Lack of correlation between in vitro antibiosis and in vivo protection against enteropathogenic bacteria by probiotic lactobacilli

Increased resistance to infection is one of the beneficial effects attributed to probiotic microorganisms. This effect may be due to several mechanisms: production of inhibitory substances, blocking of adhesion sites on the intestinal surface, competition for nutrients and stimulation of mucosal and systemic immunity. The present study aimed to investigate the correlation between in vitro and in vivo antimicrobial activity of probiotic lactobacilli. The agar spot test was used to show that twenty Lactobacillus strains were able to inhibit the enteropathogenic bacterium Yersinia enterocolitica. This inhibition was mainly attributable to a decrease in pH resulting from dextrose fermentation by lactobacilli. The inhibition of Y. enterocolitica, Salmonella enterica serovar Typhimurium and Listeria monocytogenes by two probiotic strains, Lactobacillus casei C1 and Lactobacillus plantarum C4, was also associated with the pH decrease. However, both strains lacked protective effects in mouse experimental infection models, with the exception of long-lasting pre-treatment with L. plantarum C4, which exerted a partial protective effect against S. Typhimurium that was attributable to an immunostimulatory mechanism. Our results show that in vitro antibiosis tests do not provide useful information on the probiotic potential of Lactobacillus strains.     

Anti-pathogenic and probiotic attributes of Lactobacillus salivarius and Lactobacillus plantarum strains isolated from feces of Algerian infants and adults

Sixty-seven (67) lactic acid bacteria (LAB) isolates belonging to Lactobacillus genus were isolated from human feces and tested for their auto-aggregation and cell surface hydrophobicity in order to establish their adhesion capabilities, a prerequisite for probiotic selection. Strains with the upmost auto-aggregation and cell surface hydrophobicity scores were identified by MALDI-TOF spectrometry and 16S rDNA sequencing as Lactobacillus plantarum (p25lb1 and p98lb1) and Lactobacillus salivarius (p85lb1 and p104lb1). These strains were also able to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells, with percentages ranging from 4.68 to 9.59%. They displayed good survival under conditions mimicking the gastrointestinal environment and remarkably impeded adhesion and invasion of human Caco-2 by Listeria monocytogenes and Enteropathogenic Escherichia coli. It should also be noted that Lb. plantarum p98lb1 was able to reduce in vitro cholesterol concentration by about 32%, offering an additional health attribute.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Developmental changes in intracellular pH buffering power in smooth muscle

 Intracellular pH (pH_i) is known to modulate contraction. Neonatal tissues can differ from adult tissue in contractile response to stimuli known to alter pH_i e.g. hypoxia. Changes of pH are attenuated by buffering, thus any difference in buffering power (β) between tissues could affect their functional response to pH_i perturbation. Similarly the extent to which any extracellular pH (pH_o) alteration is transmitted into a pH_i change will also influence function. We have therefore determined the intrinsic β and effect of pH_o change on pH_i in neonatal and adult ureteric, uterine and gastric smooth muscles using the pH-sensitive fluorophore carboxy-SNARF. β was found to be similar in the three adult tissues, but there were significant differences between neonatal tissues. In contrast, we found little difference in the amount of pH_i change produced by pH_o change between neonatal and adult tissues from the same smooth muscle, but a difference between smooth muscles. These data highlight significant differences between smooth muscles and their developmental state, which may contribute to different degrees of protection when pH is perturbed. Received: 17 October 1997 / Received after revision: 27 November 1997 / Accepted: 28 November 1997     

Immunomodulatory Effects of Lactobacillus salivarius LS01 and Bifidobacterium breve BR03, Alone and in Combination,on Peripheral Blood Mononuclear Cells of Allergic Asthmatics

The aim of this study was to evaluate probiotic characteristics of Lactobacillus salivarius LS01 and Bifidobacterium breve BR03 alone and in combination and their immunomodulatory activity in asthmatic subjects. Subjects affected by allergic asthma were recruited. Initially, LS01 and BR03 were analyzed for their growth compatibility by a broth compatibility assay. To study the antimicrobial activity of probiotic strains, an agar diffusion assay was performed. Finally, cytokine production by peripheral blood mononuclear cells (PBMCs) stimulated with LS01 and BR03 was determined by means of specific quantitative enzyme-linked immunosorbent assay (ELISA). The growth of some clinical pathogens were slightly inhibited by LS01 and LS01-BR03 co-culture supernatant not neutralized to pH 6.5, while only the growth of E. coli and S. aureus was inhibited by the supernatant of LS01 and LS01-BR03 neutralized to pH 6.5. Furthermore, LS01 and BR03 combination was able to decrease the secretion of proinflammatory cytokines by PBMCs, leading to an intense increase in IL-10 production. L. salivarius LS01 and B. breve BR03 showed promising probiotic properties and beneficial immunomodulatory activity that are increased when the 2 strains are used in combination in the same formulation.     

Ventral medulla pHi measured in vivo by 31P NMR is not regulated during hypercapnia in anesthetized rat

Chemoreceptors in the ventral medulla contribute to the respiratory response to hypercapnia. Do they ‘sense’ intracellular pH (pH_i)? We measured pH_i in the ventral medulla or cortex (control) using ³¹P-NMR obtained via a novel 3×5 mm² surface coil in anesthetized rats breathing air or 7% CO₂. During air breathing over 240 min, pH_i decreased slightly from 7.13±0.02 to 7.05±0.02 (SEM; n=5; 2 cortex, 3 ventral medulla). During 180 min of hypercapnia, cortical pH_i (n=4) decreased from 7.17±0.02 to 6.87±0.01 by 90 min and recovered by 150 min. Ventral medulla pH_i showed no such regulation. It decreased from 7.11±0.02 to 6.88±0.02 at 90 min and recovered only after cessation of hypercapnia (n=5), results consistent with pH_i being the chemoreceptor stimulus. However, non-chemoreceptor neurons that contribute to our medullary NMR signal also do not appear to regulate pH_i in vitro. Regional differences in pH_i regulation between cortex and ventral medulla may be due to both chemosensitive and non-chemosensitive neurons.     

An investigation of intrinsic buffering power in rat vascular smooth muscle cells

Intrinsic buffering power ( _i) has been measured in vascular strips and single cells from rat mesenteric artery. Intracellular pH (pH_i) regulation was inhibited to prevent overestimation of _i due to acid extrusion or entry via regulatory processes. At resting values of pH_i (7.0–7.2), a mean value of 41±4 mM/pH unit for _i was found. _i increased approximately fivefold from 30 to 150 mM/pH unit over the pH_i range 7.5–6.5. The mean data relating _i to pH_i could be described by relating _i to buffer concentrations and pK _a. This gave a value of 310 mM for buffer concentration and a pK _a of 6.0. As changes in pH_i are known to have marked effects on vascular tone then the increase in _i as pH_i falls may be considered as a means of attenuating pH_i decreases, before pH regulation restores pH_i to resting levels.     

Roles of pH in control of cell proliferation

Precise spatiotemporal regulation of intracellular pH (pH_i) is a prerequisite for normal cell function, and changes in pH_i or pericellular pH (pH_e) exert important signalling functions. It is well established that proliferation of mammalian cells is dependent on a permissive pH_i in the slightly alkaline range (7.0‐7.2). It is also clear that mitogen signalling in nominal absence of is associated with an intracellular alkalinization (~0.3 pH unit above steady‐state pH_i), which is secondary to activation of Na⁺/H⁺ exchange. However, it remains controversial whether this increase in pH_i is part of the mitogenic signal cascade leading to cell cycle entry and progression, and whether it is relevant under physiological conditions. Furthermore, essentially all studies of pH_i in mammalian cell proliferation have focused on the mitogen‐induced G0‐G1 transition, and the regulation and roles of pH_i during the cell cycle remain poorly understood. The aim of this review is to summarize and critically discuss the possible roles of pH_i and pH_e in cell cycle progression. While the focus is on the mammalian cell cycle, important insights from studies in lower eukaryotes are also discussed. We summarize current evidence of links between cell cycle progression and pH_i and discuss possible pH_i‐ and pH_e sensors and signalling pathways relevant to mammalian proliferation control. The possibility that changes in pH_i during cell cycle progression may be an integral part of the checkpoint control machinery is explored. Finally, we discuss the relevance of links between pH and proliferation in the context of the perturbed pH homoeostasis and acidic microenvironment of solid tumours.     

The effect of neuronal morphology and membrane-permeant weak acid and base on the dissipation of depolarization-induced pH gradients in snail neurons

Neuronal depolarization causes larger intracellular pH (pH_i) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pH_i measurements using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pH_i microdomains. In the absence of added mobile buffers, facilitated H⁺ diffusion down the length of the axon plays a critical role in determining pH_i microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membrane-permeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pH_i dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pH_i signals without perturbing steady-state pH_i. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pH_i are likely to be dominated by local pH_i-regulating mechanisms rather than simply following the soma pH_i.     

Differential effects of external pH alteration on intracellular pH in rat coronary and cardiac myocytes

Changes in extracellular pH (pH_o) induce changes in the intracellular pH (pH_i) of cardiac myocytes that are slow and attenuated. Little however is known about the effects of changing pH_o on the pH_i of the coronary smooth muscle cells. We have therefore directly compared the effects of altering pH_o on pH_i of both coronary and cardiac myocytes. Carboxy-SNARF was used in single cells to measure pH_i. Alteration of pH_o caused corresponding changes in pH_i that were large (70–80 % of pH_o) and rapid in coronary myocytes compared to cardiac myocytes. In contrast, changes of pH_i produced by weak acids or bases produced similar pH_i responses in both types of cells. It is suggested that the differential effects of pH_o on coronary and cardiac cells may be functionally significant, as it will allow rapid alteration of coronary perfusion to meet tissue needs, while maintaining cardiac output.Supported by the BHF and MRC     

Expression of the atpD gene in probiotic Lactobacillus plantarum strains under in vitro acidic conditions using RT-qPCR

F₁F₀-ATPase has been identified as an operon directly involved in the tolerance of probiotic bacteria towards a hostile acidic environment encountered in the stomach. Expression of atpD (a key part of the F₁F₀-ATPase operon) gene of the two putative probiotic Lactobacillus plantarum isolates (Lp9 and Lp91) under different in vitro pH conditions which closely mimic the physiological environment prevalent in the human gut was investigated by quantitative real-time PCR (RT-qPCR). A battery of housekeeping genes, i.e. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA, were evaluated using geNorm 3.4 Excel-based application for normalizing atpD gene expression in Lp9 and Lp91. The most stably expressed genes were found to be gapB, gyrA and ldhD. Although both putative probiotic L. plantarum isolates investigated in this study were able to survive acid stress under in vitro conditions, amongst the two, Lp91 exhibited relatively greater acid tolerance, as revealed by 4.7-fold upregulation of the atpD gene as well as higher log counts at pH 2.5 after 90 min These results clearly demonstrate that expression of the ‘atp’ operon was chiefly instrumental in in vitro survival and tolerance of test cultures at acidic conditions encountered in the stomach.     

Genetic ablation of carbonic anhydrase IX disrupts gastric barrier function via claudin‐18 downregulation and acid backflux

Aim

This study aimed to explore the molecular mechanisms for the parietal cell loss and fundic hyperplasia observed in gastric mucosa of mice lacking the carbonic anhydrase 9 (CAIX).

Methods

We assessed the ability of CAIX‐knockout and WT gastric surface epithelial cells to withstand a luminal acid load by measuring the pH_i of exteriorized gastric mucosa in vivo using two‐photon confocal laser scanning microscopy. Cytokines and claudin‐18A2 expression was analysed by RT‐PCR.

Results

CAIX‐knockout gastric surface epithelial cells showed significantly faster pH_i decline after luminal acid load compared to WT. Increased gastric mucosal IL‐1β and iNOS, but decreased claudin‐18A2 expression (which confer acid resistance) was observed shortly after weaning, prior to the loss of parietal and chief cells. At birth, neither inflammatory cytokines nor claudin‐18 expression were altered between CAIX and WT gastric mucosa. The gradual loss of acid secretory capacity was paralleled by an increase in serum gastrin, IL‐11 and foveolar hyperplasia. Mild chronic proton pump inhibition from the time of weaning did not prevent the claudin‐18 decrease nor the increase in inflammatory markers at 1 month of age, except for IL‐1β. However, the treatment reduced the parietal cell loss in CAIX‐KO mice in the subsequent months.

Conclusions

We propose that CAIX converts protons that either backflux or are extruded from the cells rapidly to CO₂ and H₂O, contributing to tight junction protection and gastric epithelial pH_i regulation. Lack of CAIX results in persistent acid backflux via claudin‐18 downregulation, causing loss of parietal cells, hypergastrinaemia and foveolar hyperplasia.     

pH aspects of transient changes in conduction velocity in isolated heart fibers after partial replacement of chloride with organic anions

Conduction velocity in isolated rabbit atrial fibers was continuously measured in solutions having a different anionic composition. When 20 mmol/l of chloride was replaced by 20 mmol/l lactate or other anions of weak organic acids at constant pH 6.8, biphasic initial transient changes in conduction velocity were observed. The produced transient changes had a greater amplitude with organic acids which have a greater pK and lipid/water partition ratio. The magnitude of the transients was also greater at pH 6.8 than at pH 7.5, and also when the buffering capacity of the superfusion solution was smaller. Measurements of intracellular pH (pH_i) in sheep Purkinje fibers and of pH at the surface (pH_s) of sheep Purkinje and rabbit atrial fibers with pH sensitive microelectrodes, showed a transient increase of pH_s and a sustained decrease of pH_i on replacement of 20 mmol/l chloride by organic anions of weak acids (at constant pH of the superfusion solution). A combined influence of the transient pH_s change and the sustained pH_i modification seems to be important in the explanation of the biphasic changes in conduction velocity.     

In Vivo Detection of Rat Colorectal Cancers by using a Dual-Wavelength Excitation Method

Hypoxia is a characteristic feature of solid neoplasms, and insufficient oxygen supply increases cellular nicotinamide adenine dinucleotide (NADH) fluorescence, which is a main component of autofluorescence of the colorectal mucosa. We investigated whether a dual-wavelength excitation method which is optimized for sensing mucosal NADH fluorescence could be applicable to the detection of rat colorectal cancers in vivo. Rat colorectal adenocarcinomas were studied by using fluorescence stereomicroscopy. After autofluorescence images at 470 nm irradiated with dual-wavelength excitation at 365 nm (F_365ex) and 405 nm (F_405ex) were acquired, ratio images were produced by dividing F_365ex by F_405ex: The excitation-emission wavelength pairs in F_365ex and F_405ex were adjusted for acquisition of NADH fluorescence and reference fluorescence. Based on observations from the luminal surface in vivo, F_365ex/F_405ex ratio images indicated a 1.57-fold higher signal value in the cancers than in the surrounding normal mucosa. The signal values in F_365ex/F_405ex ratio images were less mutually related with the hemoglobin concentration index. Small adenocarcinomas (less than 4 mm) could be detected on F_365ex/F_405ex ratio images. The results showed that NADH fluorescence measurement with little interference from tissue hemoglobin is efficient for visualizing rat colorectal cancers in vivo, suggesting that the dual-wavelength excitation method has potential for label-free endoscopic detection of diminutive colorectal neoplasms.     

Histidine 518 in the S6-CNBD linker controls pH dependence and gating of HCN channel from sea-urchin sperm

Sperm motility is a tightly regulated process. One of the crucial factors determining the swimming of the sea-urchin sperm is an elevation of intracellular pH (pH_i). The possibility that its hyperpolarisation-activated cyclic nucleotide-gated channel (SpHCN) is modulated directly by pH is addressed here. Site-directed mutagenesis showed that histidine 518 from the linker connecting the S6 helix with the cyclic nucleotide binding domain is responsible for the pH modulation of current kinetics and voltage dependence of activation. The effect of mutating histidine 518 to serine (H518S) on the time constant of activation was maximal at pH 6.4: 180±20 ms in the wild-type (wt) but only 56±10 ms in the H518S mutant channel. Furthermore, histidine 518 accounted for 31% of the shift in the voltage of half activation (V _1/2) in wt following a pH change from 6.4 to 8.4. The mutation H518S also shifted V _1/2 by 19 mV at pH 7.4 (–50.2±0.2 and –69±2 mV for H518S and wt, respectively). This indicates that histidine 518 couples voltage sensing to gating. The wt and H518S channels had a different affinity for cyclic adenosine monophosphate (cAMP) (IC₅₀ 1.0±0.02 and 2.5±0.06 µM, respectively). Changes in pH_i also modulated channel selectivity.     

A novel method for absolute calibration of intracellular pH indicators

In this paper we present methods to measure intracellular pH (pH_i) with fluorescent indicators. These methods are based on the change in intracellular pH following the addition of weak acids and weak bases to the extracellular medium. The first method requires that the fluorescence of the indicator is proportional to the change in pH_i that follows the addition of a weak acid or weak base to the extracellular medium. The second is a null method which uses a mixture of weak acid and weak base that does not change the fluorescent signal. This null method can be used in situations in which the fluorescent signal is a monotonic but non-linear function of pH. The first method depends upon four assumptions. (i) That only the uncharged forms of the weak acids and bases cross the surface membrane. (ii) That the pK_a is the same inside and outside the cell. (iii) That the buffering power is constant. (iv) That there is no significant pH regulation on the time scale of the change in pH_i. The null method only requires the first two assumptions. We have made estimates of pH_i in four different cell types and compared the results obtained with these methods with those obtained from other methods of pH_i calibration.     

pH sensitivity of the cardiac gap junction proteins,connexin 45 and 43

Intercellular communication through gap junction channels can be regulated by changes in intracellular pH (pH_i). This regulation may play an important role in ischemic heart tissue. Using the dual voltage-clamp technique, we compared the pH_i sensitivity of gap junction channels composed of connexin 43 (Cx43) and Cx45, two of the gap junction proteins that are expressed in heart. We made use of SKHep1 cells, endogenously expressing low levels of Cx45 and SKHep1 cells stably transfected with rat Cx43. To manipulate the pH_i we applied the NH₃/NH₄ ⁺ pH-clamp method. At pH_i 6.7 the g_j of Cx45 channels was reduced to 20% of control values (pH_i 7.0) and at pH_i 6.3 all channels closed. The g_j of Cx43 channels was 70% of control values at pH_i 6.7 and 40% at pH_i 6.3. Cx43 channels closed at pH_i 5.8. Single channel conductances were 17.8 pS for Cx45 and 40.8 pS for Cx43 at pH_i 7.0 and did not change significantly at lower pH_i. This suggests that the decrease in macroscopic conductance observed at low pH_i results from the decrease in open probability of gap junctional channels rather than from a decrease in single channel conductance. Our results demonstrate that gap junction channels built of Cx45 are far more pH sensitive than channels built of Cx43.     

Simultaneous measurement of intracellular pH and contraction in uterine smooth muscle

Changes in intracellular pH (pH_i) are thought to produce large changes in force production in the uterus. There have however, been no simultaneous measurements of pH_i and force in the uterus and therefore no direct information is available about the relation between the two. We have used carboxy-SNARF (a pH-sensitive fluorophore) in small strips of longitudinal myometrium and obtained simultaneous measurements of pH_i and force. SNARF did not alter contractile function, and continuous measurements of pH_i could be made for 2 hours. The mean resting pH_i (7.16) was similar to that reported previously. Application of weak bases rapidly raised pH_i, in a concentration-dependent manner, followed by a gradual restoration of pH_i to resting levels. Alkalinization greatly increased the frequency of contractions, often accompanied by a small increase in their amplitude. Removal of base produced a rebound acidification which transiently abolished contractions. Direct acidification of the cytoplasm, by application of weak acid, also abolished contractions. However the alkalinization which accompanied removal of acid, produced variable effects on force.Supported by the M.R.C.     

Cytoplasmic alkalinization increases high-threshold calcium current in chick dorsal root ganglion neurones

Changes of calcium currents with intracellular pH (pH_i) were investigated in chick dorsal root ganglion (DRG) neurones. High-threshold calcium currents decreased after extracellular application of a permeable weak acid, sodium acetate (CH₃COONa), and increased when applying a permeable weak base, ammonium chloride (NH₄Cl), whereas both compounds were ineffective against the low-threshold calcium current. These weak electrolytes, employed to change pH_i, did not alter the kinetic and steady-state parameters of activation and inactivation of the calcium current. Extracellular application of concanavalin A (Con A) and wheat germ agglutinin to elevate pH_i increased the high-threshold calcium current. Their effect developed within 2–5 min and was independent of lectin concentration varied from 0.1 to 1 mg/ml. The lectin effects were greatly diminished if Na/H exchange was blocked by amiloride or suppressed by low external sodium. Succinilated Con A and Con A in the presence of d-mannose were less effective. Calcium currents were recorded simultaneously with the pH_i, monitored with a proton-sensitive microelectrode. It was found that 50% inhibition of the calcium current occured at pH_i of 6.5. Histidine-specific reagents — diethylpyrocarbonate, Rose Bengal and Methylene Blue — prevented the modulation of the calcium conductance by CH₃COONa and NH₄Cl. Extracellular baclofen and theophylline or intracellular phorbol esters, staurosporine, calmodulin antagonists R24571 and W-13, and neomycine failed to prevent the modulation of the calcium current by weak electrolytes. These observations are consistent with an interaction between intracellular protons and calcium channels.     

Administration of Lactobacillus casei and Lactobacillus plantarum affects the diversity of murine intestinal lactobacilli, but not the overall bacterial community structure

Lactobacilli are normal inhabitants of the gastrointestinal tract (GIT) of many mammalian hosts. Their administration as probiotics in functional foods is currently a frequent practice, mainly because of their benefits to host health. It is therefore of interest to study the impact of administration of exogenous strains of Lactobacillus normally used as probiotics upon endogenous microbial populations. For this purpose, fecal and intestinal tissue samples were analyzed in a mouse model fed with a mixture of Lactobacillus plantarum and Lactobacillus casei isolated from commercially available dairy products. The murine intestinal microbiota was studied by means of cultivation-independent 16S rRNA gene-targeted techniques, namely denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of clone libraries. Multivariate statistical analysis was used to integrate datasets obtained from the different techniques applied. Whereas no differences were detected in the composition of the overall fecal bacterial community, changes were observed for intestinal tissue samples. Moreover, an increase in the diversity of gut lactobacilli was observed in fecal as well as intestinal tissue samples when mice received the mixture of L. casei and L. plantarum.