According to the definition by the World Health Organization (7
), a probiotic strain is “a live microorganism which, when administered in adequate amounts, confers a health benefit on the host.” Potential probiotic applications are numerous but often lack clinical practicalities as well as proper selection procedures that allow clinicians and microbiologists to safely promote unrestricted recommended use in severely ill or debilitated patients (39
). The aim of this study was to evaluate some aspects of the safety and functionality of three probiotic lactobacilli by using in vitro techniques and variations of the mouse model of TNBS-induced in vivo colitis. To this purpose, in our study, we have also included the nonprobiotic Lactobacillus paracasei
strain YS8866441, previously isolated from a patient with endocarditis.
We first compared the persistence of the LAB in the mouse GIT to that of L. plantarum
NCIMB8826 because its survival and persistence in mouse and human GITs was well documented (33
). Our results showed that LAB strains from different origins were able to persist for more than a week in the GIT of mice. All strains showed some resistance to the in vitro-tested conditions and adhered to Caco-2 cells, except L. paracasei
YS8866441, which was shown to persist in mice despite its in vitro sensitivity to bile salts and acid. These results indicate that in vitro tests cannot always be predictive of the in vivo behavior of strains. Similar observations have been made by other authors who showed that strains of lactobacilli, which have a documented ability to survive and reproduce in the human gut, scored poorly when challenged in vitro. Furthermore, when data obtained for different strains by in vitro experiments are compared, it appears that the lactobacilli of the Lactobacillus casei
group of species are the most sensitive (11
). In a second step, we have shown that oral administration of these LAB strains to healthy mice did not induce adverse effects or abnormal translocation of the bacteria administered.
We then compared the prophylactic capacity of LAB in a mouse model of TNBS-induced acute colitis and investigated the potential risk of bacterial translocation using the same mouse model. Although the relationship of the model of TNBS-induced colitis to human disease is imperfect, hapten-induced colitis displays Crohn's disease-like features, notably, transmural mononuclear inflammation, lymphocyte infiltration, and a Th1-dominated cytokine profile (30
). Ls-33 is the only strain that had significant anti-inflammatory effects under both conditions tested, while L. paracasei
YS8866441 exacerbated the inflammatory score under severe inflammatory conditions and translocated to extraintestinal organs. These results confirmed previous findings that probiotic activities are largely strain specific and that not all lactobacilli have similar effects on intestinal inflammation (9
). Other probiotics were shown to have anti-inflammatory effects on experimental colitis (20
), but very few studies have actually demonstrated strain-specific responses for different Lactobacillus
strains in the model of TNBS-induced colitis (10
). Our results also indicate that in vitro adherence to Caco-2 cells and in vivo persistence in the GIT do not correlate directly with anti-inflammatory effects.
It is likely that multiple properties contribute to the anti-inflammatory nature of a particular strain like L. salivarius
Ls-33 in the mouse model of TNBS-induced colitis used, but the exact mechanisms have yet to be established. The selection of Ls-33 was based on previous in vitro studies that showed its ability to induce high levels of anti-inflammatory cytokines together with low levels of proinflammatory cytokines after stimulation of immunocompetent cells (9
). The in vitro anti-inflammatory capacity seems to be closely correlated with its in vivo protection capacity, suggesting that Ls-33 could downregulate an early Th1 proinflammatory immune response evoked by the colonic instillation of TNBS. Inhibition of bacterial translocation and reinforcement of barrier function could also contribute indirectly to the observed anti-inflammatory properties of L. salivarius
Ls-33. Similar observations have been made by Llopis et al., who showed that L. casei
administration exerts a protective effect by preventing barrier disruption by TNBS, as translocation of bacteria to extraintestinal organs was reduced in rats colonized with L. casei
). It has also been shown that a mixture of probiotic bacteria, in addition to decreasing proinflammatory cytokines, reinforces barrier function by the secretion of soluble factors that enhance barrier integrity and by the regulation of tight junctions (23
). Two other L. salivarius
strains have been shown to have anti-inflammatory effects in two different models of experimental colitis (25
). Attenuation of colitis in both cases was associated with a reduced ability to produce proinflammatory cytokines at the mucosal level. Sheil et al. challenged the conventional hypothesis of probiotics by administering L. salivarius
UCC118 subcutaneously to interleukin-10 knockout mice (40
). The anti-inflammatory effect of subcutaneous administration was not specific, as it was also seen in a murine model of arthritis, suggesting that probiotics have more than a local anti-inflammatory effect (9
We have shown for the first time a detrimental effect of an L. paracasei
strain in extreme experimental conditions of intestinal inflammation. We needed these conditions of inflammation to exacerbate the risk of bacterial translocation and in which the safety of strains could be challenged. While these extreme conditions in mice may mimic the integrity of an impaired human intestinal barrier to some extent, these conditions are probably too severe to allow proper conclusions on the bacterial efficiency in colitis protection in mice and hence on the efficiency in humans. We previously established (10
) the optimal experimental settings by using doses varying from 100 to 125 mg/kg to accurately compare the various protective capacities of LAB strains. Using this standardized mouse model of TNBS-induced colitis, it has previously been shown that L. casei
BL23 and L. paracasei
IPL111 were able to reduce 69 and 48% of colon inflammation, respectively, compared to the nontreated TNBS control group (26
). These two strains were considered “protective,” in contrast to L. paracasei
strain YS8866441, illustrating that taxonomically related strains can have substantially different behaviors in response to TNBS-induced inflammation.
Our results have shown that Ls-33, NCFM, and Lp-115 have an acceptable safety profile, while certain risks may exist for YS8866441 under conditions of extensive mucosal damage. We selected L. paracasei
YS8866441 as a positive control in our bacterial translocation experiments out of 12 other L. paracasei
and Lactobacillus rhamnosus
strains isolated from endocarditis patients or patients with bacteremia (2
) (results not shown). Only L. paracasei
YS8866441 and another L. rhamnosus
strain were found to exacerbate intestinal inflammation and translocate to different extraintestinal organs under conditions of extensive mucosal damage, indicating that (i) LAB strains very rarely translocate actively to extraintestinal organs and (ii) further studies may be necessary to identify the parameters or properties that allow a LAB strain to cross the intestinal mucosal barrier and/or persist in the extraintestinal organs or circulation.
As more studies involving the administration of probiotics to critically ill patients are done, the safety of LAB should be evaluated under extreme conditions, and preferably, clinicians and microbiologists should give priority to strains with a proven safety profile, remaining vigilant concerning the use of nonscreened LAB in such patients (41
). This is especially important in light of recent demonstrations that under metabolic stress, enteric epithelia will react even towards members of the host's own commensal flora by increased interleukin-8 production, loss of barrier function, and increased translocation (29
). These host-related effects could further exacerbate severe intestinal inflammatory conditions, as observed with L. paracasei
YS8866441, and might lead to unexpected in vivo translocation towards extraintestinal organs and may hence explain its initial isolation from a critically ill patient (15
). The strain-related differences observed remain to be clarified.
Our results showed that an evaluation of the safety and functionality of new probiotics in healthy as well as in extreme conditions is recommended. The study supports the use of selected probiotic strains, such as L. salivarius Ls-33, as possible candidates in the prevention or treatment of intestinal mucosa inflammation, as it was the only strain that could significantly attenuate colitis and reduce the translocation of the endogenous flora. Our study thus provides new insights into the efficacy of Ls-33 in suppressing acute mucosal inflammation, and our efforts should now be directed to study the exact mechanisms explaining its strain-specific anti-inflammatory capacity.