Immunomodulatory function of lactic acid bacteria

Using mice, we found that oral administration of Bifidobacterium breve YIT4064 (B. breve) activated the humoral immune system, augmented anti-rotavirus IgA production or anti-influenza virus (IFV) IgG production and protected against rotavirus infection or influenza infection, respectively. Furthermore, when the B. breve was given to infants from an infant home, there was a significant reduction of the frequency of rotavirus shedding in stool samples during the administration of the bacteria. It was also found, again using mice, that oral administration of Lactobacillus casei strain Shirota (LcS) stimulated type 1 helper T (Th1) cells, activated the cellular immune system and inhibited incidence of tumors and IgE production. These results demonstrated that these two strains of lactic acid bacteria modulated the different immune systems each in its own way and prevented against various diseases.     

Differential effects of two probiotic strains with different bacteriological properties on intestinal gene expression, with special reference to indigenous bacteria

Probiotics are used for the improvement of gut disorders. To explore the potential of probiotics, a gnotobiotic study using BALB/c mice to analyze epithelial gene expression was performed. Microarray analysis of probiotic strain-monoassociated mice showed that Lactobacillus casei Shirota and Bifidobacterium breve Yakult noticeably affected gene expression in the ileal and colonic epithelial cells, respectively, although to a smaller extent than segmented filamentous bacteria (SFB). Lactobacillus casei Shirota enhanced the gene expression involving defense/immune functions and lipid metabolism more strongly than B. breve Yakult. In the colon, expression of a chloride transporter was slightly enhanced, although downregulation of many genes, such as guanine nucleotide-binding protein, was evident in mice with B. breve Yakult compared with the ones with L. casei Shirota. SFB affected gene expression more strongly than the probiotic strains. In particular, alpha(1-2) fucosyltransferase and pancreatitis-associated protein were significantly enhanced only in SFB-monoassociated mice but not probiotic strain-monoassociated mice. Gene expression of SFB-monoassociated mice was either stimulated or repressed in a manner similar to or opposite that of conventional colonized mice. Taken together, probiotic strains of L. casei Shirota and B. breve Yakult differentially affect epithelial gene expression in the small intestine and colon, respectively.     

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.     

NF-kappa B is required within the innate immune system to inhibit microflora-induced colitis and expression of IL-12 p40

We have previously presented evidence demonstrating that mice deficient in NF-kappaB subunits are susceptible to colitis induced by the pathogenic enterohepatic Helicobacter species, H. hepaticus. However, it has not been determined whether NF-kappaB is required within inhibitory lymphocyte populations, within cells of the innate immune system, or both, to suppress inflammation. To examine these issues, we have performed a series of adoptive transfer experiments using recombination-activating gene (Rag)-2(-/-) or p50(-/-)p65(+/-)Rag-2(-/-) mice as hosts for wild-type (WT) and p50(-/-)p65(+/-) lymphocyte populations. We have shown that although the ability of H. hepaticus to induce colitis in Rag-2(-/-) mice is inhibited by the presence of either WT or p50(-/-)p65(+/-) splenocytes, these splenocyte populations are unable to suppress H. hepaticus-induced colitis in p50(-/-)p65(+/-)Rag-2(-/-) mice. Colitis in these animals is characterized by increased expression of inflammatory cytokines including IL-12 p40, and depletion of IL-12 p40 from p50(-/-)p65(+/-) mice ameliorates H. hepaticus-induced disease. Consistent with a primary defect in the regulation of IL-12 expression, H. hepaticus induced markedly higher levels of IL-12 p40 in p50(-/-)p65(+/-) macrophages than in WT macrophages. These results suggest that inhibition of H. hepaticus-induced IL-12 p40 expression by NF-kappaB subunits is critical to preventing colonic inflammation in response to inflammatory microflora.     

Differential cytokine response from dendritic cells to commensal and pathogenic bacteria in different lymphoid compartments in humans

Resident host microflora condition and prime the immune system. However, systemic and mucosal immune responses to bacteria may be divergent. Our aim was to compare, in vitro, cytokine production by human mononuclear and dendritic cells (DCs) from mesenteric lymph nodes (MLNs) and peripheral blood mononuclear cells (PBMCs) to defined microbial stimuli. Mononuclear cells and DCs isolated from the MLN (n = 10) and peripheral blood (n = 12) of patients with active colitis were incubated in vitro with the probiotic bacteria Lactobacillus salivarius UCC118 or Bifidobacterium infantis 35624 or the pathogenic organism Salmonella typhimurium UK1. Interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and IL-10 cytokine levels were quantified by ELISA. PBMCs and PBMC-derived DCs secreted TNF-alpha in response to the Lactobacillus, Bifidobacteria, and Salmonella strains, whereas MLN cells and MLN-derived DCs secreted TNF-alpha only in response to Salmonella challenge. Cells from the systemic compartment secreted IL-12 after coincubation with Salmonella or Lactobacilli, whereas MLN-derived cells produced IL-12 only in response to Salmonella. PBMCs secreted IL-10 in response to the Bifidobacterium strain but not in response to the Lactobacillus or Salmonella strain. However, MLN cells secreted IL-10 in response to Bifidobacteria and Lactobacilli but not in response to Salmonella. In conclusion, commensal bacteria induced regulatory cytokine production by MLN cells, whereas pathogenic bacteria induce T cell helper 1-polarizing cytokines. Commensal-pathogen divergence in cytokine responses is more marked in cells isolated from the mucosal immune system compared with PBMCs.     

Probiotic bacteria produce conjugated linoleic acid locally in the gut that targets macrophage PPAR γ to suppress colitis

Background

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis.

Conclusions/Significance

Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis.     

Helicobacter hepaticus infection triggers inflammatory bowel disease in T cell receptor alphabeta mutant mice

The T cell receptor alpha chain-deficient (TCR alpha-/-) and TCR beta chain-deficient (TCR beta-/-) mice develop chronic intestinal inflammation that resembles inflammatory bowel disease by 3 to 4 months of age. The objective of the study reported here was to determine the role of infection with the bacterial pathogen Helicobacter hepaticus in the pathogenesis of disease in TCR alphabeta mutant mice. The H. hepaticus-infected TCR alphabeta mutant mice were rederived by use of embryo transfer to produce Helicobacter-free animals. Helicobacter-free TCR alpha-/-, TCR beta-/-, and TCR alpha-/- beta-/- mice were inoculated with H. hepaticus. Experimentally infected mice and uninfected control mice were examined for intestinal lesions at 3, 6, and 9 months after inoculation. The TCR alphabeta mutant mice inoculated with H. hepaticus developed intestinal epithelial cell hyperplasia and mucosal inflammation. By 6 months after inoculation, infected animals had moderate cecal and colonic lesions. Helicobacter-free TCR alpha-/- mice, but not TCR beta-/- or TCR alpha-/- x beta-/- mice, also developed H. hepaticus-independent colitis by 9 months after inoculation. Infection with H. hepaticus is sufficient to cause chronic proliferative intestinal inflammation in TCR alphabeta mutant mice. However, H. hepaticus infection is not necessary for intestinal disease in TCR alpha-/- mice.     

Helicobacter-induced inflammatory bowel disease in IL-10- and T cell-deficient mice

Inflammatory bowel disease (IBD) is thought to result from a dysregulated mucosal immune response to luminal microbial antigens, with T lymphocytes mediating the colonic pathology. Infection with Helicobacter spp has been reported to cause IBD in immunodeficient mice, some of which lack T lymphocytes. To further understand the role of T cells and microbial antigens in triggering IBD, we infected interleukin (IL)-10(-/-), recombinase-activating gene (Rag)1(-/-), T-cell receptor (TCR)-alpha(-/-), TCR-beta(-/-), and wild-type mice with Helicobacter hepaticus or Helicobacter bilis and compared the histopathological IBD phenotype. IL-10(-/-) mice developed severe diffuse IBD with either H. bilis or H. hepaticus, whereas Rag1(-/-), TCR-alpha(-/-), TCR-beta(-/-), and wild-type mice showed different susceptibilities to Helicobacter spp infection. Proinflammatory cytokine mRNA expression was increased in the colons of Helicobacter-infected IL-10(-/-) and TCR-alpha(-/-) mice with IBD. These results confirm and extend the role of Helicobacter as a useful tool for investigating microbial-induced IBD and show the importance, but not strict dependence, of T cells in the development of bacterial-induced IBD.     

Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization

BACKGROUND: Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. MATERIALS AND METHODS: Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. RESULTS: Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. CONCLUSION: We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.     

Adoptive transfer of NK 1.1+ lymphocytes in immune-mediated colitis: a pro-inflammatory or a tolerizing subgroup of cells?

T lymphocytes expressing NK1.1 marker (NKT) have been suggested to play crucial roles in immune modulation. AIM: To determine the role of NK1.1+ cells in induction and maintenance of pro-inflammatory and/or tolerizing responses. METHODS: Colitis was induced in C57/B6 donor mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Donor mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Depletion of NK1.1+ lymphocytes was performed before lymphocyte harvesting. Splenocytes were harvested and separated into T-cell subpopulations, and transplanted into recipient mice before intracolonic instillation of TNBS. Standard clinical, macroscopic, and microscopic scores, and intracellular staining, flow cytometry, and cytotoxicity assays were performed. RESULTS: The adoptive transfer of CD4+ and NK1.1+ cells harvested from tolerized mice markedly ameliorated the colitis in recipient mice. In contrast, the adoptive transfer of CD8+ and double negative lymphocytes failed to transfer the tolerance. Recipients of splenocytes from tolerized mice exhibited an increase in CD4+ IL4+/CD4+ IFNgamma+ ratio. In contrast, recipients of splenocytes from NK1.1-depleted-tolerized mice exhibited severe colitis with a significant decrease of the CD4+ IL4+/CD4+ IFNgamma+ ratio. However adoptive transfer of splenocytes from non-tolerized NKT-depleted mice led to an alleviation of colitis with a relative increase of the CD4+ IL4+/CD4+ IFNgamma+ ratio. CONCLUSIONS: NK1.1+ lymphocytes play a critical role in immune regulation. They may be accountable for an alteration of the inflammatory response and the CD4+ IL4+/CD4+ IFNgamma ratio immune-mediated colitis and in peripheral tolerance induction.     

长双歧杆菌TTF菌株增强机体免疫活性研究

通过长双岐杆菌TTF活菌、菌体破碎物和发酵上清液3种处理物灌胃正常小鼠和免疫功能低下小鼠试验发现,长双歧杆菌TTF的3种受试物对环磷酰胺（Cy）造成的免疫功能低下小鼠模型（IDMM）3项免疫指标均有显著（P〈0．05）或极显著（P〈0．01）影响,且在本试验选取的3个剂量范围内基本呈量效关系。B．longum TTF的3种受试物对IDMM的非特异性免疫功能的影响普遍大于对小鼠的细胞免疫和体液免疫的影响;对正常小鼠免疫功能影响的水平低于其对IDMM各项免疫指标的影响,且无论活菌、菌体破碎物、发酵上清液均不会改变小鼠自身正常的免疫功能。急性毒性试验表明,B．longum,TTF3种处理物对健康小鼠无急性毒性。就3种处理物来讲,发酵上清液组的效果高于菌体破碎物和活菌。     

Thin layer chromatographic determination of organic acids for rapid identification of bifidobacteria at genus level

This study presents a simple and fast method for the identification of bifidobacteria using a thin layer chromatographic (TLC) analysis of the short chain fatty acids in a culture broth. When the chromatogram was sprayed with the indicator solution (methyl red-bromophenol blue in 70% ethanol), lactic acid exhibited two red spots, and acetic acid, propionic acid, and butyric acid all produced blue spots. Succinic acid and citric acid produced yellow and dark yellow spot, respectively. In addition, these organic acids showed different R(f) values. The total time taken to analyze the organic acids in the 10 bacterial culture broths using the proposed method was approximately 50 min. The proposed TLC method was used to analyze the organic acids in culture broths of the following strains, five Bifidobacterium species. (Bifidobacterium longum, B. breve, B. infantis, B. bifidum, and B. adolescentis) and five other lactic acid bacteria strains (Lactobacillus casei, L. bulgaricus, L. acidophilus, Streptococcus thermophilus, and S. lactis). Both spots of lactic acid and acetic acid were detected on all the TLC plates from the five bifidobacterial culture broths. The five other lactic acid bacterial culture broths, however, only exhibited lactic acid spots. Accordingly, the proposed TLC method would appear to be a useful tool for rapid identification of Bifidobacterium spp. at the genus level.     

Molecular profiling of Lactobacillus, Streptococcus, and Bifidobacterium species in feces of active racehorses

Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equinus was predominant in the Streptococcus group. The Bifidobacterium group was not detected by single-PCR but atypical species of the group were found in three of the six Thoroughbreds tested by nested-PCR. Calculation and estimation of lactic acid bacteria and bifidobacteria revealed that lactic acid bacteria were predominant in the feces and bifidobacteria were minor. These results indicate that the community of lactic acid bacteria and bifidobacteria in horse feces are unique because of the presence of specific species for horse feces and a minority of the Bifidobacterium group. Repeated tests of the feces from the same horse over 3 months showed that the diversity and composition of lactic acid bacteria and bifidobacteria in the feces was basically stable throughout the test period.     

Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates

We observed recently that colonic fermentation of lactose might be a major factor in the pathophysiology of lactose intolerance. Proteomic techniques could be helpful in interpreting the metabolic pathways of lactose fermentation. The objective of this study was to explore proteomic methodologies for studying bacterial lactose metabolism that can be used to detect and identify proteins associated with the onset of intolerance symptoms. Differential expression of cytoplasmic proteins of Bifidobacterium animalis, Bifidobacterium breve and Bifidobacterium longum grown on different carbohydrates (lactose, glucose, galactose) was analyzed with surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins were identified with LC-MS/MS. The three strains grown on the same carbohydrate or the same strain grown on glucose or lactose showed differences in SELDI-TOF MS protein profiles. Differences in protein expression were observed in B. breve grown on glucose, galactose or lactose as analyzed with SDS-PAGE. With LC-MS/MS, proteins from Bifidobacterium were identified, which included enzymes for metabolism of lactose, glucose and galactose. In conclusion, the applied techniques can discern differences in protein expression of bacteria metabolizing different carbohydrates. These techniques are promising in studying metabolism of lactose and other substrates in a complex bacterial ecosystem such as the colonic microbiota.     

Helicobacter spp. Other Than Helicobacter pylori

Non- H. pylori Helicobacter species (NHPHS) are associated with several important human and animal diseases. In the past year research into this group of bacteria has continued to gain attention, and novel species have been described in new niches owing to improvements in detection methods. Polymerase chain reaction and/or sequencing remain the gold standard for the detection of this genus. New insights into the pathogenesis of the NHPHS in hepatobiliary, gastric, and intestinal diseases were gained. In particular, data revealed interaction between hepatic steatosis and infectious hepatitis in the development of hepatocellular carcinoma. Evidence of an association between hepatitis C virus and Helicobacter spp. in hepatocarcinoma development was also provided; and male sex hormone signaling appeared to influence infectious hepatitis induced by Helicobacter hepaticus . More findings support an association between Helicobacter heilmannii and gastric adenocarcinoma; and in mice, mucins MUC4 and MUC5 but not MUC1 influence the colonization and pathogenesis of Helicobacter felis . Data indicated that the roles of the adaptive immune system in H. hepaticus -induced intestinal tumorigenesis are different in the small and large intestines, and environmental factors, such as bile acids may modulate H. hepaticus carcinogenic potential. New reports in the prevention and eradication of NHPHS showed a protective response against Helicobacter suis induced by vaccine administration, and a successful cross-foster rederivation method successfully eradicated Helicobacter spp. from contaminated mice litters. Overall, the studies provided insights into the pathophysiology of Helicobacter species other than Helicobacter pylori.     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

Detection of Helicobacter hepaticus in Human Bile Samples of Patients with Biliary Disease

Background:  Since the discovery of Helicobacter pylori , various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown.
Materials and Methods:  We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis.
Results:  Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher ( p  = .029) prevalence of H. hepaticus infection than samples from patients with other diseases.
Conclusion:  Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.     

Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus

Genome-wide association studies of inflammatory bowel diseases identified susceptible loci containing an autophagy-related gene. However, the role of autophagy in the colon, a major affected area in inflammatory bowel diseases, is not clear. Here, we show that colonic epithelial cell-specific autophagy-related gene 7 (Atg7) conditional knock-out (cKO) mice showed exacerbation of experimental colitis with more abundant bacterial invasion into the colonic epithelium. Quantitative PCR analysis revealed that cKO mice had abnormal microflora with an increase of some genera. Consistently, expression of antimicrobial or antiparasitic peptides such as angiogenin-4, Relmβ, intelectin-1, and intelectin-2 as well as that of their inducer cytokines was significantly reduced in the cKO mice. Furthermore, secretion of colonic mucins that function as a mucosal barrier against bacterial invasion was also significantly diminished in cKO mice. Taken together, our results indicate that autophagy in colonic epithelial cells protects against colitis by the maintenance of normal gut microflora and secretion of mucus.     

Fecal microflora of Greek healthy neonates

This study aimed to explore, in our geographical region, the development of intestinal microflora and the colonization patterns of lactic acid bacteria and bifidobacteria during the first three months of life and to investigate the effect of the mode of delivery. Fecal specimens from 82 healthy, full-term infants were collected prospectively 4, 30 and 90 days after delivery and subcultured on nonselective and selective media. Identification of isolates was performed by microbiological and molecular methods. For the delivery effect, two groups of vaginally or caesarean-delivered exclusively breast-fed infants were studied. Despite the early high total counts of aerobes and anaerobes, colonization of lactobacilli and bifidobacteria was overall limited until 3 months of age. Furthermore, caesarean-delivered infants were less often colonized with lactobacilli at day 4 (4% vs. 59%, p = 0.000) and with bifidobacteria at day 4 (0% vs. 23%, p = 0.015) and 30 (0% vs. 35%, p = 0.042) compared to vaginally delivered ones. No bacterial populations differences were detected to compare colonized infants. Identification results indicated the predominance of Lactobacillus rhamnosus, Lactobacillus johnsonii and Lactobacillus paracasei species in neonatal gut microflora up to the first month of life and diversity of Lactobacillus species in vaginally delivered, colonized newborns, at fourth day. Furthermore, Bifidobacterium longum and Bifidobacterium breve were the most frequently detected Bifidobacterium species in vaginally delivered, breast-fed infants. In conclusion our study revealed a restricted colonization pattern of lactic acid bacteria in Greek infants and a delay in the development of Lactobacillus and Bifidobacterium spp. microbiota after caesarean section. Further analysis of potential consequences of these findings is required.