Takashi Asahara

Perioperative Synbiotic Treatment to Prevent Postoperative Infectious Complications in Biliary Cancer Surgery A Randomized Controlled Trial

Summary Background Data:Use of synbiotics has been reported to benefit human health, but clinical value in surgical patients remains unclear. Objective:To investigate the effect of perioperative oral administration of synbiotics upon intestinal barrier function, immune responses, systemic inflammatory responses, microflora, and surgical outcome in patients undergoing high-risk hepatobiliary resection. Methods:Patients with biliary cancer involving the hepatic hilus (n = 101) were randomized before hepatectomy, into a group receiving postoperative enteral feeding with synbiotics (group A); or another receiving preoperative plus postoperative synbiotics (group B). Lactulose-mannitol (L/M) ratio, serum diamine oxidase (DAO) activity, natural killer (NK) cell activity, interleukin-6 (IL-6), fecal microflora, and fecal organic acid concentrations were determined before and after hepatectomy. Postoperative infectious complications were recorded. Results:Of 101 patients, 81 completed the trial. Preoperative and postoperative changes in L/M ratio and DAO activity were similar between groups. Preoperatively in group B, NK activity, and lymphocyte counts increased, while IL-6 decreased significantly (P < 0.05). Postoperative serum IL-6, white blood cell counts, and C-reactive protein in group B were significantly lower than in group A (P < 0.05). During the preoperative period, numbers of Bifidobacterium colonies cultured from and total organic acid concentrations measured in feces increased significantly in group B (P < 0.05). Postoperative concentrations of total organic acids and acetic acid in feces were significantly higher in group B than in group A (P < 0.05). Incidence of postoperative infectious complications was 30.0% (12 of 40) in group A and 12.1% (5 of 41) in group B (P < 0.05). Conclusions:Preoperative oral administration of synbiotics can enhance immune responses, attenuate systemic postoperative inflammatory responses, and improve intestinal microbial environment. These beneficial effects likely reduce postoperative infectious complications after hepatobiliary resection for biliary tract cancer.

Probiotic Bifidobacteria Protect Mice from Lethal Infection with Shiga Toxin-Producing Escherichia coli O157:H7

ABSTRACT The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539T did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.

Sensitive quantitative detection of commensal bacteria by rRNA-targeted reverse transcription-PCR.

ABSTRACT A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group- or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10−3 cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 100 to 105 CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4′,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n = 38) at a level of 103 cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10 −/−, Tlr2 −/−, and Myd88 −/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra −/− T cells. B. breve treatment of Tlr2 −/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4+ T cells from wild-type mice, but not Il10 −/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Synbiotics reduce postoperative infectious complications: a randomized controlled trial in biliary cancer patients undergoing hepatectomy

Background and aimsThe clinical value of synbiotics in surgical patients remains unclear. The aim of this study was to investigate the effect of synbiotics on intestinal integrity and microflora, as well as on surgical outcome, in patients undergoing high-risk hepatectomy.MethodsFifty-four patients with biliary cancer were randomly allocated to two groups before hepatectomy. One group received postoperative enteral feeding that included synbiotics; the other received enteral feeding only. Lactulose/mannitol (L/M) ratio, serum diamine oxidase (DAO) activity, and fecal microflora and organic acid concentrations were determined. Postoperative infectious complications were recorded.ResultsOf the 54 patients, 44 completed the trial (21 receiving synbiotics and 23 others as controls). Postoperative changes in L/M ratios and serum DAO activities were identical between the two groups. Numbers of beneficial bacteria increased in the synbiotics group after surgery but decreased in controls. Numbers of harmful microorganisms decreased in the synbiotics group but increased in controls. Total organic acid concentrations increased in the synbiotics group but decreased in controls. Incidence of infectious complications was 19% (4/21) in the synbiotics group and 52% (12/23) in controls (P<0.05). All study patients tolerated surgery (mortality 0%).ConclusionsSynbiotics, combined with early enteral nutrition, can reduce postoperative infections. This beneficial effect presumably involves correction of an intestinal microbial imbalance induced by surgical stress.

Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent

Background: Enterobacteria and cytokines both play roles in the pathophysiology of NSAID-induced enteropathy. Toll-like receptor (TLR) 4 recognises lipopolysaccharide (LPS), resulting in activation of an inflammatory cascade via the accessory protein MyD88. Aims: To investigate role of TLR4 in inflammatory responses in indomethacin-induced enteropathy. Methods: Indomethacin was administered p.o. to non-fasting rats and mice to induce small intestinal damage. The extent of such damage was evaluated by measuring the injured area stained dark blue with Evans blue. Rats were given antibiotics (ampicillin, aztreonam or vancomycin) p.o., or intraperitoneal LPS (a TLR4 ligand) or neutralising antibodies against neutrophils, tumour necrosis factor (TNF)-α, or monocyte chemotactic protein (MCP)-1. Furthermore, the intestinal ulcerogenicity of indomethacin was examined in TLR4-mutant, TLR4−/−, and MyD88−/− mice. Results: Indomethacin induced small intestinal damage with an increase in expression of TNF-α and MCP-1 in both rats and mice. Antibodies against neutrophils, TNF-α and MCP-1 inhibited the damage by 83%, 67% and 63%, respectively, in rats. Ampicillin and aztreonam also inhibited this damage, and decreased the number of Gram-negative bacteria in the small intestinal contents of the rat. However, vancomycin, which exhibited no activity against Gram-negative bacteria, had no preventive effect against this damage. Administration of LPS 1 h after indomethacin aggravated the damage, whereas LPS pretreatment inhibited it with reduction of expression of TLR4 and cytokines. In TLR4-mutant mice, the damage and cytokine expression were markedly inhibited. TLR4−/− and MyD88−/− mice were also resistant to the damage. Conclusions: Indomethacin may injure the small intestine through a TLR4/MyD88-dependent pathway.

The value of bile replacement during external biliary drainage : an analysis of intestinal permeability, integrity, and microflora

Objective:To investigate the effect of bile replacement following percutaneous transhepatic biliary drainage, ie, external drainage, on intestinal permeability, integrity, and microflora in a clinical setting. Summary Background Data:Several authors have reported that internal biliary drainage is superior to external drainage. However, it is unclear whether bile replacement following external drainage is beneficial. Methods:Twenty-five patients with biliary cancer underwent percutaneous transhepatic biliary drainage (PTBD) as a part of presurgical management. All externally drained bile was replaced either per os or by administration through a nasoduodenal tube. The interval between PTBD and the beginning of bile replacement was 21.3 ± 19.7 days, and the length of bile replacement was 20.7 ± 9.6 days. The lactulose-mannitol test, measurement of serum diamine oxidase (DAO) activity, and analyses of fecal microflora and organic acids were performed before and after bile replacement. Results:The volume of externally drained bile varied widely from patient to patient, ranging from 220 ± 106 mL/d to 1616 ± 394 mL/d (mean, 714 ± 346 mL/d). Biliary concentrations of bile acids, cholesterol, and phospholipids increased significantly after bile replacement. The lactulose-mannitol (L/M) ratio decreased from 0.063 ± 0.060 before bile replacement to 0.038 ± 0.032 after bile replacement (P < 0.05). Serum DAO activity increased from 3.9 ± 1.4 U/L before bile replacement to 5.1 ± 1.6 U/L after bile replacement (P < 0.005), and the magnitude of change in serum DAO activity correlated with the length of bile replacement (r = 0.483, P < 0.05). Neither the L/M ratios nor serum DAO activities before bile replacement correlated with the interval between PTBD and the beginning of bile replacement. Fecal microflora and organic acids were unchanged. Conclusion:Impaired intestinal barrier function does not recover by PTBD without bile replacement. Bile replacement during external biliary drainage can restore the intestinal barrier function in patients with biliary obstruction, primarily due to repair of physical damage to the intestinal mucosa. Our results support the hypothesis that bile replacement during external drainage is beneficial.

Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by synbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides

Aims: The anti‐infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT‐2 in an opportunistic antibiotic‐induced murine infection model in mice was examined.

Probiotic Lactobacillus casei strain Shirota prevents indomethacin-induced small intestinal injury: involvement of lactic acid

Inflammatory responses triggered by activation of the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway are a key mechanism in nonsteroidal anti-inflammatory drug-induced enteropathy. The aim of this study was to investigate the probiotic effect of Lactobacillus casei strain Shirota (LcS) on indomethacin-induced small intestinal injury. Rats pretreated with viable LcS or heat-killed LcS once or once daily for a week were administered indomethacin by gavage to induce injury. Anti-inflammatory effects of L-lactic acid (1-15 mM) were evaluated in vitro by use of THP-1 cells. One-week treatment with viable LcS prevented indomethacin-induced intestinal injury with increase in the concentration of lactic acid in small intestinal content and inhibited increases in myeloperoxidase activity and expression of mRNA for tumor necrosis factor-alpha (TNF-alpha) while affecting neither TLR4 expression nor the number of gram-negative bacteria in intestinal content, whereas neither heat-killed LcS nor a single dose of viable LcS inhibited intestinal injury. Prevention of this injury was also observed in rats given l-lactic acid in drinking water. Both L-lactic acid and LcS culture supernatant containing 10 mM lactic acid inhibited NF-kappaB activation and increases in TNF-alpha mRNA expression and TNF-alpha protein secretion in THP-1 cells treated with LPS. Western blot analyses showed that both L-lactic acid and LcS culture supernatants suppressed phosphorylation and degradation of I-kappaB-alpha induced by LPS without affecting expression of TLR4. These findings suggest that LcS exhibits a prophylactic effect on indomethacin-induced enteropathy by suppressing the LPS/TLR4 signaling pathway and that this probiotic effect of LcS may be mediated by L-lactic acid.