aureus has to interact with LAB, such as in ecosystems and in contexts of disease, the pathogenesis of S. aureus related diseases (toxic shock syndrome toxin and enterotoxin production) indeed relies on the temporal expression of virulence factors. The expression of virulence factors in S. aureus is tightly controlled and regulated by a complex network of regulation systems including the accessory gene regulator (agr, a quorum sensing system that plays a central role in the regulation of virulence), the staphylococcal accessory regulator (sar), and several two-component systems or transcription factors (e.g. arl, sae, srrAB, rot, mgr) (Novick, 2000). LAB antagonism to S. aureus may act at more than one level: i) by hampering its growth physiology thus disturbing its growth rate and/or survival and ii) by interfering with the modulation of the expression of virulence factors, thus impairing its pathogenic potential. This aspect of antagonism by LAB was not addressed here even though some studies have observed a “modulating” effect on staphylococcal enterotoxin production in mixed cultures in laboratory media or in food matrices (Meyrand and VernozyRozand, 1999). Recently, Laughton et al. (2006) reported that Lb. reuteri produces a small-sized soluble compound which is able to interfere with the expression of an exotoxin gene in S. aureus. Molecular approaches must now be used in order to increase our understanding of the mechanisms involved in the inhibition of S. aureus growth and the expression of its virulence in complex microbial community interactions (mixed cultures and food context). Whole genome sequences of 14 S. aureus strains and those of at least one strain of most LAB species are now publicly available (Genome online database, February 2008; http://genomesonline.org/). These sequences may be used to detect the Achilles' heels in S. aureus physiology (Fraser and Rappuoli, 2005) or putative antimicrobial compounds in LAB species (Nes and Johnsborg, 2004). The multiplication of sequencing projects have also stimulated the development of new molecular tools such as DNA microarrays allowing for a comprehensive approach to gene expression profiles in diverse conditions such as physiological stresses like pH (Weinrick et al., 2004; Bore et al., 2007), osmolarity (Pane-Farre et al., 2006), or temperature (Anderson et al., 2006). However, until now, very few gene expression studies have been carried out to evaluate the impact of bacteria–bacteria interactions at the gene expression level. For instance, in a recent study of S. aureus–Pseudomonas aeruginosa interactions, only the gene expression profile of P. aeruginosa was studied (Mashburn et al., 2005). One bottleneck for these microarray approaches is the phylogenic proximity between S. aureus and most LAB species (Gram positive, low G + C content), which may result in non-specific cross-hybridisation. Efforts will have to be dedicated to the development of species-specific DNA microarrays in order to minimise cross-hybridisation and to allow for the use of microarrays in a mixed-culture context. These new approaches will enable the characterisation of the bacterial interactions at a molecular level in

37

order to accurately define the nature of the interactions and the complex molecular mechanisms involved in the LAB impact on S. aureus physiology and virulence factors expression. Molecular and global approaches including transcriptomic and proteomic approaches appear as powerful tools to investigate the antagonistic potential of LAB with regard to S. aureus. Increasing our knowledge of the mechanisms involved in LAB–S. aureus interactions will open new avenues for the biocontrol of S. aureus with nonantibiotic means which are not deleterious to bacterial ecosystems or for biotechnological applications in a global context where the emergence and spread of antibiotic resistant strains becomes a major concern for public health. In particular, this should lead to the rational selection of probiotic strains effective in preventing urogenital infections as well as to the selection of starter strains ensuring food safety without altering typicity of products. Acknowledgements Authors warmly thank John McCulloch for helpful discussion and critical reading of the manuscript. Cathy Charlier was recipient of a Ph. D. fellowship from Institut National de la Recherche Agronomique (INRA) and Région Bretagne. Marina Cretenet is recipient a PhD fellowship from Centre National Interprofessionnel de l'Economie Laitière (CNIEL). Part of this work was supported by Agence Nationale de la Recherche (GenoFerment Project). Reference Aguirre, M., Collins, M.D., 1993. Lactic acid bacteria and human clinical infection. J. Appl. Bacteriol. 75, 95–107. Anderson, K.L., Roberts, C., Disz, T., Vonstein, V., Hwang, K., Overbeek, R., Olson, P.D., Projan, S.J., Dunman, P.M., 2006. Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover. J. Bacteriol. 188, 6739–6756. Aroutcheva, A.A., Simoes, J.A., Faro, S., 2001. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis. Infect. Dis. Obstet. Gynecol. 9, 33–39. Aslim, B., Kilic, E., 2006. Some probiotic properties of vaginal lactobacilli isolated from healthy women. Jpn. J. Infect. Dis. 59, 249–253. Banville, R.R., 1964. Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87, 1192–1197. Barber, L.E., Deibel, R.H., 1972. Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage. Appl. Microbiol. 24, 891–898. Bauer, G., 2001. Lactobacilli-mediated control of vaginal cancer through specific reactive oxygen species interaction. Med. Hypotheses 57, 252–257. Becker, S.A., Palsson, B.O., 2005. Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation. BMC. Microbiol. 5, 8. Ben Zakour, N., Sturdevant, D.E., Even, S., Guinane, C.M., Barbey, C., Alves, P.D., Cochet, M.F., Gautier, M., Otto, M., Fitzgerald, J.R. and Le Loir, Y., in press. Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome. J. Bacteriol. Besselink, M.G., van Santvoort, H.C., Buskens, E., Boermeester, M.A., van, G.H., Timmerman, H.M., Nieuwenhuijs, V.B., Bollen, T.L., van, R.B., Witteman, B.J., Rosman, C., Ploeg, R.J., Boermeester, M.A., Schaapherder, A.F., Dejong, C.H., Wahab, P.J., van Laarhoven, C.J., van der, H.E., van Eijck, C.H., Cuesta, M.A., Akkermans, L.M., Gooszen, H.G., 2008. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet 371, 651–659. Bore, E., Langsrud, S., Langsrud, O., Rode, T.M., Holck, A., 2007. Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis. Microbiology 153, 2289–2303. Boskey, E.R., Telsch, K.M., Whaley, K.J., Moench, T.R., Cone, R.A., 1999. Acid production by vaginal flora in vitro is consistent with the rate and extent of vaginal acidification. Infect. Immun. 67, 5170–5175. Brasca, M., Morandi, S., Lodi, R., Tamburini, A., 2007. Redox potential to discriminate among species of lactic acid bacteria. J. Appl. Microbiol. 103, 1516–1524. Charlier, C., Even, S., Gautier, M., Le Loir, Y., 2008. Acidification is not involved in the early inhibition of Staphylococcus aureus growth by Lactococcus lactis in milk. Int. Dairy. J. 18, 197–203. Chervaux, C., Ehrlich, S.D., Maguin, E., 2000. Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium. Appl. Environ. Microbiol. 66, 5306–5311. Chestnutt, I.G., MacFarlane, T.W., Stephen, K.W., 1994. An in vitro investigation of the cariogenic potential of oral streptococci. Arch. Oral Biol. 39, 589–593. Clements, M.O., Foster, S.J., 1999. Stress resistance in Staphylococcus aureus. Trends Microbiol. 7, 458–462. Cotter, P.D., Hill, C., 2003. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol. Mol. Biol. Rev. 67, 429–453.

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