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Keywords:

  • anti-infectious activity;
  • Bifidobacterium breve;
  • methicillin-resistant Staphylococcus aureus;
  • synbiotics

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

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.

List of Abbreviations: 
MRSA

methicillin-resistant Staphylococcus aureus

5-FU

5-fluorouracil

B. breve strain Yakult

Bifidobacterium breve strain Yakult

GOS

galactooligosaccharides

MSSA

methicillin-sensitive S. aureus

NICU

neonatal intensive-care unit

KM

kanamycin sulfate

MTN

metronidazole

CBPZ

cefbuperazone sodium

RPLA

reversed passive latex agglutination

SE

Staphylococcal enterotoxin

TSST-1

toxic shock syndrome toxin-1

BHI

brain heart infusion

CFU

colony-forming units

MLN

mesenteric lymph nodes

SD

standard deviation

HPLC

high-performance liquid chromatography

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.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Animals

Specific-pathogen-free 6-week-old male BALB/c mice were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Groups of 5 or 6 mice were housed in polypropylene cages (CLEA Japan, Tokyo, Japan) with sterilized bedding under controlled lighting (12 hr light, 12 hr dark), temperature (24°C) and relative humidity (55%) conditions. The mice were maintained on an MF diet (Oriental Yeast, Tokyo) and sterilized water (126°C for 30 min) containing Cl2 at a final concentration of 1.5 ppm (μg/ml), ad libitum. Kanamycin sulfate (KM, Sigma Chemical, St. Louis, MO, USA), metronidazole (MTN, Sigma) and cefbuperazone sodium (CBPZ, Toyama Chemical, Tokyo) were dissolved in the drinking water at concentrations of 1 mg/ml, 0.2 mg/ml and 0.02 mg/ml, respectively. The water bottles were exchanged with freshly prepared bottles every 3 days. All experimental procedures were performed in accordance with the standards set forth in the Guide for the Care and Use of Laboratory Animals (18).

Murine model of methicillin-resistant Staphylococcus aureus infection

MRSA strains Nos. 36, 92, 108 and O-MR-12 were isolated from patients at medical departments such as pediatric surgery and critical care medicine. MRSA strains were identified by their characteristic growth morphologies, Gram stain characteristics, reaction to catalase, coagulase production, results of API Staph (Biomerieux, Marcy l’Etoile, France) and results of the latex agglutination test with protein A- and PBP2′-specific monoclonal antibody–sensitized latex particles (Staph LA, Staph MRSA (Denka Seiken, Tokyo)). MRSA strains were resistant to KM (MICs: >200 μg/ml), MTN (MICs: >200 μg/ml) and CBPZ (MICs: 100 μg/ml). The presence of relevant genes was examined by PCR using specific primers for sea, seb, sec, sed and tst in accordance with the method of Johnson et al. (19). When quantifying the toxin production (ng/ml BHI broth) of an MRSA strain in vitro using the reversed passive latex agglutination (RPLA) test (SET-RPLA kit and TSST-RPLA kit, Denka Seiken, Tokyo), the results for MRSA strain No. 36 were Staphylococcal enterotoxin (SE)C: 800 and toxic shock syndrome toxin-1 (TSST-1): 800, those for No. 92 were SEC: 25 and TSST-1: 50, that for No. 108 was TSST-1: 400, and that for No. O-MR-12 was SEB: 51,200. The cells were grown overnight in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI) at 37°C. MRSA cells were suspended at a concentration of 1 × 108 colony-forming units (CFU)/ml in saline and a 100 μl portion of the suspension was administered orally to the mice using a gastric probe (Fuchigami, Kyoto, Japan). Mice were infected orally with MRSA on day 0. 5-fluorouracil (5-FU, Kyowa Hakko Kogyo, Tokyo) at a dose of 400 mg/kg body weight was injected intraperitoneally on day 7 after MRSA infection as a treatment for immunosuppression.

To assess the MRSA viable counts in the feces, intestinal contents, the liver and mesenteric lymph nodes (MLN), the samples were removed aseptically from the mice and then homogenized in 1 ml (5 ml for liver) of sterile saline solution using a Teflon grinder. The number of viable MRSA cells was determined by their growth on Sta-HI-OX agar (20) at 37°C for 24 hr.

Probiotics (Bifidobacteria)

Bifidobacterium breve strain Yakult, B. breve ATCC 15700T, B. dentium ATCC 27534T and B. pseudocatenulatum ATCC 27919T were used after the selection of the strains that had been confirmed by growth in PY broth (21) containing KM (0.2 mg/ml), MTN (0.05 mg/ml) and CBPZ (0.01 mg/ml). All bifidobacterial strains were identified by a PCR assay using the corresponding species-specific primers for 16S ribosomal RNA (22). Each bifidobacterial strain was cultivated separately in GAM broth (Nissui Pharmaceutical, Tokyo) for 24 hr at 37°C and washed with saline twice; then, it was suspended in saline at a concentration of 109 CFU/ml. Colonization by bifidobacteria was established by the daily administration of the bacteria (1–3 × 108 CFU/mouse/day) to separate groups of mice receiving KM, MTN and CBPZ in their drinking water. Periodic examinations of viable counts of bifidobacteria in stool specimens were performed in subsets of 6 mice from each group. Briefly, fresh stool specimens (1–2 pellets) were weighed and placed in an Eppendorf tube containing 1 ml of sterilized anaerobic buffer solution (0.0225% (w/v) KH2PO4, 0.0225% (w/v) K2HPO4, 0.045% (w/v) NaCl, 0.00225% (w/v) (NH4)2SO4, 0.0225% (w/v) CaCl2, 0.00225% (w/v) MgSO4, 0.3% (w/v) Na2C03, 0.05% (w/v) L-cysteine hydrochloride and 0.0001% (w/v) resazurin) and then homogenized with a pestle. TOS agar (23) supplemented with 0.625 g/ml streptomycin sulfate (Sigma) and 1 μg/ml carbenicillin disodium salt (T-CBPC agar, Sigma) was used for the quantitation of the B. breve strain Yakult and CPLX agar (24) was used for the selective isolation of other Bifidobacterium strains. The media were cultured anaerobically in an atmosphere of 7% H2 and 5% CO2 in N2 at 37°C for 72 hr and then the colonies on the plates were counted.

Prebiotics

Galactooligosaccharides (GOS, purity > 99%) were prepared as described previously (25). The GOS preparation was dissolved in distilled water (100 mg/ml) and a 0.1 ml portion (10 mg/mouse) was administered once daily to the mice.

Examination of cecal bacterial flora

To determine the cecal contents, six ether-anesthetized mice per group per period were killed by cervical dislocation. The cecal contents were removed, placed in grinding tubes containing 1 ml of sterilized anaerobic buffer solution and homogenized with a Teflon grinder. After serial dilution of the cecal suspensions with anaerobic buffer solution, 50 μl portions of the diluents were spread onto the following culture media. Modified NBGT agar was used for selective isolation of Bacteroidaceae (16). CPLX agar was used for selective isolation of Bifidobacterium (24). LBS agar (Becton Dickinson and Company, Cockeysville, MD), supplemented with 0.8% (w/v) Lab Lemco powder (Oxoid Ltd., Basingstoke, UK), 0.1% (w/v) sodium acetate-trihydrate and 0.37% (w/v) acetate, was used for selective isolation of Lactobacillus. COBA agar was used for selective isolation of Enterococcus (26). DHL agar (Nissui Pharmaceutical Co. Ltd., Tokyo) was used for selective isolation of Enterobacteriaceae. Staphylococcus medium No. 110 agar (Nissui) was used for selective isolation of Staphylococcus and Bacillus. T-CBPC agar was used for quantification of B. breve strain Yakult. Modified VL-G roll tube agar was used to determine the total anaerobe counts (16). Modified NBGT agar, CPLX agar, LBS agar and T-CBPC agar media were cultured anaerobically in an atmosphere of 7% H2 and 5% CO2 in N2 at 37°C for 72 hr. After incubation, the colonies on the plates were counted and Gram-stained. Species and biotypes of the bacteria were identified with API systems (BioMerieux S. A., Montalieu-Vercieu, France): Rapid ID 32 A for the Bacteroidaceae and Lactobacillus, API 20 STREP for the Enterococcus, API 20 E for the Enterobacteriaceae and API 20 STAPH for the Staphylococcus. The lower limit of bacterial detection with this procedure was 100 CFU/g feces.

Scanning for anaerobic fusiform bacteria was carried out by performing microscopic bacterial counts (27). For quantification, 10 μl portions of the diluents were placed on a 10-well immunofluorescence slide (Flow Laboratories, Inc., McLean, VA, USA), fixed and Gram-stained. Fusiform bacteria were counted with the aid of an ocular grid containing 100 squares (calibrated with a stage micrometer), a 100× objective and a 10× ocular. Acceptable slides considered for analysis met the following two criteria: (i) the bacteria appeared to be evenly distributed and (ii) the number of bacteria per 100 grid squares ranged between 20 and 300. The counts were made in 10 randomly chosen fields. The results are expressed as the means ± standard deviation (SD) numbers of CFU per 1 g of cecal contents.

Detection of organic acids in cecal contents

The cecal contents were homogenized in 1 ml of distilled water and the homogenate was centrifuged at 13,000 ×g at 4°C for 10 min. A mixture of 0.9 ml of the resulting supernatant and 0.1 ml of 1.5 mM perchloric acid was mixed well in a glass tube and allowed to stand at 4°C for 24 hr. The suspension was then passed through a filter with a pore size of 0.45 μm (Millipore Japan, Tokyo). The organic acid content of the sample was analyzed by high-performance liquid chromatography (HPLC) as described in a previous report (28). The HPLC was performed using a Waters system (Waters 432 Conductivity Detector; Waters, Milford, MA) equipped with two columns (Shodex Rspack KC-811; Showa Denko, Tokyo). The concentrations of organic acids were calculated using external standards.

Toxin assay

To determine the toxin concentration in the intestinal contents, 6 mice per group were killed after being anesthetized with ether.

SEA, SEB, SEC, SED and TSST-1 in the intestinal contents were extracted as follows. Briefly, the contents of the gastrointestinal tracts were prepared as described above. After homogenization, the samples were centrifuged at 30,000 ×g for 10 min. The supernatants were filtered through a 0.45 μm membrane filter and then subjected to ultrafiltration (MW cut-off: 20,000; 5,000 ×g for 60 min) to remove low–molecular-weight substances, such as KM, MTN and CBPZ, which can affect toxin quantification using both the SET-RPLA kit and the TSST-RPLA kit (Denka Seiken). After centrifugation, the resulting fraction on the membrane in the tube was reconstituted in the original volume of PBS and then serially two-fold diluted with PBS supplemented with 0.5% (w/v) bovine serum albumin and 0.1% (w/v) NaN3. SEA, SEB, SEC, SED and TSST-1 were quantified using the RPLA test and then the toxin concentrations were expressed as the mean and the standard deviation in micrograms per tissue weight.

Statistical analysis

The average number of bacteria was analyzed using Dunnett's test to determine any significant differences between the treatment and control groups. Differences in the survival ratios were determined using Fisher's exact probability test followed by correction with the Bonferroni inequality equation. A significant difference was defined as a value of P < 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

Inhibition of lethal MRSA intestinal infection by B. breve colonization in the KM + MTN + CBPZ-treated mice

The continuous treatment of mice with KM + MTN + CBPZ in drinking water resulted in selective intestinal flora decontamination: the indigenous species such as Bifidobacterium, Enterobacteriaceae and Staphylococcus decreased to undetectable levels, whereas there were no significant differences in the colonization levels of the other bacterial genera tested (Table 1). In the KM + MTN + CBPZ-treated mice, 108 orally administered MRSA colonized the intestines at a high population level (Fig. 1a). 5-FU treatment significantly reduced the body weights of all the 108 MRSA-infected mice under continuous treatment with KM + MTN + CBPZ and, as a result, they all died (Fig. 1c,d). However, the same phenomenon was not seen in either those with single antibiotics + 5-FU or those infected with 108 MRSA only (data not shown). The fecal viable count of MRSA decreased in the mice treated with B. breve alone to less than 1/10 of that in the controls (Fig. 1a). The synbiotic treatment (B. breve+ GOS) contributed to the reduction more effectively whereas no mice survived with GOS alone. The B. breve administration produced a population level of 108 CFU/g (Fig. 1b) and additional GOS tended to increase the level. B. breve contributed to a reduction of malnutrition or mortality in the mice infected with MRSA followed by the 5-FU treatment whereas none survived without the probiotics (Fig. 1c,d). Interestingly, synbiotic treatment (B. breve+ GOS) yielded more favorable results. In neither the B. breve-administered group nor the group given B. breve in combination with GOS were any significant differences observed in the bacterial flora species levels in comparison to those of the KM + MTN + CBPZ-treated control group, with the exception of the detection of the B. breve being given daily (Table 1).

Table 1.  Effect of Bifidobacterium breve strain Yakult colonization on indigenous cecal microflora under KM + MTN + CBPZ treatment in mice
OrganismsNumber of bacteria in cecal contents
NormalKM + MTN + CBPZKM + MTN+ CBPZ +B. breve‡,§KM + MTN + CBPZ + GOS‡,¶KM + MTN+ CBPZ +B. breve+ GOS‡,§,¶
  1. The results are expressed as mean Log10 CFU ± SD per g of the cecal contents for 6 mice.

  2. KM, MTN and CBPZ were given in drinking water to mice from day −7 until day 0.

  3. §B. breve strain Yakult was administered to the mice once daily from day −7 until day 0.

  4. GOS was administered to the mice once daily from day −7 until day 0.

  5. #Methicillin sensitive.

Total10.5 ± 0.110.4 ± 0.110.4 ± 0.110.4 ± 0.110.4 ± 0.1
Fusiform bacteria10.5 ± 0.110.4 ± 0.110.4 ± 0.110.4 ± 0.110.4 ± 0.1
Bacteroidaceae9.2 ± 0.19.1 ± 0.19.1 ± 0.19.0 ± 0.19.1 ± 0.1
Bifidobacterium6.9 ± 0.2<2.08.0 ± 0.2<2.08.2 ± 0.3
Lactobacillus9.0 ± 0.28.1 ± 0.48.2 ± 0.38.2 ± 0.28.1 ± 0.3
Enterococcus4.4 ± 0.44.3 ± 0.74.3 ± 0.84.3 ± 0.64.1 ± 0.2
Enterobacteriaceae4.2 ± 0.8<2.0<2.0<2.0<2.0
Staphylococcus#3.5 ± 0.3<2.0<2.0<2.0<2.0
Bacillus3.1 ± 0.33.3 ± 0.33.4 ± 0.43.4 ± 0.43.2 ± 0.2
B. breve strain Yakult<2.0<2.08.0 ± 0.3<2.08.2 ± 0.2
image

Figure 1. Inhibition of lethal MRSA intestinal infection by B. breve colonization in KM + MTN + CBPZ-treated mice. Feces for bacteriological analysis were obtained from 6 mice in each group, and the counts of (a) viable MRSA and (b) B. breve strain Yakult were examined using the selective media. The MRSA (strain No. 92)-infected mice (10 mice/group) were (c) examined for changes in body weight and (d) observed for survival for 28 days after the challenge. Symbols: •, infection control (saline); ○, B. breve; ▵, GOS; □, B. breve+ GOS. A significant difference was observed between the treated mice and the untreated control mice (*P < 0.05, **P < 0.01).

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The extra-intestinal translocation of MRSA to the MLN and the hepatic tissues was markedly inhibited by the administration of B. breve (Table 2) and this effect was augmented by the synbiotic treatment (B. breve+ GOS). In contrast, GOS itself did not contribute to the prevention of the bacterial translocation by MRSA.

Table 2.  Inhibition of extra-intestinal translocation of MRSA by B. breve colonization in KM + MTN + CBPZ-treated mice
GroupsNumber of MRSA in organ
Cecal contentsMLNLiver
  1. Mice (Fig. 1a and b) were dissected on day 16 after MRSA (strain No. 92) infection, and the results are expressed as the mean Log10 CFU and SD per g cecal contents or entire MLN or liver of 6 mice.

  2. The number of organs in which MRSA were detected/number of organs tested.

  3. Significant difference between the treated mice and the untreated control mice (*P < 0.05, **P < 0.01).

Infection control (Saline)8.2 ± 0.2 (6/6)4.2 ± 1.4 (6/6)4.6 ± 1.3 (6/6)
B. breve-treated6.4 ± 1.1* (6/6)3.1 (1/6)3.6 (1/6)
GOS-treated8.2 ± 0.2 (6/6)4.7 ± 1.8 (6/6)4.7 ± 1.7 (6/6)
B. breve+ GOS-treated5.8 ± 0.3** (6/6)<1.3 (0/6)<2.0 (0/6)

Changes in the intestinal pH and concentrations of organic acids after MRSA infection by B. breve colonization

In the KM + MTN + CBPZ-treated control group, the acetate concentration in the cecal contents markedly decreased to less than half the level in normal mice, thus reflecting the reduction in total organic acid concentration (Fig. 2a,b). The pH of the cecal contents increased markedly as a result of KM + MTN + CBPZ treatment (Fig. 2c). The B. breve resulted in an improvement of the intestinal environment due to a decline in the cecal pH and an increase in the intestinal dose of total organic acids including acetic acid (Fig. 2). This ecological benefit seemed to be more effective when given to the synbiotic group than to the probiotic group.

image

Figure 2. Changes in the intestinal pH and concentrations of organic acids after MRSA infection by B. breve colonization in KM+MTN+ CBPZ -treated mice. The cecal contents were obtained from the mice (6 mice/group) both at the time of MRSA (strain No. 92) infection (day 0) and at day 16 after MRSA infection. Organic acid (total organic acids (a) and acetic acid (b)) concentrations and pH (c) were determined as described in the text. The results are expressed as the means and standard deviations of 6 mice. Columns: dotted, untreated normal; black, infection control (saline); white, B. breve; grey, GOS; hatched, B. breve+ GOS. A significant difference was observed between the treated mice and the infection control mice (*P <0.05, **P <0.01).

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Disinfection effect of B. breve administration on MRSA intestinal infection

The daily administration of B. breve from the day following exposure to MRSA contributed to reductions in malnutrition (Fig. 3a) and mortality (Fig. 3b) in the post-5-FU-treated mice. In the B. breve-treated group in which effects of treatment on the infection were observed, the colonization level of MRSA in the intestinal tract significantly decreased from 7 days after infection with MRSA when B. breve was collected from the feces at the 108 CFU level (Fig. 3c,d). This effect was magnified with the passage of time with B. breve. The therapeutic values for the combination of B. breve and GOS also produced the most satisfactory results.

image

Figure 3. Disinfection effect of B. breve administration on MRSA intestinal infection. B. breve strain Yakult and GOS were administered to mice once daily from day 1 until day 33. The MRSA (strain No. 92)-infected mice (10 mice/group) were (a) examined for changes in body weight and (b) observed for survival for 33 days after the challenge. Feces for bacteriological analysis were obtained from mice in each group after the MRSA infection, and the counts of (c) viable MRSA and (d) B. breve strain Yakult were examined using the selective media. Symbols: •, infection control (saline); ○, B. breve; ▵, GOS; □, B. breve+ GOS. A significant difference was observed between the treated mice and the untreated control mice (*P < 0.05, **P < 0.01).

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The numbers of various MRSA strains in organs and concentrations of their toxins in the cecal contents of mice treated with B. breve and GOS compared with those of controls are summarized in Table 3. When KM + MTN + CBPZ-treated mice were orally infected with 4 strains of MRSA at 108 CFU each, MRSA was detected at 108 CFU/g from the feces of every mouse in the infected control groups by 20 days after infection (Table 3). High lethality was observed even in non-producer strains such as SEB, SEC and TSST-1 when inoculating 5-FU into mice in each infected control group under conditions in which there was no difference in the MRSA colonization level within the intestines, and there was no relationship between the production quantities and the lethalities of SEB, SEC and TSST-1. No other toxins such as SEA and SED were detected in the cecum of all mice (data not shown). On the other hand, in the group in which B. breve and GOS were concurrently administered daily from the day after infection with MRSA, a high level of MRSA colonization in the appendix and in vivo invasion, which were observed in mice in each infected control group with all MRSA strains, were largely inhibited, and the quantity of MRSA-producing toxin in the appendix was reduced (Table 3).

Table 3.  Summary of numbers of various MRSA strains in organs and concentrations of their toxins in cecal contents in mice treated with B. breve and GOS compared with controls
MRSAGroupNo.of deaths/Total no. of mice§Number of MRSA in organStaphylococcal enterotoxins and toxic shock syndrome toxin-1 in cecal contents#
Cecal contentsMLNLiverSEBSECTSST-1
  1. The mice (16 mice/group) were infected orally with MRSA strain Nos. 36 (1.2 × 108 CFU), 92 (1.2 × 108 CFU), 108 (1.0 × 108 CFU) or O-MR-12 (1.2 × 108 CFU) at an inoculum of 0.1 ml/mouse on day 0. The MRSA-infected mice were treated intraperitoneally with 5-FU on day 12 after MRSA infection.

  2. B. breve strain Yakult and GOS were administered to the mice once daily from day 1 until day 33.

  3. §The mice were observed to determine survival for 33 days after the infection.

  4. The mice were dissected on day 20 after MRSA infection, and the results are expressed as the mean Log10 CFU and SD per g cecal contents or entire MLN or liver of 6 mice.

  5. #The mice were sacrificed on day 20 after MRSA infection, and the results are expressed as the mean ng and SD per g cecal contents of 6 mice.

  6. oThe numbers of organs in which MRSA were detected/number of organs tested.

  7. The incidence of toxin (number of cecain which toxins were detected/number of ceca tested).

  8. Significant difference between the treated mice and the untreated control mice (*P < 0.05, **P < 0.01).

36Infection control10/108.2 ± 0.4 (6/6)o4.9 ± 1.5 (6/6)4.8 ± 1.0 (6/6)458 ± 292 (6/6)42 ± 24  (6/6)
B. breve+ GOS3/10*6.2 ± 0.6** (6/6)3.7 (2/6)3.9 (2/6)16 (2/6)<8 (0/6)*
92Infection control10/108.4 ± 0.8 (6/6)4.5 ± 1.4 (6/6)4.7 ± 1.1 (6/6)417 ± 129 (6/6)21 ± 8   (6/6)
B. breve+ GOS3/10*6.5 ± 0.4** (6/6)3.4 (2/6)3.5 (2/6)<8 (0/6)*<8 (0/6)*
108Infection control10/108.0 ± 0.6 (6/6)4.4 ± 1.0 (6/6)4.2 ± 0.8 (6/6)107 ± 82 (6/6)
B. breve+ GOS2/10*5.9 ± 0.9** (6/6)3.1 (1/6)*3.0 (1/6)*39 (2/6)
O-MR-12Infection control10/108.2 ± 0.9 (6/6)4.3 ± 1.2 (6/6)4.6 ± 1.0 (6/6)115 ± 73 (6/6)
B. breve+ GOS3/10*6.5 ± 0.4** (6/6)3.4 (1/6)*3.7 (1/6)*31 (2/6)

Comparison of disinfection effect among several strains of bifidobacteria with spontaneous resistance to KM, MTN and CBPZ

Six strains of naturally resistant bifidobacteria, which were orally administered to KM + MTN + CBPZ-treated mice (concomitantly used with GOS) were each detected at 108 CFU/g according to the contents of the appendix, and there was no difference in the colonization level among the strains of bifidobacteria (Table 4). However, the treatment effects of Bifidobacterium on the lethal intestinal infection of MRSA showed a significant difference among the strains. Four strains—B. breve strain Yakult, B. breve ATCC 15700T, B. dentium ATCC 27534T and B. pseudocatenulatum ATCC 27919T—largely inhibited mortality, MRSA colonization in the appendix and MRSA invasion in the MLN and the liver, which were observed in mice in the infected control groups. On the other hand, 2 strains—B. catenulatum ATCC 27539T and B. infantis ATCC 15697T—showed no effect of treatment on the infection.

Table 4.  Comparison of disinfection effect among several strains of bifidobacteria with natural resistance to KM, MTN and CBPZ
GroupNo. of deaths/Total no. of miceNumber of MRSA in organ§Number of given bifidobacteria in cecal contents§pH and concentrations of organic acids in cecal contents
Cecal contentsMLNLiverpHTotal organic acidAcetic acid
  1. The MRSA (strain No. 92)-infected mice (16 mice/group) were treated intraperitoneally with 5-FU on day 12 after MRSA infection. Bifidobacterium strains and GOS were administered to mice once daily from day 1 until day 33.

  2. Mice were observed for survival for 33 days after the infection.

  3. §Mice were dissected on day 20 after MRSA infection, and the results are expressed as the mean Log10 CFU and SD per g cecalcontents or entire MLN or liver of 6 mice.

  4. Mice were dissected on day 20 after MRSA infection, and the results are expressed as the mean Π moland SD per g cecal contents of 6 mice.

  5. #Number of organs in which MRSA were detected/number of organs tested.

  6. Significant difference between the treated mice and the infection control mice (*P < 0.05).

Saline (Infection control)10/108.2 ± 0.3 (6/6)#4.8 ± 0.9 (6/6)4.6 ± 1.1 (6/6)7.3 ± 0.133 ± 523 ± 5
GOS10/108.2 ± 0.2 (6/6)5.0 ± 0.7 (6/6)4.7 ± 0.7 (6/6)7.3 ± 0.133 ± 624 ± 5
B. breve strain Yakult3/106.6 ± 0.7* (6/6)3.5 (2/6)3.5 (2/6)8.2 ± 0.2 (6/6)6.9 ± 0.2*64 ± 12*53 ± 12*
B. breve ATCC 15700T3/106.8 ± 0.8* (6/6)3.7 (2/6)3.7 (2/6)8.2 ± 0.4 (6/6)6.9 ± 0.3*63 ± 12*51 ± 11*
B. catenulatum ATCC 27539T10/108.2 ± 0.2 (6/6)5.0 ± 1.3 (6/6)4.9 ± 0.9 (6/6)8.2 ± 0.1 (6/6)7.5 ± 0.331 ± 823 ± 6
B. dentium ATCC 27534T3/106.7 ± 0.7* (6/6)4.0 (2/6)3.8 (2/6)8.1 ± 0.2 (6/6)6.9 ± 0.3*62 ± 13*51 ± 14*
B. infantis ATCC 15697T10/108.2 ± 0.3 (6/6)5.0 ± 0.7 (6/6)4.9 ± 0.8 (6/6)8.1 ± 0.2 (6/6)7.5 ± 0.231 ± 1024 ± 7
B. pseudocatenulatum ATCC 27919T5/106.8 ± 0.7* (6/6)4.0 ± 0.6 (3/6)4.0 ± 0.3 (3/6)8.1 ± 0.2 (6/6)6.9 ± 0.3*55 ± 12*47 ± 13*

In the appendix of mice in the bifidobacteria (4 strains)-treated group in which an effect of treatment on the infection was observed, abnormalities in the intestinal environment such as an increase in pH and changes in organic acid concentration (total organic acid and decrease in acetic acid concentration) in the infected control groups were largely improved (Table 4). On the other hand, in the intestines of bifidobacteria (2 strains)-treated mice in which no effect of treatment on the infection was observed, the above-mentioned improvements were not observed. Furthermore, in the GOS-treated group, the above-mentioned effect of treatment on the infection and improvements in the intestinal environment were not observed.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

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.

ACKNOWLEDGMENT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES

We would like to thank Akira Takahashi for valuable assistance in performing the animal experiments.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENT
  7. REFERENCES
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