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- MATERIALS AND METHODS
The anti-infectious activity of synbiotics against methicillin-resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 108 CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5-fluorouracil (5-FU) was injected intraperitoneally on day 7 after the infection. A dose of 108 CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 108 CFU/g of intestinal contents after oral MRSA infection and the subsequent 5-FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.
Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to multiple drugs and is an important cause of opportunistic infections in post-operative patients and compromised hosts (1–3). The first reported case of MRSA enteritis occurred in the early 1980s (4). Since then, numerous case reports and series have arisen worldwide, and, in the majority of cases, it appears that MRSA enteritis is associated with gastric surgery and antibiotic use, which allows the colonization and subsequent overgrowth of MRSA (5–7). Methicillin-sensitive S. aureus (MSSA) has been replaced by MRSA as the primary cause of severe enteritis (7). Outbreaks of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in the neonatal intensive-care unit (NICU) setting have been well documented (8–12). MRSA infection is difficult to prevent and treat with antimicrobial drugs, so a new approach is desirable.
Probiotics are viable cell preparations or foods containing viable bacterial cultures or components of bacterial cells that have beneficial effects on the health of the host (13). Many of these probiotics are lactic acid bacteria and anaerobic bifidobacteria have been reported to be useful in the treatment of disturbed intestinal microflora and diarrheal diseases (for a review, see Ref. 13). On the other hand, the term prebiotics has been defined as a non-digestive food constituent that selectively alters the growth and/or activity of one or a limited number of bacteria in the colon, thus potentially improving the health of the host (14, 15). The combined use of probiotics and prebiotics is called synbiotics (15). There have been some reports demonstrating that the oral administration of probiotics (Bifidobacterium breve strain Yakult) or synbiotics (B. breve strain Yakult and galactooligosaccharides) can improve an imbalance of microflora or intestinal ecology resulting in the prevention of intestinal Gram-negative bacterial infection in mouse models induced by multiple antibiotics (16, 17). Therefore, synbiotics are expected to be useful to prevent MRSA intestinal infections; however, the effectiveness of this modality has yet to be verified in animal models.
The main purpose of these experiments was, therefore, to evaluate the anti-infectious activity of synbiotics against MRSA infection in a novel mouse infection model.
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- MATERIALS AND METHODS
Animal models may be useful for evaluating the preventive effects of agents and foods on intestinal infection by MRSA. However, thus far there has not been a useful animal model, with the exceptions of a rat model developed by Takahata et al. in 2004 by corrupting the microflora with plural cephalosporins (29) and a mouse model that continuously excreted MRSA in feces infected via the nasal mucosa under treatment with cyclophosphamide (30). No lethal model has been produced. Therefore, a new animal model was clearly required. On the other hand, KM, MTN and CBPZ are known to induce intestinal MRSA infections resistant to those antibiotics used to protect against post-operative infections in the gastroenterological field (31). Such antibiotics may cause a colonization resistance disorder, as suggested by a reduction in intestinal organic acid production and a tendency for increased intestinal pH leading to massive intestinal colonization by MRSA. In fact, the detection level of MRSA in the feces of MRSA-infected mice treated with KM + MTN + CBPZ was 108 CFU/g intestinal contents, which is more than 1,000 times higher than that in the uninfected mice.
Nomoto et al. demonstrated that all of the specific pathogen-free mice inoculated with a large amount of 5-FU (>338 mg/kg) died of systemic infection induced by endogenous E coli proliferating dramatically in the intestine (32). The intestinal colonization by orally administered MRSA could occur under KM + MTN + CBPZ treatment; however, it did not give rise to any symptoms in this model (Fig. 1). Because the endogenous intestinal E. coli were highly sensitive to CBPZ, the KM + MTN + CBPZ treatment could completely eliminate them in the intestine of mice (Table 1). Therefore, KM + MTN + CBPZ could lead to the development of a lethal MRSA infection by the corruption of endogenous E coli after the inoculation of 5FU. As such, the current mouse model appears to be a novel model that accurately mimics a clinically lethal MRSA infection by bacterial alterations induced by multiple antibiotics. There have so far been few clinical reports regarding the effect of synbiotics on MRSA intestinal infection. Kanamori (33) demonstrated that anaerobic dominant flora is reconstructed by synbiotics using the Bifidobacterium breve strain Yakult, Lactobacillus casei strain Shirota and GOS after treatment with vancomycin in an infant with Down's syndrome suffering from MRSA enteritis. The intestinal environment improved by an increase in the intestinal dose of total organic acids, thus leading to the suppression of an exacerbation of MRSA infection in this infant. Therefore, a mouse model of MRSA provides a clinical model that yields valuable information about the efficacy of probiotics.
The cecal population levels of MRSA in the 104 or 106 MRSA-infected mice were 1/100 and 1/10, respectively, and the levels of the 108 MRSA-infected mice showed hardly any extra-intestinal translocations (data not shown). Therefore, the prevention of systemic MRSA infection might depend on the extent to which the intestinal MRSA colonization could be reduced. Furthermore, B. breve contributed to reductions in extra-intestinal translocation, malnutrition and mortality in the mice infected with MRSA followed by 5-FU treatment, whereas no mice could survive without the probiotics and the synbiotics increased this benefit. In clinical practice, if it is anticipated that an MRSA carrier would be immunodeficient in surgery or chemotherapy, it is particularly important to reduce the bacterial level of MRSA in the intestines as much as possible, and the results obtained in this study suggest that synbiotics may be useful for this purpose.
TSST-1 and staphylococcal exotoxins possessing super-antigenic properties produced by S aureus (34–36) cause toxic shock syndrome (TSS) (37), neonatal toxic-shock-like exanthematous diseases (38) and lethal septic shock in invasive surgical cases (39, 40). In this MRSA intestinally infected mouse model, high lethality was observed, even in non-producer strains such as staphylococcal exotoxin (SEB, SEC and TSST-1), and there was no relationship between the production quantities and the lethalities of SEB, SEC and TSST-1. On the other hand, most studies on the lethal effects of TSST-1 and the staphylococcal enterotoxins in vivo have been conducted with mice, which are highly resistant to the lethal effects of TSST-1 and the staphylococcal enterotoxins. For example, many strains of mice do not develop a disease resembling TSS even after high-dose injections (4 mg/mouse) or continuous infusions (500 μg/mouse) of TSST-1, although they may develop massive splenomegaly (41, 42). The quantity of staphylococcal exotoxin in the intestines of mice immediately before death in this study was 500 ng or less per 1 g of intestinal content. As such, in this mouse model, it is strongly suggested that the involvement of MRSA-producing toxin in lethality was very low.
It has become a major concern that MRSA causes intractable chronic infection in compromised hosts and serious infections such as septicemia due to immunodepression or surgical treatments (43). In the mice immediately before death after infection with the 4 MRSA strains in this study, the infected MRSA colonization levels in the intestines were at the same level, and the invasive MRSA counts in the MLN and the liver were also at the same level. This suggests that multiple organ failure due to serious seismic infection with MRSA was the cause of death in this mouse model and strongly reflects the clinical conditions.
When orally administering B. breve strain Yakult and GOS daily from the day after infection with MRSA, it was elucidated that the same level of effect of treatment was observed against all 4 MRSA strains of the lethal intestinal infection. In clinical practice, if it is anticipated that an MRSA carrier would be immunodeficient in surgery or chemotherapy, it is extremely important to reduce the bacterial level of MRSA in the intestines as much as possible, and the results obtained in this study suggest that synbiotics may be useful for such a purpose. In the intestines of KM + MTN + CBPZ-treated mice, it is believed that, because endogenous bifidobacteria in the intestines were removed, there was no effect of treatment on the infection by solely administering GOS, which is a selective growth factor of bifidobacteria.
When examining the effect of treatment on MRSA lethal intestinal infection by concurrently administering GOS with B. breve strain Yakult and 5 strains of other bifidobacteria at similar levels of intestinal colonization, it was elucidated that there was a significant difference in the effects of treatment among the strains. In the intestines of mice in the bifidobacteria-strain-treated group in which an effect of treatment was observed, total intestinal organic acid and acetic acid concentrations were significantly increased whereas such effects were not observed in the strain-treated group in which no effect of treatment was observed.
Indigenous bifidobacteria were removed from the mouse intestines owing to sensitivity to KM + MTN + CBPZ, suggesting that, in the bifidoba-cteria-strain-treated group in which an effect of treatment was observed, the administered bifidobacteria that colonized the intestines may produce acetic acid aggressively by utilizing GOS. On the other hand, dissociated acetic acid produced by B breve is antibacterial to S aureus and most of the Gram-positive and -negative microbes in vitro (44–47). On the basis of these findings, a high concentration of acetic acid and a low pH in the intestine are important factors for intestinal colonization with resistance to MRSA.
This may be the first report to describe the efficacy of a combination therapy of probiotics and prebiotics, a combination known as synbiotics, for the prevention of systemic MRSA infection from MRSA enterocolitis in a lethal animal model. This model is therefore considered to be appropriate for conducting investigations of clinical MRSA enterocolitis.