Gerald C. O'Sullivan

In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings

The enteric flora comprises approximately 95% of the total number of cells in the human body and can elicit immune responses while protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract may also be implicated in the pathogenesis of diseases such as inflammatory bowel disease (ulcerative colitis and Crohn disease). The objectives of the Probiotic Research Group based at University College Cork were to isolate and identify lactic acid bacteria exhibiting beneficial probiotic traits, such as bile tolerance in the absence of deconjugation activity, acid resistance, adherence to host epithelial tissue, and in vitro antagonism of pathogenic microorganisms or those suspected of promoting inflammation. To isolate potentially effective probiotic bacteria, we screened the microbial population adhering to surgically resected segments of the gastrointestinal tract (the environment in which they may subsequently be reintroduced and required to function). In total, 1500 bacterial strains from resected human terminal ilea were assessed. From among these organisms, Lactobacillus salivarius subsp. salivarius strain UCC118 was selected for further study. In mouse feeding trials, milk-borne L. salivarius strain UCC118 could successfully colonize the murine gastrointestinal tract. A human feeding study conducted in 80 healthy volunteers showed that yogurt can be used as a vehicle for delivery of strain UCC118 to the human gastrointestinal tract with considerable efficacy in influencing gut flora and colonization. In summary, we developed criteria for in vitro selection of probiotic bacteria that may reflect certain in vivo effects on the host such as modulation of gastrointestinal tract microflora.

Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. Aims: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice. Methods: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 109 CFU/ml, B infantis 35624 at 108 CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers’ patches and splenocytes was measured in vitro by ELISA. Results: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers’ patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor β (TGF-β) levels were maintained. Conclusion: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-β are maintained.

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.

Probiotic impact on microbial flora, inflammation and tumour development in IL‐10 knockout mice

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.

Laparoscopic management of generalized peritonitis due to perforated colonic diverticula

PURPOSE The use of laparoscopic peritoneal lavage in conjunction with parenteral fluids and antibiotic therapy in the management of generalized peritonitis secondary to perforated diverticular disease of the colon was assessed. PATIENTS AND METHODS This cohort comprised 8 patients with generalized peritonitis secondary to perforated diverticular disease of the left colon that was diagnosed laparoscopically. All the patients had purulent peritonitis, but no fecal contamination. They were treated with laparoscopic peritoneal lavage and intravenous fluids and antibiotics. RESULTS All patients made a complete recovery, with resumption of normal diet within 5 to 8 days. No patient has required surgical intervention during a 12- to 48-month follow-up. This approach merits further assessment as an alternative to the traditional open surgical management.

Probiotic bacteria: myth or reality?

With the emergence of a more health-conscious society, the role of probiotic bacteria in human health has gained considerable attention from both the consumer and the producer. Considerable controversy has been associated with the lack of scientific evidence for the probiotic concept. However, current scientifically based research efforts worldwide are now focusing on the development of high-quality probiotic products containing microorganisms preselected for specific probiotic characteristics.

Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.

Culture‐independent analysis of the gut microbiota in colorectal cancer and polyposis

A role for the intestinal microbiota is routinely cited as a potential aetiological factor in colorectal cancer initiation and progression. As the majority of bacteria in the gut are refractory to culture we investigated this ecosystem in subjects with colorectal cancer and with adenomatous polyposis who are at high risk of developing colorectal cancer, using culture-independent methods. Twenty colorectal cancer and 20 polypectomized volunteers were chosen for this analysis. An exploration of the diversity and temporal stability of the dominant bacteria and several bacterial subgroups was undertaken using 16S rRNA gene denaturing gradient gel electrophoresis and ribosomal intergenic spacer analysis (RISA). Metabonomic analysis of the distal gut microbiotas environment was also undertaken. A significantly reduced temporal stability and increased diversity for the microbiota of subjects with colorectal cancer and polyposis was evident. A significantly increased diversity of the Clostridium leptum and C. coccoides subgroups was also noted for both disease groups. A clear division in the metabonome was observed for the colorectal cancer and polypectomized subjects compared with control volunteers. The intestinal microbiota and their metabolites are significantly altered in both colorectal cancer and polypectomized subjects compared with controls.

Bacterial DNA within Granulomas of Patients with Crohn's Disease—Detection by Laser Capture Microdissection and PCR

OBJECTIVES:We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohns disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohns disease.METHODS:Archival tissue from 15 surgical cases of Crohns disease and 10 non-Crohns granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene.RESULTS:E. coli DNA was detected in microdissected granulomas in 12/15 Crohns disease patients and in 1/10 non-Crohns control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohns full-thickness sections and in 4/10 control full-thickness sections.CONCLUSIONS:E. coli DNA may be detected more frequently in Crohns granulomas than in other non-Crohns bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohns granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohns disease.

Immune privilege or inflammation? Insights into the Fas ligand enigma

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.