Safety studies on probiotic strains Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, and Bifidobacterium lactis HN019 : a thesis submitted for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand

Content removed due to copyright restrictions: Appendix 5 - Zhou, J. S., Gopal, P. K., & Gill, H. S. (2001). Potential probiotic lactic acid bacteria lactobacillus rhamnosus (HN001), lactobacillus acidophilus (HN017) and bifidobacterium lactis (HN019) do not degrade gastric mucin in vitro. International Journal of Food Microbiology, 63(1-2), 81-90. Zhou, J. S., Shu, Q., Rutherfurd, K. J., Prasad, J., Gopal, P. K., & Gill, H. S. (2000). Acute oral toxicity and bacterial translocation studies on potentially probiotic strains of lactic acid bacteria. Food and Chemical Toxicology, 38(2-3), 153-161 Zhou, J. S., Shu, Q., Rutherfurd, K. J., Prasad, J., Birtles, M.J., Gopal, P. K., & Gill, H. S. (2000). Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HNOOl, Lb. acidophilus HNOI7, and Bifidobacterium lactis HN019 in BALB/c mice, International Journal of Food Microbiology, 56, 87-96. Shu, Q., Zhou, J. S., Rutherfurd, K. J., Prasad, J., Birtles, M.J., Gopal, P. K., & Gill, H. S. (2000). Probiotic lactic acid bacteria (Lactobacillus acidophilus HNOI7, Lb. rhamnosus HNOOl and Bifidobacterium lactis HNOI9) have no adverse effects on the health of mice, International Dairy Journal, 9,831-836Lactic acid bacteria (LAB) have been consumed in foods by human beings for several centuries without any obvious adverse effects. But the safety of consumption of these organisms, especially novel strains, which are added to foods as probiotics, has been questioned recently due to occasionally reported infections implicated with some particular LAB strains. Evaluation of the safety or potential toxicity of probiotic candidate strains, especially novel strains for which no prior safety data exist, is highly recommended. The LAB strains Lactobacillus rhamnosus HN001 (DR20TM), Lb, acidophilus HN017 and Bifidobacterium lactis HN019 (DR10TM) are three newly identified probiotic organisms with immune-enhancing properties. Their safety/potential toxicity was investigated in this study through a series of both in vitro and in vivo experiments. The mucus layer coating the surface of the gastrointestinal tract plays an important role in the gut mucosal defence system. Platelet activation and /or aggregation is a critical factor in the pathogenesis of infective endocarditis (IE). In the first part of this study, the potential pathogenicity of LAB strains was examined by in vitro mucin degradation (HN001, HN017, and HN019) and platelet aggregation (HN001 and HN019) assays. Following incubation with hog gastric mucin (HGM) in a minimal medium, the mucin degradation activity of test strains was determined via changes in the carbohydrate and protein concentration of the culture media and molecular weight changes of mucin glycoproteins (SDS-polyacrylamide gel electrophoresis, SDS-PAGE). The mucinolytic activity of test strains was also measured in an agarose petri dish assay. The results from these experiments suggested that HN001, HN017 and HN019 had no ability to degrade HGM in vitro. Flow cytometry analysis using platelet specific monoclonal antibodies demonstrated an inability of the test strains HN001 and HN019 to induce or enhance human platelet aggregation. These experiments indicated that the test strains are unlikely to degrade the mucin layer of the gastrointestinal mucosal surface or participate in the pathogenesis of endocarditis. Resistance of LAB strains to commonly used antibiotics has caused safety concerns regarding the genetic stability of these resistance properties. The antibiotic susceptibility and plasmid profiles of test organisms were investigated in another series of experiments. The susceptibility of the test strains to 18 antibiotics in common clinical use was examined by disk diffusion method. No extraordinary antimicrobial resistance was detected among the test strains (HN001, HN017, HN019, and HN067), and there were several antibiotics that efficiently suppressed the growth of test bacterial cells. A plasmid screening experiment demonstrated that all LAB strains examined were plasmid-free, this was verified by Southern blotting and DNA hybridisation techniques. These results indicate that the probiotic organisms tested here do not express or carry plasmid-associated antibiotic resistance, so their antibiotic resistance attributes are unlikely to disseminate to other clinically significant strains. To investigate the oral toxicity of test strains (HN001, HN017, and HN019), conventional BALB/c mice were inoculated with a high dose (10 11cfu/mouse/day) of the test probiotic LAB strains for 8 consecutive days. The feed and water intake, body weight gain, and general health status, of the mice were monitored. The potential translocation of inoculated LAB strains and gut mucosal histological changes following feeding were also investigated. Random amplified polymorphic DNA (RAPD) fingerprinting techniques were used for bacterial identification. Results showed that the test LAB strains had no adverse effects on the parameters observed; no viable bacteria were recovered from blood or tissue samples (mesenteric lymph nodes, liver, and spleen). These results suggest that the test strains had no acute toxicity and had no potential to result in infection in normal mice at the high dose applied in this study. To observe the consequences of longer-term consumption of test LAB strains, groups of BALB/c mice were orally administered with test LAB strains (HN001, HN017 and HN019) at doses of 5 x 10 7, 10 9 or 5 x 10 10 cfu/mouse/day for 4 weeks. In addition to the indicators observed in the acute toxicity study, the animals' haematological parameters; total and differential leucocyte counts; and blood biochemistry (plasma total protein, albumin, cholesterol, and glucose) were also investigated. Similar results to those of the acute toxicity study were obtained, i.e. 4 weeks consumption of HN001, HN017, and HN019 had no significant effects on the animals' general health status, haematology, blood biochemistry, or gut mucosal histological parameters. No dose-related effects were detected for any of the observed indicators. Translocation of test LAB strains was not observed. These results suggest that longer-term consumption of test strains is unlikely to cause any obvious health problems in host animals. In the final stage of this study, the potentially detrimental effects of HN001 and HN019 on hosts with sub-optimal immune functions were tested. To characterise the potential infectivity of test strains in immune deficient hosts, a group of adult male BALB/c mice pre-treated with dexamethasone (200µg/mouse/48 hrs) were fed with freshly cultured living HN001 or HN019 at doses of 1.5 ~ 2.5 x 10 7 cfu/mouse/day for 7 days; similar safety indicators to those outlined above were monitored. Results showed that no significant changes were noted in any of the safety parameters measured. No translocation of dietary LAB or systemic infection was detected. These findings suggest that HN001 and HN019 are well tolerated in immunocompromised mice without any significant safety concerns. To investigate the effects of consumption of test LAB strains in hosts with a preexisting immunological dysfuction, a group of female CBA/CaH mice (6 to 8 weeks) with experimentally induced autoimmune thyroiditis (EAT) were fed with freshly prepared probiotic preparations (HN001 4.2 x 10 8 cfu/mouse/day; HN019 2.16 x 10 8 cfu/mouse/day) for 5 to 8 weeks. Probiotic feeding was commenced one week prior to the immunization with auto antigens (MTg, mouse thyroglobulin). Antibody titres and spleen cell proliferative responses to the autoimmune inducing antigens (MTg) were determined via in vitro immunoassays. Lymphocyte (or mononuclear leucocyte) infiltration into thyroid tissue was also examined. Results showed that HN001 or HN019 feeding did not exacerbate spleen cell proliferative responses to MTg or lymphocyte infiltrations in thyroid tissues. These results indicate that feeding of HN001 or HN019 had no adverse effect on the induction or progress of autoimmune responses in CBA/CaH mice. Overall, the combined results from these studies suggest that the probiotic LAB strains HN001, HN017, and HN019 are non-pathogenic for experimental animals and are likely to be safe for human consumption