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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Background:

The enteric bacterial flora has been implicated in the pathogenesis of enterocolitis and colon cancer in C57BL/6 IL-10 knockout mice. Probiotic Lactobacilli modify the enteric flora and are thought to have a beneficial effect on enterocolitis. We conducted a controlled feeding trial in IL-10 knockout mice using the probiotic Lactobacillus salivarius ssp. salivarius UCC118.

Aim:

To determine the effect of probiotic consumption on the gastrointestinal microflora, tumour development and colitis in IL-10 knockout mice.

Methods:

Twenty IL-10 knockout mice were studied (10 consumed probiotic organisms in milk and 10 consumed unmodified milk) for 16 weeks. Faecal microbial analysis was performed weekly to enumerate excretion of the probiotic UCC118, total lactobacilli, Clostridium perfringens, bacteroides, coliforms, bifidobacteria and enterococci. At sacrifice, the small and large bowel were microbiologically and histologically assessed.

Results:

L. salivarius UCC118 was detected in faeces from all mice in the probiotic fed group, but not the control group. Faecal coliform and enterococci levels were significantly reduced in probiotic fed animals compared to the controls (P < 0.05). At sacrifice, a significant reduction in C. perfringens numbers was observed in the test mice (P < 0.05). There were no fatalities in the test group compared to two deaths from fulminant colitis in the control group. Only one test mouse developed colonic adenocarcinoma compared to five in the control group. Test animal mucosal inflammation consistently scored lower than that of the control mice.

Conclusion:

In this placebo controlled trial, modification of enteric flora in IL-10 knockout mice by probiotic lactobacilli was associated with reduced prevalence of colon cancer and mucosal inflammatory activity.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aberrant immune responses to the indigenous microflora have been implicated in certain disease states, such as inflammatory bowel disease.1, 2 Antigens associated with the normal flora usually lead to immunological tolerance and failure to achieve this tolerance is a major mechanism of mucosal inflammation.3 Evidence for this breakdown in tolerance includes an increase in antibody levels directed against the gut flora in patients with inflammatory bowel disease.4 In addition, healthy human lamina propria mononuclear cells do not proliferate in response to bacterial sonicates from autologous intestine. However, lamina propria mononuclear cells isolated from the inflamed intestine of inflammatory bowel disease patients proliferate rapidly to bacterial sonicates.5 Furthermore, certain murine models predisposed to inflammatory lesions of the gastrointestinal tract remain disease-free when housed in germ-free conditions or when treated with antibiotics.6[7]–8 One such murine model is the C57BL/6 interleukin-10 knockout mouse which is predisposed to developing enterocolitis in the presence of an enteric bacterial flora, but remains disease-free when maintained under germ-free conditions.6

Not all members of the gastrointestinal flora are considered proinflammatory. Mice mono-associated with Bacteroides vulgatus developed colitis, whereas mono-association with E. coli has no effect.9 Probiotic bacteria are non-pathogenic lactic acid bacteria, which have been an integral component of the human diet for centuries. Probiotic bacteria have been used as bio-therapeutic agents in the treatment of gastrointestinal disorders for almost a century.10 However, there is a lack of convincing in vivo evidence for the use of probiotic bacteria in inflammatory models. Exceptions include a non-placebo-controlled trial using a probiotic cocktail, which had considerable efficacy in the maintenance of remission in patients with pouchitis, and a lactobacillus species that attenuated inflammation in an IL-10 knockout murine model.11, 12 However, different probiotic bacteria have different effects and claims made for one probiotic strain cannot readily be applied to another.13 Thus, probiotic bacterial strains must be evaluated individually.

One such probiotic organism, Lactobacillus salivarius ssp. salivarius UCC433118 (UCC118), was originally isolated from a healthy human gastrointestinal tract. This bacterium was demonstrated to be bile and acid resistant, and survives passage through the gastrointestinal tract.14 UCC118 is non-pathogenic and following consumption in humans, consistent modulation of the gut flora was noted.15 Because the pathogenesis of gastrointestinal inflammatory diseases has been linked with the enteric flora, elimination of specific components of this flora may have a beneficial effect on disease severity. Thus, the primary objective of this study was to assess the ability of Lactobacillus salivarius ssp. salivarius UCC433118 in modulating disease activity of enterocolitis in the IL-10 knockout mice in a controlled pilot study.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

IL-10 knockout mice

Ten C57BL/6 J-IL10 and 10 C57BL/10 J-IL10 knockout male mice, aged 4–8 weeks, were used in this study (Jackson Laboratories, Maine, USA). These mice were maintained on a homozygous background and were housed under specific pathogen-free conditions. Mice were housed in a 24-h light/dark cycle. Following initiation of this study, all mice consumed a standard non-sterile diet.

Probiotic strain

Lactobacillus salivarius ssp. salivarius UCC118 (NCIMB 40829) was originally isolated from the ileal–caecal region of a human adult undergoing reconstructive surgery. UCC118 was isolated on the basis of having desirable probiotic properties.14, 16 Briefly, these properties included: being of human origin; being non-pathogenic; being resistant to intestinal acid and bile; having the ability to adhere to human epithelial cells; and being able to temporarily colonize and be metabolically active with the human gastrointestinal tract. The probiotic UCC118 was routinely cultured in MRS broth at 37 °C in an anaerobic environment for 24 h. A spontaneous rifampicin-resistant variant of UCC118 was isolated, prior to initiation of this study, in order to facilitate uncomplicated identification of this bacterium from all other lactobacilli.

Feeding trial

The 20 IL-10 knockout mice were randomized to one of two groups. The probiotic-fed group consumed 1 × 109Lactobacillus salivarius ssp. salivarius UCC118 per day in pasteurized milk, while the control group consumed unmodified pasteurized milk only. The probiotic UCC118 was initially grown to a 10-mL volume in de Man, Rogosa, Sharpe (MRS) broth (Oxoid, UK) by incubating overnight at 37 °C under anaerobic conditions. A 1% inoculum (v/v) was transferred to 400 mL of fresh MRS broth and incubated as before. UCC118 was pelleted by centrifugation and re-suspended at 1 × 109 cells/mL in 10% pasteurized skimmed milk. UCC118 was administered to the IL-10 knockout mice in water bottles and the mice consumed this mix ad libitum.

Murine faecal samples were collected prior to feeding (week 0) and at weekly intervals over the trial period. The trial was completed after 16 weeks of feeding, at which time all surviving mice were sacrificed by cervical dislocation. Blood samples were obtained from all mice, by cardiac puncture, for serological analysis. Intraluminal samples were removed under sterile conditions from the ileum, caecum and colon for microbiological analysis, while tissue segments of the ileum, caecum and colon were fixed in formaldehyde for histopathological analysis.

Serology

Serum levels of UCC118 specific antibodies were measured using a standard agglutination assay. Briefly, serum from IL-10 knockout mice were diluted 1/10, 1/20, 1/40, 1/80 and 1/160 with sterile phosphate buffer saline. Sera was incubated overnight with the probiotic UCC118 and examined for evidence of agglutination. The reciprocal of the lowest dilution to result in agglutination was used as a measure of antibody levels specific to UCC118.

Microbial analysis

Faecal samples were collected weekly, weighed and dispersed in 10 mL of phosphate buffer saline. Microbial analysis of the faecal samples involved the enumeration of Lactobacillus salivarius ssp. salivarius UCC118, total Lactobacilli, Bifidobacteria, Enterococci, Bacteroides and Coliforms. This analysis was performed by pour plating or spread plating onto MRS agar plus rifampicin, MRS agar, MRS agar supplemented with 5% sheep blood, 0.2% LiCl2, 0.3% Na Propionate and 0.05% cysteine, Slanetz and Bartley agar, Wilkins Chalgren agar supplemented with supplement SR108 and 5% horse blood, and violet red blood agar (VRBA), respectively (Oxoid, UK). In addition to the faecal specimens, luminal samples from the ileum, caecum and colon were assessed for the presence of the same bacteria, as outlined above, plus Clostridium perfringens, as measured by OPSP agar supplemented with supplements A SR76 and B SR77 (Oxoid, UK). VRBA and Slanetz and Bartley agar were incubated aerobically for 24 h and 48 h, respectively, while all other plates were incubated anaerobically for 48 h at 37 °C. Anaerobic environments were created using CO2 generating kits (Anaerocult A, Merck) in sealed gas jars (BBL).

Histopathology

At sacrifice, segments of the small intestine, caecum and colon were fixed in 10% formalin and assessed histologically. Two blinded independent observers, using a histological index ranging from 0 to 5, graded the severity of damage due to inflammatory activity within the murine gastrointestinal tract. This index was based on the degree of epithelial layer erosion, goblet cell depletion and inflammatory cell infiltrate. In addition, these tissues were inspected for the presence of neoplastic cells.

Statistical analysis

Analysis of variance, for differences between groups in inflammatory activity, was carried out using ANOVA analysis, while differences between groups in microbial numbers were estimated using area under the curve analysis. The Fischer exact test was used to determine the statistical differences in tumour development and murine mortality between the probiotic-fed and control groups.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Serology

Following consumption of the probiotic bacterium UCC118 for 16 weeks, peripheral blood antibody levels, specific to this bacterium, were quantified. There was no difference in antibody levels for the mice consuming UCC118 compared to mice consuming the placebo product alone (10.1 + 4.1 vs. 9.7 + 2.5, respectively). This suggests that UCC118 was not perceived systemically by the immune system of the IL-10 knockout mice.

Microbiology

The probiotic UCC118 was detected in faeces from all mice in the probiotic group within 1 week of feeding. During the 16 weeks of feeding, UCC118 was recovered at approximately 1 × 106 bacteria per gram of faeces (Figure 1). UCC118 was not cultivated from any of the mice in the placebo group. Faecal coliform and enterococci levels were decreased in mice consuming UCC118, compared to mice consuming pasteurized milk only (Figure 2). Total lactobacilli, Bifidobacteria and Bacteroides levels remained unchanged between the two groups (Figure 2).

image

Figure 1. Faecal detection of the probiotic UCC118. The probiotic L. salivarius ssp. salivarius UCC118 was consumed daily for 16 weeks and faecal excretion of this bacterium averaged 1 × 106 cells per gram of faecal matter. Thus, UCC11. 8 survives passage through the murine gastrointestinal tract in high numbers. Results are presented as mean colony forming units (cfu) per gram (g) ± standard error for each week of feeding.

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image

Figure 2.  Microbiological assessment of the faecal flora. Faecal enterococci, coliform, lactobacilli, bifidobacteria and bacteroides levels were measured weekly over the trial period. Enterococci and coliform levels were significantly lower in the probiotic-fed mice, compared to the placebo group (P < 0.05). No differences were noted in bacteroides, lactobacilli and bifidobacteria numbers between the two groups. Results are expressed as the mean cfu/g of faeces per group for each week of the study.

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In addition to weekly microbial analysis, lumen samples from the ileum, caecum and colon were extracted post-sacrifice. C. perfringens numbers were significantly decreased from mice consuming UCC118, particularly within the colon (P < 0.05), compared to mice consuming the placebo product alone (Figure 3). There were no significant differences noted for total lactobacilli, Bifidobacteria, coliforms, enterococci or Bacteroides numbers.

image

Figure 3.  Intraluminal C. perfringens levels in the probiotic and placebo groups. Following 16 weeks of feeding, all mice were sacrificed and their gastrointestinal contents were removed aseptically. C. perfringens was significantly reduced in the mice consuming UCC118 compared to the placebo group, particularly from within the colon (P < 0.05). Results are plotted as the mean log values ± standard error for each of the groups.

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Histopathology

Post sacrifice, inflammatory activity of the caecum, ileum and colon from both groups of mice was graded (Figure 4). There was a strong trend towards reduced inflammatory activity in all sites but this did not reach conventional statistical significance (Table 1). The most substantial difference in inflammatory activity between the two groups was seen in the colon (P=0.09). There was a substantial reduction in the development of cancer between the two groups. Gastrointestinal neoplastic transformation was evident in 50% of mice from the placebo group, while only 10% of the UCC118 group demonstrated any evidence of neoplastic change (P=0.07). Furthermore, 20% of the placebo group died before the conclusion of the trial, while all mice in the UCC118 group survived.

image

Figure 4.  Histological sections from IL-10 knockout mice. Representative gastrointestinal histological sections from IL-10 knockout mice are illustrated. (A) Normal mucosa from an IL-10 knockout mouse consuming the probiotic UCC118. (B) Severe gastrointestinal inflammation from an IL-10 knockout mouse in the placebo group. (C) High grade dysplasia of the colon. (D) Early invasive adenocarcinoma of the colon.

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Table 1.   Gastrointestinal inflammatory scores. Inflammatory scores for the ileum, caecum and colon were compared for mice consuming the probiotic UCC118 and placebo product. Mice consuming UCC118 consistently scored lower than the placebo group at all three sites, particularly in the colon (P = 0.09). Indeed, when the scores of all three sites are combined, inflammatory activity is considerably reduced in the gastrointestinal tracts of probiotic fed mice (P = 0.06). Results are expressed as mean ± standard error Thumbnail image of

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The results of this study demonstrate survival of the probiotic Lactobacillus salivarius ssp. salivarius UCC118 following gastrointestinal transit and a significant probiotic-associated alteration of the gastrointestinal flora. Consumption of the probiotic was associated with a trend towards reduced tumour development and attenuation of gastrointestinal inflammation. These results warrant a large-scale probiotic study in order to reliably assess the statistical significance of these observations.

A number of mechanisms are proposed for the modulation of the gastrointestinal flora by this probiotic lactobacillus. UCC118 produces an antimicrobial factor in vitro that is antagonistic to a wide range of Gram-positive and Gram-negative microorganisms.16 Production of this antimicrobial factor in vivo could eliminate competing organisms conferring a survival advantage to the newly ingested bacterium. Furthermore, UCC118 is strongly adherent to gastrointestinal epithelial cells in vitro.17 Thus, the competitive exclusion of other microorganisms from this niche environment would affect the composition of the faecal flora.

Consumption of UCC118 attenuates gastrointestinal inflammation in this murine model. Colonization of the gastrointestinal tract by this probiotic bacterium also results in modification of the gut flora, possibly eliminating proinflammatory species. Thus, removal of the inflammatory insult is one potential mechanism by which probiotic bacteria may modulate disease severity. In addition, probiotic bacteria may have a more direct influence on inflammatory responses within the gastrointestinal tract via interaction with the mucosal immune system.18, 19 The correct balance of Th1/Th2 responses in the gut is critical in maintaining intestinal integrity.20 Consumption, in high numbers, of certain bacterial species may help restore the correct Th1/Th2 balance in IL-10 knockout mice.21 Consumption of UCC118 has previously been shown to induce the production of sIgA antibodies, demonstrating interaction of this strain with the mucosal immune system.19 Thus, direct interaction between UCC118 and the mucosal immune system may induce Th2 type responses resulting in a restoration of the Th1/Th2 balance and attenuation of inflammation in this model.

In addition, we examined the role of the probiotic UCC118 in reducing the rate of neoplastic change within the gastrointestinal tract. Inflammation within the gastrointestinal tract profoundly influences mucosal integrity and its ability to resist injury induced by luminal factors, thus increasing the risk of developing neoplastic disease. Additionally, certain inflammatory mediators can promote the growth of gastrointestinal tumour cells.22, 23 The enteric bacterial flora themselves have been implicated as contributing factors to the pathogenesis of gastrointestinal malignancies. Germ-free rats treated with the carcinogen 1,2-dimethylhydrazine have a lower incidence of colon tumours than similarly treated rats with a normal microflora.24 Intestinal bacteria can produce, from dietary compounds, substances with genotoxic, carcinogenic and tumour-promoting activity, and gut bacteria can activate pro-carcinogens to DNA-reactive agents.25 In general, lactic acid bacteria have not been implicated as causative factors in these disease states and may in fact antagonize harmful elements of the gastrointestinal flora.26[27]–28 Thus, the UCC118 probiotic could delay the development of gastrointestinal malignancies by attenuating inflammatory activity within the gastrointestinal tract, or by modification of the bacterial flora.

Certain probiotic bacteria are attractive bio-therapeutic agents for the treatment of gastrointestinal inflammation due to their effects on the composition of the gut flora and activity of the immune system.29 Consumption of the probiotic UCC118 successfully modified the gastrointestinal flora, attenuated inflammation and reduced neoplastic lesions in this mouse model. While considerable caution must be exercised in extrapolating data from animal experiments, further evaluation of the therapeutic role of probiotic bacteria seems appropriate. Perhaps our most intriguing and clinically relevant finding was the reduced prevalence of colonic carcinoma in probiotic-fed animals. In this respect, the reported beneficial effects of probiotics in human ulcerative colitis may have special application to long-term reduction of dysplasia/neoplasia in such patients.30, 31

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The Health Research Board and HEA, Ireland, and the European Union provided financial support for this study.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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