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1 Functional Foods Forum, Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland
2 Department of Environmental Engineering and Biotechnology, Tampere University of Technology, Tampere, Finland
3 Danisco Innovations, Kantvik, Finland
Correspondence
Satu Vesterlund
satu.vesterlund{at}utu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The nasal carriage of Staph. aureus is common, 20–50 % of the population (Cespedes et al., 2005), but also intestinal carriage appears to be increased among hospitalized patients (Dupeyron et al., 2001; Ray et al., 2003; Rimland & Roberson, 1986; Squier et al., 2002) and infants (Lindberg et al., 2000). Lindberg et al. (2000) showed that over 75 % of Swedish infants have Staph. aureus in their stools while Bjorksten et al. (2001) showed that 65 % of infants have these bacteria in their stools. Towards adulthood the intestinal carriage of Staph. aureus decreases due to increased complexity of the adult microbiota and so-called ‘colonization resistance’, meaning that the indigenous intestinal microbiota provides protection against colonization of the gastrointestinal tract by exogenous micro-organisms (Lindberg et al., 2004; van der Waaij et al., 1971).
As the microbiota covering the intestinal epithelium has a protective role in preventing colonization of ingested bacteria, certain bacterial strains belonging to the healthy intestinal microbiota can be isolated and used as probiotics. Probiotics are ‘live micro-organisms which when administered in adequate amounts confer a health benefit on the host’ (WHO, 2001). There are several reports showing that specific probiotic strains protect against gastrointestinal infections (Gorbach et al., 1987; Saavedra et al., 1994; Vanderhoof et al., 1999). Different mechanisms for this have been suggested, such as overall reduction of the gut pH, a direct antagonism against pathogens (production of antimicrobial components such as hydrogen peroxide and bacteriocins), competition for the same binding sites as pathogens, stimulation of the immune system and competition for nutrients (Collins & Gibson, 1999).
The aim of the present study was to assess whether Staph. aureus can adhere to healthy human colonic mucus and whether adhesion and viability of potentially adherent Staph. aureus can be reduced by specific lactic acid bacteria; a preliminary investigation was made of the possible mechanisms for such effects.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and a bioluminescent variant of the same strain, Staph. aureus RN4220/pAT19 (Vesterlund et al., 2004). Salmonella enterica serovar Typhimurium ATCC 14028 was used as a negative control in adhesion assays, as in previous experiments it has exhibited low adhesion (Vesterlund et al., 2005). The 11 strains of lactic acid bacteria (LAB) used are listed in Table 1. All bacterial stocks were stored at –86 °C in 40 % (v/v) glycerol. Staph. aureus and Sal. enterica serovar Typhimurium were plated first and subsequently cultured by inoculating one colony into Luria–Bertani broth (LB; yeast extract and tryptone were from Pronadisa). When adhesion was measured, 10 µl ml–1 of [5'-3H]thymidine (16.7 Ci mmol–1; 618 GBq mmol–1) was added to the cultures to metabolically radiolabel the bacteria. In the case of the bioluminescent Staph. aureus strain, broth and plates were supplemented with 10 µg erythromycin ml–1. Staph. aureus and Sal. enterica serovar Typhimurium cultures were grown for 16 h without agitation at 30 °C to reach stationary growth phase. LAB strains were grown in de Man, Rogosa and Sharpe (MRS) broth (Oxoid) and they were inoculated directly as a 0.5 % inoculum from the glycerol stocks. When adhesion kinetics was measured, the LAB cultures were supplemented with 10 µl [5'-3H]thymidine ml–1. In the case of Lb. reuteri 40 mM glycerol was added as a substrate for production of reuterin in the culture broth (Talarico et al., 1988). LAB were grown in anaerobic conditions for 20 h at 37 °C (except for Lc. lactis subsp. lactis and P. freudenreichii subsp. shermanii JS, which were grown for 2 days at 30 °C) in order to reach the late exponential growth phase. All bacterial strains were harvested by centrifugation and washed twice with 1 ml phosphate-buffered saline (PBS; pH 7.2). The OD600 of the bacterial suspensions was adjusted with PBS to 0.5±0.02, corresponding to 0.5x108 c.f.u. ml–1 for Lb. acidophilus La5, 1–2x108 c.f.u. ml–1 for Lb. casei Shirota, Lb. johnsonii LA1, Lb. rhamnosus GG and Lc. lactis subsp. lactis and 2–4x108 c.f.u. ml–1 for the remaining strains. Although the number of added bacteria varied from 0.5x108 to 4x108 c.f.u. ml–1, the bacterial concentrations used led to a linear relationship between added and bound bacteria and thus constant adhesion percentages. Moreover, the saturation level, when the numbers of added bacteria are too high, leading to underestimation of the percentage bound bacteria, was not reached.
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). In short, resected material was collected on ice within 20 min and processed immediately by washing gently with PBS containing 0.01 % gelatin. Mucus was collected by gently scraping with a rubber spatula into a small amount of HEPES-Hanks buffer (10 mmol HEPES l–1; pH 7.4) and centrifuged (13 000 g, 10 min). After measurement of the protein content, the mucus was stored at –20 °C. In adhesion assays the mucus was diluted to a protein concentration of 0.5 mg ml–1 with HEPES-Hanks buffer. Mucus was passively immobilized on a polystyrene microtitre plate (Maxisorp, Nunc; and in bioluminescence measurements B&W Isoplate 1450-581, PerkinElmer) as a volume of 100 µl by incubating overnight at 4 °C (Ouwehand et al., 2003).
Adhesion assay.
After overnight incubation the mucus-coated microtitre plate wells were washed three times with 250 µl HEPES-Hanks buffer. Then radiolabelled Staph. aureus bacteria were added to the wells in a volume of 100 µl (in competition assays in a volume of 50 µl, i.e. 50 µl of Staph. aureus incubated alone or together with 50 µl of LAB). Four parallel wells were used in each experiment. Bacteria were allowed to adhere for 1 h at 37 °C and the wells were washed three times with 250 µl HEPES-Hanks buffer to remove the nonadherent bacteria. In exclusion assays LAB were incubated first with the mucus, then washed away and followed by incubation with radiolabelled Staph. aureus. Similarly in displacement assays radiolabelled Staph. aureus was incubated first with the mucus, then washed away and followed by incubation with LAB. The bacteria bound to mucus were released and lysed with 1 % SDS/0.1 M NaOH by incubation at 60 °C, followed by measurement of radioactivity by liquid scintillation. Sal. enterica serovar Typhimurium was used as a negative control in adhesion assays. The adhesion ratio (%) of bacteria was calculated by comparing the radioactivity of the adhered bacteria to that of the added bacteria.
Viability of adherent bacteria.
The bioluminescent indicator strain has been used earlier in the screening of antimicrobial substances produced by LAB against Staph. aureus (Vesterlund et al., 2004). In short, this indicator strain allowed stable light production since it harboured luxAB genes responsible for light production as well as luxCDE genes responsible for the production of the substrate (long-chain fatty aldehyde) for the reaction. The effect of adherent LAB on the viability of adherent Staph. aureus was determined in a competition assay. This ensured that the number of adherent Staph. aureus was the same regardless of the presence or absence of LAB. After adhesion and washings, the wells were covered either with HEPES-Hanks or with LB supplemented with 1 % glucose. HEPES-Hanks was used as it is used in adhesion assays, whereas LB supplemented with glucose allows the effect of available nutrients on viability to be observed. Results were calculated after 2 h incubation by comparing the viability of the sample to the viability of the adherent Staph. aureus incubated without LAB.
Antimicrobial substances produced by LAB.
The production of antimicrobial substances by those strains which were able to reduce viability of Staph. aureus was studied. A newly developed assay was used for this purpose (Vesterlund et al., 2004). This assay allows detection of organic acids, hydrogen peroxide or bacteriocins produced by LAB. In short, LAB were grown as described above and the culture supernatants were collected by centrifugation, filter-sterilized (0.22 µm pore size) and supplemented with erythromycin. Erythromycin was used as the used indicator strain carries an erythromycin resistance marker. When the production of hydrogen peroxide and bacteriocins was determined, the supernatants were neutralized to pH 7.2 with 4 M NaOH and phosphate buffer (pH 7.2; 0.1 M phosphate final concentration). To determine possible production of hydrogen peroxide by LAB, the supernatants were treated with catalase; to determine possible effects of bacteriocins, the supernatants were treated with proteinase K (both enzymes were purchased from Sigma and used at a concentration of 1 mg ml–1). MRS was used as a negative control and nisin (10 IU ml–1) as a positive control in the assay and they were treated in a similar way as supernatants.
Determination of maximum number of adhered bacteria on mucus and dissociation constants of bacteria
Theory.
Michaelis–Menten-type dissociation kinetic models have been used to describe adhesion kinetics of bacteria (Lee et al., 2000). Briefly, the equation:
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Hence, plots of 1/ex against 1/x give straight lines, in which the intercepts on the ordinate give the values of 1/em and those on the abscissa give the values of –1/kd.
Assay.
The adhesion assay was performed with twofold dilution series from each bacterium and followed the protocol described above.
Statistical analysis.
Pair-wise Student's t-test was used to determine the significance (P<0.05) of differences between the control and the samples. Results shown are from three or four independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Staph. aureus showed similar adhesion as Lb. acidophilus La5, 4.4 % and 4.0 %, respectively (Table 2), and showed higher adhesion (P<0.05) than the rest of the tested strains. Three of the LAB strains, Lb. casei Shirota, Lb. paracasei-33 and E. faecium adhered poorly, expressing similar adhesion as the negative Salmonella control (0.4 %).
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). Furthermore, a trend of reduced adhesion of Staph. aureus was seen with Lb. rhamnosus in competition (15 %; P=0.24), with Lc. lactis in exclusion (14 %; P=0.21) and in competition (20 %; P=0.32), and with P. freudenreichii in exclusion (21 %; P=0.23) after 1 h, but statistical significance was not reached due to relatively high variation between experiments.
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) and P. freudenreichii subsp. shermanii JS, two linear regions were observed. This may mean that two binding mechanisms are involved for these bacteria, one for a high bacterial concentration (lower affinity and thus probably non-specific adhesion when the adhesion sites are masked due to a high number of bacteria; forces such as van der Waals and hydrophobic interactions included) and one for a lower concentration, which implies higher affinity and thus probably specific adhesion. Thus at low bacterial concentration, the adhesion of bacterial cells on mucosa involves the maximum number of adhesion sites, and at high bacterial concentration there is self-competition for the adhesion and thus minimum numbers of adhesion sites are involved (Lee et al., 2000).
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. By using equation 1, the maximum number of adhered bacteria on mucus (em) and dissociation constants (kd) for each strain were calculated, and are summarized in Table 4. The values were calculated as c.f.u. per well, which compares a mucus area of 0.1 cm2. As shown in Table 4, among the 12 bacterial strains tested, Lb. plantarum had the highest amount of adhered bacteria on mucus (1.4x107 c.f.u. per well) and the em was 170 times higher compared to Lb. paracasei-33, which had the lowest em (8.4x104 c.f.u. per well). However, Lb. plantarum showed an adhesion ratio of only 1.7 % (Table 2
); this is likely to be due to its having the highest kd among the tested strains, 1.2x109 c.f.u. per well (Table 5), implying low affinity for adhesion to mucus. Staph. aureus had the third highest em among the tested strains and this also explained its relatively high adhesion ability (4.4 %; Table 2). The em for Staph. aureus was 3.3x106 c.f.u. per well and only Lb. plantarum (1.4x107 c.f.u. per well) and Lc. lactis subsp. lactis (6.7x106 c.f.u. per well) had a higher em. However, the kd of Staph. aureus was relatively high, 7.7x107 c.f.u. per well, indicating that Staph. aureus dissociates from mucus more easily compared to six tested LAB: Lb. acidophilus La5 (5.5x106 c.f.u. per well; P<0.001), Lb. johnsonii LJ1 (8.3x106 c.f.u. per well; P<0.001), Lb. rhamnosus (1.2x106 c.f.u. per well; P<0.001), Lc. lactis (5.1x107 c.f.u. per well; P<0.05), E. faecium SF68 (2.2x107 c.f.u. per well; P<0.05) and P. freudenreichii (2.4x107 c.f.u. per well; P<0.05). With lower bacterial concentrations, Lb. casei Shirota and P. freudenreichii also showed tighter binding (P<0.001) to mucus compared to Staph. aureus: 1.2x106 c.f.u. per well, 6.3x105 c.f.u. per well and 7.7x107 c.f.u. per well, respectively. However, these lower bacterial concentrations were not used in displacement, exclusion and competition assays (Table 3).
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). This has been proven also with several other bacterial strains (Beard et al., 2002; Rocchetta et al., 2001; Unge et al., 1999). When bacteria were allowed to adhere to the mucus, the non-bound bacteria were washed away and the microtitre plate wells were covered with HEPES-Hanks, Lb. acidophilus La5 was able to reduce the viability of Staph. aureus (Table 5). However, when the wells were filled with culture medium, Lb. acidophilus had no effect on Staph. aureus. Although most of the strains were able to reduce viability of Staph. aureus in the presence of culture medium, a statistically significant reduction (27–36 %; P<0.05) was obtained with Lb. reuteri, Lc. lactis and P. freudenreichii.
Antimicrobial substances produced by LAB
Supernatants of Lb. reuteri, Lc. lactis and P. freudenreichii were collected, neutralized and treated with catalase or proteinase K. Proteinase K treatment did not cause recovery of bioluminescence when compared to non-proteinase-treated but neutralized supernatant, indicating that LAB were not producing bacteriocins against Staph. aureus. However, either catalase treatment or neutralization caused recovery, indicating that hydrogen peroxide and organic acids had antimicrobial activity against Staph. aureus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Treatment of these infections has become difficult due to emergence of antibiotic-resistant strains, and new agents to treat and especially prevent staphylococcal infections are needed. The possible intestinal carriage of Staph. aureus may have negative health effects; for example during antibiotic treatment it can lead to the overgrowth of bacteria in the intestine and thus antibiotic-associated diarrhoea (Ackermann et al., 2005; Boyce & Havill, 2005). Also association of Staph. aureus with inflammatory bowel disease has been suggested, as lumen-derived Staph. aureus superantigens have been shown to elicit inflammation in a mouse model (Lu et al., 2003). Moreover, there is accumulating evidence that the colon may serve as a reservoir of antibiotic-resistance genes (Salyers et al., 2004), for example vancomycin-resistant Staph. aureus (VRSA) (Ray et al., 2003). Although it is unclear whether Staph. aureus belongs to the normal human colon microbiota, it seems that at least among hospitalized patients colonization is possible (Donskey, 2004). The hypothesis of intestinal colonization is also supported by a recent study showing that the caecal mucus layer may provide an important niche for intestinal colonization by Staph. aureus (Gries et al., 2005).
As Staph. aureus has been found to adhere to nasal mucin (Shuter et al., 1996), we hypothesized here that adhesion to intestinal mucus, of which the main components are mucins, would be possible as well. Moreover, in earlier studies several bacteria have been found to adhere to intestinal mucin oligosaccharides (Moncada et al., 2003). In the present study we used a model based on human intestinal mucus obtained from resected colonic tissue to assess whether Staph. aureus adheres to mucus. Human cell-lines Caco-2 and HT-29 do not produce mucus and the mucus-producing cell line HT-29-MTX (Lesuffleur et al., 1990) produces mainly mucins with gastric immunoreactivity (MUC3 and MUC5C) and only few mucins with colonic immunoreactivity (MUC2 and MUC4) (Lesuffleur et al., 1993). Intestinal epithelial cells offer an important model for studying adhesion of bacteria to intestinal areas without a mucus layer, such as Peyer's patches, or areas where the mucus is eroded due to disease, but they can not be used as models for adhesion to mucus. Another advantage of the use of mucus is that the colon's own mucosa-associated microbiota is present and its effect on adhesion is also taken into account. A drawback is the availability of the mucus and also the need to process it immediately.
Here we show for the first time that Staph. aureus can adhere to human colonic mucus but can be displaced by specific LAB. Lb. rhamnosus GG, Lc. lactis subsp. lactis and P. freudenreichii subsp. shermanii were able to displace Staph. aureus from human colonic mucus by 39–44 %. Interestingly, the displacement capability was restricted to the LAB with relatively high adhesion ability, Lb. rhamnosus GG, Lc. lactis subsp. lactis and P. freudenreichii subsp. shermanii JS, with adhesion ratios of 11.5 %, 10.1 % and 11.3 %, respectively (Table 2). Mathematical modelling including determination of the maximum number of adhered bacteria on mucus (em) and the binding affinity (kd) to mucus as well as measurement of viability of adherent Staph. aureus were used to explain the mechanism of displacement. Staph. aureus showed the third highest em among the tested bacteria. Only Lb. plantarum and Lc. lactis had higher em values. This also explained the relatively high binding of Staph. aureus to mucus. However, the binding affinity of Staph. aureus to mucus was only moderate (7.7x107 c.f.u. per well; Table 4), and the highest affinity to mucus was obtained with Lb. rhamosus GG (1.2x106 c.f.u. per well). This indicates that Staph. aureus can be outcompeted by probiotics which have higher affinity to the mucus. This is likely to explain why Lb. rhamnosus, Lc. lactis and P. freudenreichii displaced Staph. aureus from mucus. Similarly under in vivo conditions, Staph. aureus would probably be washed out more easily from the intestinal mucus surface than for example Lb. rhamnosus GG. However, in competition assays, LAB showing higher affinity than Staph. aureus to mucus were not able to reduce its adhesion. This may have been due to the amounts of bacteria used: in displacement the adherent pathogens were covered with LAB and outnumbered whereas in competition the amounts of bacteria were similar. In exclusion assays there was no effect of LAB on adhesion of Staph. aureus, indicating that the bacteria do not use same adhesion receptors.
When viability of adherent Staph. aureus was measured in the presence of adherent LAB, the LAB had an effect only when nutrients were available. Adherent Lb. reuteri, Lc. lactis and P. freudenreichii significantly reduced the viability of Staph. aureus by 27–36 % within 2 h. The reduction of viability was not due to competition for nutrients (which were present in excess) but rather to the in situ production of organic acids and hydrogen peroxide, and in the case of Lb. reuteri possibly reuterin (Arques et al., 2004; Vesterlund et al., 2004). Uehara et al. (2001) showed that colonization of meticillin-resistant Staph. aureus (MRSA) in the oral cavities of newborns was inhibited by the viridans group of streptococci, and that this was probably due to the production of hydrogen peroxide by these streptococci. However, it is unclear whether LAB can produce antimicrobial substances against Staph. aureus in vivo. It is also possible that the hydrogen peroxide produced is degraded by the metabolism of other bacteria (Ryan & Kleinberg, 1995).
The emergence of antibiotic resistance among Staph. aureus strains and possibly increased intestinal colonization of these bacteria require alternative methods for prevention and treatment of staphylococcal diseases. Our results show that Staph. aureus adheres to human colonic mucus and that certain LAB could have antiadhesive and antimicrobial effects against this bacterium. However, it remains for further studies to show that other virulent Staph. aureus strains can adhere to colonic mucus in vitro and in vivo, and to show that LAB have antiadhesive and antimicrobial effects against Staph. aureus also in vivo.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 14 September 2005;
revised 3 January 2006;
accepted 10 January 2006.
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 S. Nuding, K. Fellermann, J. Wehkamp, and E. F Stange Reduced mucosal antimicrobial activity in Crohn's disease of the colon Gut, September 1, 2007; 56(9): 1240 - 1247. [Abstract] [Full Text] [PDF]
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, Low bacterial concentration; 
, high bacterial concentration.
, P. freudenreichii; x, Staph. aureus.