Disturbances in the ocular surface microbiome by perioperative antimicrobial eye drops.

We aimed to elucidate the effects of antimicrobial eye drops used in the perioperative period of ophthalmic surgery on the ocular surface microbiome by metagenomic analysis. Twenty-eight eyes from 15 patients (mean age 74.1 years) with no history of eye drop use within 3 months before cataract surgery were included in this study. Gatifloxacin eye drops were used in all patients in the perioperative period. The antimicrobial eye drops were started 3 days before surgery. They were discontinued after conjunctival sac specimen collection for 2 weeks after the surgery. Conjunctival sac specimens were collected to investigate the alterations in the ocular surface microbiome by meta-16S analysis targeting the V3-V4 region of the bacterial 16S rRNA gene. Principal coordinate analysis showed that the bacterial composition tended to be different before and 2 and 4 weeks after surgery. Individual observations on six eyes showed that the bacterial composition at 12 weeks after surgery was closer to that before surgery than to that at 4 weeks after surgery in two eyes, while the bacterial composition in the remaining four eyes was different at various time points. Before surgery, Firmicutes, Proteobacteria, and Bacteroidetes were predominant; however, 2 weeks after surgery, the proportion of Proteobacteria increased and that of Firmicutes decreased. A similar trend was noticed 4 weeks after surgery, although antibacterial eye drops had been discontinued 2 weeks after surgery. The Shannon-Weaver coefficient showed a decreasing trend at 2-, 4-, and 12-weeks post operation compared to that before operation. The diversity of the microbiome decreased significantly at 2- and 4-weeks after surgery when compared to that before surgery (p < 0.05). The ocular surface microbiome is easily disrupted by antimicrobial eye drops, and it needs recovery time. In such cases, the ocular surface microbiome is presumed to contain many antimicrobial-resistant bacteria. In some cases, it may not recover, and a new microbiome is formed.

Partially hydrolyzed guar gum alleviates hepatic steatosis and alters specific gut microbiota in a murine liver injury model.

PURPOSE: The gut microbiota, via the gut-liver axis, plays an important role in the development of intestinal failure-associated liver disease. Here, we investigated whether partially hydrolyzed guar gum (PHGG), a dietary fiber could alleviate liver damage and modulate the gut microbiota in a murine liver injury (LI) model. METHODS: Liver injury was induced in 6-week-old male C57BL/6 mice using an enteral liquid diet composed of parenteral nutrition (LI group) and treated with 5% PHGG (LI/PHGG group). Liver histopathology was examined using oil red O and a tumor necrosis factor-α (TNF-α) labeling. The gut microbiota was examined using 16S rRNA gene sequencing. RESULTS: Lipid accumulation was significantly decreased in the LI /PHGG group when compared with that of the LI group. The area of TNF-α-positive cells was significantly higher in the LI group when compared with that of the control. The principal coordinate analysis (PCoA) revealed pronounced changes in the gut microbiota after PHGG treatment. Linear discriminant analysis of effect size showed that PHGG treatment significantly increased cecal abundance of Parabacteroides. CONCLUSIONS: PHGG alleviated hepatic steatosis following liver injury in mice. The protective effect of PHGG treatment could be associated with increased abundance of Parabacteroides in the cecum.

Inactivation of human norovirus by chlorous acid water, a novel chlorine-based disinfectant.

INTRODUCTION: Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces. Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type 2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV. METHODS: HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-qPCR) after pretreatment with RNase. In addition, the log10 reduction of HuNoV RNA copy number by CA and NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone, meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions. RESULTS: In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log10 reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log10 reduction in HuNoV RNA copy number while less than 1.0 log10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5% polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino acids tested. CONCLUSIONS: CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions.

Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

Antiviral and Virucidal Activities of Umesu Phenolics on Influenza Viruses.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Partially hydrolyzed guar gum alleviates small intestinal mucosal damage after massive small bowel resection along with changes in the intestinal microbiota.

PURPOSE: Short bowel syndrome is associated with intestinal mucosal inflammation and microbial dysbiosis, leading to intractable complications. Partially hydrolyzed guar gum (PHGG) has trophic and anti-inflammatory effects on the intestine. We investigated whether PHGG ameliorates small intestinal mucosal damage and alters the intestinal microbiota using a rat small bowel resection (SBR) model. METHODS: Sprague Dawley rats were divided into sham operation (Sham), Sham/PHGG, SBR, and SBR/PHGG groups. On day 21, all rats were euthanized. To assess small intestinal mucosal damage, the degeneration rate was morphometrically evaluated and immunohistochemically examined using anti-CD45 antibodies. Analyses of fecal microbiota using 16S rRNA and short-chain fatty acid production were also performed. RESULTS: The mucosal degeneration rate was significantly higher in the SBR group than in the Sham or SBR/PHGG groups. The number of CD45-positive cells was significantly higher in the SBR group than in the Sham, Sham/PHGG, or SBR/PHGG groups. The relative abundance of family Lachnospiraceae was significantly higher in the SBR/PHGG group than in the SBR group. CONCLUSIONS: PHGG administration alleviated small intestinal mucosal damage which could be associated with modulation of the intestinal microbiota.

TLR2-stimulating contaminants in glycoconjugate fractions prepared from Bacteroides fragilis.

Bacteroides fragilis is a member of the normal intestinal flora and is involved in host immunostimulation via TLR2. On the bacterial cell surface, glycoconjugates, such as LPS and capsular polysaccharide A (PSA), have been reported to participate in host immunostimulation via TLR2. Previously, we identified a TLR2-stimulating lipoprotein in B. fragilis cells. In this study, we demonstrated that TLR2-stimulating principal molecules in glycoconjugate fractions prepared from B. fragilis are contaminating proteinous molecules, which may also be lipoproteins. The glycoconjugate fractions were prepared by phenol-hot water extraction of B. fragilis wild type and PSA-deficient strains, followed by hydrophobic interaction chromatography. TLR2-stimilating activities of the fractions were not affected by PSA deficiency. By in-gel TLR2-stimulation assay, molecules in high-molecular-mass area, where capsular polysaccharides were migrated, were found not to stimulate TLR2, but those in the range of 15-40 kDa were active. Further, proteinase K could digest the latter molecules and the TLR2-stimulating activities were migrated to the area of below 15 kDa. These results support that proteinous molecules, which are estimated to be lipoproteins, are responsible for almost all TLR2-stimulating activity in the glycoconjugate fractions prepared from B. fragilis.

Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions.

Sanitation of environmental surfaces with chlorine based-disinfectants is a principal measure to control outbreaks of norovirus or Clostridium difficile. The microbicidal activity of chlorine-based disinfectants depends on the free available chlorine (FAC), but their oxidative potential is rapidly eliminated by organic matter. In this study, the microbicidal activities of weakly acidified chlorous acid water (WACAW) and sodium hypochlorite solution (NaClO) against feline calcivirus (FCV) and C. difficile spores were compared in protein-rich conditions. WACAW inactivated FCV and C. difficile spores better than NaClO under all experimental conditions used in this study. WACAW above 100 ppm FAC decreased FCV >4 log10 within 30 sec in the presence of 0.5% each of bovine serum albumin (BSA), polypeptone or meat extract. Even in the presence of 5% BSA, WACAW at 600 ppm FAC reduced FCV >4 log10 within 30 sec. Polypeptone inhibited the virucidal activity of WACAW against FCV more so than BSA or meat extract. WACAW at 200 ppm FAC decreased C. difficile spores >3 log10 within 1 min in the presence of 0.5% polypeptone. The microbicidal activity of NaClO was extensively diminished in the presence of organic matter. WACAW recovered its FAC to the initial level after partial neutralization by sodium thiosulfate, while no restoration of the FAC was observed in NaClO. These results indicate that WACAW is relatively stable under organic matter-rich conditions and therefore may be useful for treating environmental surfaces contaminated by human excretions.

Strain-specific transmission in an outbreak of ESBL-producing Enterobacteriaceae in the hemato-oncology care unit: a cohort study.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing bacteria are resistant to several types of antibiotics excluding carbapenems. A transmissibility of ESBL-producing Enterobacteriaceae would be depending on each bacterial property, however, that has not been elucidated in clinical setting. In this study, we attempted to identify the source of an outbreak of ESBL-producing bacteria in a medical oncology and immunology care unit. METHODS: An ESBL-producing Enterobacteriaceae (ESBL-E) outbreak observed between July 2012 and August 2012 in Kagawa University Hospital was surveyed using various molecular microbiology techniques. We used Pulsed-field gel electrophoresis (PFGE), PCR-based ESBL gene typing, and direct sequence of ESBL gene as molecular microbiology typing method to distinguish each strain. RESULTS: The typical prevalence of ESBL-E isolation in the unit was 7.0 per month (1.7 per week). The prevalence of ESBL-E isolation during the target research period was 20.0 per month (5.0 per week). In total, 19 isolates (11 K. pneumoniae and 8 E. coli) were obtained from clinical samples, including four control strains (two each of both bacteria), that were physically different from those obtained from other inpatient units in our hospital. Pulsed-field gel electrophoresis (PFGE) for K. pneumoniae (digested by XbaI) produced similar patterns excluding one control strain. PCR classification of the ESBL gene for K. pneumoniae revealed that all strains other than the control strain carried SHV and CTX-M-9. This result was reconfirmed by direct DNA sequencing. Although the outbreak of K. pneumoniae was considered to be "clonal," PFGE and PCR classification of the ESBL genes for E. coli uncovered at least six different "non-clonal" strains possessing individual ESBL gene patterns. According to the result of an antibiogram, the pattern of antimicrobial susceptibility was more variable for K. pneumoniae than for E. coli. CONCLUSIONS: Typing by PFGE and ESBL gene PCR analysis is practical for discriminating various organisms. In our cohort, two outbreaks were concomitantly spread with different transmission strategies, namely clonal and non-clonal, in the same unit. This might represent clinical evidence that transmissibility differs according to the type of strain. We speculated that patient-to-patient transmission of ESBL-E occurred according to the properties of each individual strain.

Acute keratoconjunctivitis due to contamination of contact lens care solution with histamine-producing Raoultella species: A case report.

RATIONALE: Contact lens storage cases are known to be contaminated by a significant number of bacteria. However, histamine-producing Raoultella species has not been reported to contaminate contact lens storage case. PATIENT CONCERNS: A 27-year-old woman with keratoconjunctivitis that developed in the left eye owing to a cosmetic contact lens and poor hygiene was referred to our hospital. The corrected visual acuity was hand motion. DIAGNOSES: Corneal infection other than Acanthamoeba keratitis (AK) and corneal hypoxia were excluded. INTERVENTIONS: We initiated empirical therapy for AK, although no cysts or trophozoites were detected in the cornea and in the lens care solution. Analysis of 16S rDNA sequences from the lens care solution yielded the highest homology with Raoultella species, which are histamine-producing bacteria. Histamine was estimated to be 492 ng/mL in the lens care solution. OUTCOMES: Her clinical course was distinct from that of usual AK cases. The corrected visual acuity increased up to (1.2) only 5 days after initiating empirical therapy. LESSONS: To our knowledge, this is the first report to indicate an association between histamine-producing bacteria and keratoconjunctivitis. We should pay an attention to the microbial contamination of contact lens storage cases by histamine producing bacteria.

Achromobacter buckle infection diagnosed by a 16S rDNA clone library analysis: a case report.

BACKGROUND: In clinical settings, bacterial infections are usually diagnosed by isolation of colonies after laboratory cultivation followed by species identification with biochemical tests. However, biochemical tests result in misidentification due to similar phenotypes of closely related species. In such cases, 16S rDNA sequence analysis is useful. Herein, we report the first case of an Achromobacter-associated buckle infection that was diagnosed by 16S rDNA sequence analysis. This report highlights the significance of Achromobacter spp. in device-related ophthalmic infections. CASE PRESENTATION: A 56-year-old woman, who had received buckling surgery using a silicone solid tire for retinal detachment eighteen years prior to this study, presented purulent eye discharge and conjunctival hyperemia in her right eye. Buckle infection was suspected and the buckle material was removed. Isolates from cultures of preoperative discharge and from deposits on the operatively removed buckle material were initially identified as Alcaligenes and Corynebacterium species. However, sequence analysis of a 16S rDNA clone library using the DNA extracted from the deposits on the buckle material demonstrated that all of the 16S rDNA sequences most closely matched those of Achromobacter spp. We concluded that the initial misdiagnosis of this case as an Alcaligenes buckle infection was due to the unreliability of the biochemical test in discriminating Achromobacter and Alcaligenes species due to their close taxonomic positions and similar phenotypes. Corynebacterium species were found to be contaminants from the ocular surface. CONCLUSIONS: Achromobacter spp. should be recognized as causative agents for device-related ophthalmic infections. Molecular species identification by 16S rDNA sequence analysis should be combined with conventional cultivation techniques to investigate the significance of Achromobacter spp. in ophthalmic infections.

Infectious conjunctivitis caused by Pseudomonas aeruginosa isolated from a bathroom.

BACKGROUND: The elucidation of the routes of transmission of a pathogen is crucial for the prevention of infectious diseases caused by bacteria that are not a resident in human tissue. The purpose of this report is to describe a case of suture-related conjunctivitis caused by Pseudomonas aeruginosa for which we identified the transmission route using pulsed-field gel electrophoresis (PFGE). CASE PRESENTATION: A 38-year-old man, who had undergone surgery for glaucoma 2 years ago previously, presented with redness, discomfort, and mucopurulent discharge in the right eye. A 9-0 silk suture had been left on the conjunctiva. A strain of P. aeruginosa was isolated from a culture obtained from the suture, and the patient was therefore diagnosed with suture-related conjunctivitis caused by P. aeruginosa. The conjunctivitis was cured by the application of an antimicrobial ophthalmic solution and removal of the suture. We used PFGE to survey of the indoor and outdoor environments around the patient's house and office in order to elucidate the route of transmission of the infection. Three strains of P. aeruginosa were isolated from the patient's indoor environment, and the isolate obtained from the patient's bathroom was identical to that from the suture. CONCLUSION: The case highlights the fact that an indoor environmental strain of P. aeruginosa can cause ocular infections.

Effects of indole-3-carbinol and phenethyl isothiocyanate on bile and pancreatic juice excretion in rats.

Bile and pancreatic juice contain a number of parameters for cancer chemoprevention. Indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC), which are hydrolytic products of brassica plants, have been established to be anti-cancer agents. Here, we developed a method for the continuous and selective sampling of bile and pancreatic juice, and the effects of I3C and PEITC on bile and pancreatic excretion and γ-glutamyl transpeptidase (γ-GTP) activity in the samples were investigated. Male Fisher 344 rats (eight weeks of age) were challenged intragastrically with I3C (150 mg/kg) or PEITC (160 mg/kg) for five days. Twenty-four hours after the final administration, cannulation was undertaken into the rats' bile and pancreatic ducts, and the bile and pancreatic juice were separately collected for 48 h. In this rat model, bile was stably excreted, and the bile and pancreatic excretion of the control rats was 21.9 ± 1.4 ml/48 h and 12.8 ± 1.7 ml/48 h, respectively. Bile excretion for the first 24 h significantly increased in the I3C- or PEITC-treated rats compared with the control rats. In the case of pancreatic juice, excretion during the first 24 h significantly increased in the PEITC-treated rats. In bile, γ-GTP activity was significantly increased for the first 24 h in the I3C- and PEITC-treated rats, but no difference was observed in the pancreatic juice. Increases of bile excretion and γ-GTP activity in bile might be a factor involved in the anti-cancer effect of I3C and PEITC. Our rat model described here is a useful tool for the study of cancer chemoprevention.

Bifidobacterium kashiwanohense sp. nov., isolated from healthy infant faeces.

Strains HM2-1 and HM2-2(T) were isolated from the faeces of a healthy infant and were characterized by determining their phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive, obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile rods. They did not grow at 15 or 45 °C in anaerobic bacterial culture medium, and their DNA G+C content was in the range 56-59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2(T) were positive for α/ß-galactosidases and α/ß-glucosidases but negative for ß-glucuronidase and cystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2(T) revealed the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate phosphoketolase shows that isolates HM2-1 and HM2-2(T) are members of the genus Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium. Strain HM2-2(T) was found to be related to Bifidobacterium catenulatum JCM 1194(T) (97.4 % 16S rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200(T) (97.2 %: 1472/1514 bp), Bifidobacterium dentium ATCC 27534(T) (96.7 %: 1459/1509 bp) and Bifidobacterium angulatum ATCC 27535(T) (96.5 %: 1462/1515 bp). The predominant cellular fatty acids of strains HM2-1 and HM2-2(T) were 16 : 0 and 18 : 1ω9c, with proportions greater than 18 % of the total. Phylogenetic analyses involving phenotypic characterization, DNA-DNA hybridization and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed. The type strain is HM2-2(T) ( = JCM 15439(T) = DSM 21854(T)).

Production of apoptosis-inducing substances from soybean protein by Clostridium butyricum: characterization of their toxic effects on human colon carcinoma cells.

Microbial metabolism of soybean constituents is known to produce novel active substances as a chemopreventive agent during the fermentation, and enterobacteria are expected to produce chemopreventive agents as a consequence of metabolizing soybean constituents in the intestinal tract. Then, the conditioned medium was prepared by culturing an enterobacterium Clostridium butyricum (C. butyricum) with soybean protein, and its direct effect on human colon carcinoma HCT116 cells was examined. The conditioned medium was shown to induce the cell death, and suggested to contain novel apoptosis-inducing substances. Thus, enterobacteria are predicted to produce anti-tumor substances from food-derived proteins within the intestinal tract.

Suppressive effect of Clostridium perfringens-produced heat-stable substance(s) on proliferation of human colon adenocarcinoma HT29 cells in culture.

Clostridium perfringens has been regarded as one of the intestinal bacteria increasing colon cancer risk. In previous studies, we have shown that the oral administration of C. perfringens culture medium can inhibit the mutagen-induced formation of pre-neoplastic lesions in rat colon, thus proposing the existence of factor(s) preventing colon tumorigenesis in this culture medium. However, the properties of effective factor(s) and the mechanism of this inhibitory action still remain to be investigated. Then, the effect of C. perfringens culture medium on human colon adenocarcinoma HT29 cells was examined. The exposure of HT29 cells to C. perfringens culture medium was found to suppress the proliferation of these cells probably through the reduction of immediate early gene egr-1 expression. These observations suggest that C. perfringens culture medium has a cytostatic action on colon tumor cells, which may be responsible for the prevention of pre-neoplastic formation in rat colon.

Inhibitory effects of asiatic acid and CPT-11 on growth of HT-29 cells.

Asiatic acid is a pentacyclic triterpene contained in medicinal plants. The cytotoxic effect of this compound and its augmentative effect on the anticancer drug irinotecan hydrochloride (CPT-11) were investigated in the human colon adenocarcinoma cell line HT-29. Asiatic acid dose-dependently showed cytotoxicity in HT-29 cells. DNA fragmentation, annexin-positive apoptotic cells, and caspase-3 activation were observed in a dose-dependent manner. A caspase-3 inhibitor suppressed the DNA ladder formation in a concentration-dependent manner. Bcl-2 and Bcl-XL proteins were decreased by asiatic acid treatment. These results indicate that asiatic acid induced apoptosis in HT-29 cells via caspase-3 activation. Cytotoxic effects of combined treatment with CPT-11 and asiatic acid on HT-29 cells were further examined. Simultaneous treatment or sequential exposure first to asiatic acid and then to CPT-11 showed an additive effect. Synergism was observed when cells were first exposed to CPT-11 and then to asiatic acid. These results suggest that asiatic acid can be used as an agent for increasing sensitivity of colon cancer cells to treatment with CPT-11 or as an agent for reducing adverse effects of CPT-11.

Effects of high amylose maize starch and Clostridium butyricum on metabolism in colonic microbiota and formation of azoxymethane-induced aberrant crypt foci in the rat colon.

High amylose maize starch (HAS) is not digested in the small intestine and most of it reaches the large intestine. In the large intestine, HAS is fermented by intestinal bacteria, resulting in production of short-chain fatty acids (SCFA), particularly butyrate. Clostridium butyricum can utilize HAS and produce butyrate and acetate. It has been proposed that butyrate inhibits carcinogenesis in the colon. In this study, we examined the inhibitory effects of HAS and C. butyricum strain MIYAIRI588 (CBM588) on azoxymethane-induced aberrant crypt foci (ACF) formation in rats. In the group of rats administered only CBM588 spores, the concentration of butyrate in the cecum increased, but there was no decrease in the number of ACF. In the group of rats fed an HAS diet, a decrease in the number of ACF was observed, and in the group of rats administered HAS and CBM588, the number of ACF decreased significantly. In these two groups, the concentrations of acetate and propionate in intestinal contents significantly increased, but the concentration of butyrate did not change. It was found that the beta-glucuronidase activity level of colonic contents decreased significantly in the two groups of rats fed HAS. This study showed that HAS and CBM588 changed the metabolism of colonic microbiota and decreased the level of beta-glucuronidase activity, phenomena that may play a role in the inhibition of ACF formation in the rat colon.

Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice.

Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity.

Antimutagenicity of Murdannia loriformis in the Salmonella mutation assay and its inhibitory effects on azoxymethane-induced DNA methylation and aberrant crypt focus formation in male F344 rats.

An 80% ethanol extract of Murdannia loriformis, a Thai medicinal plant, was examined for antimutagenic activity and cancer chemopreventive activity. In the Salmonella mutation assay, the extract showed antimutagenicity against 2-amino-3-methylimidazo [4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, 2-aminodipyrido[1,2-a:3',2'-d]imidazole, 2-aminoanthracene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol acetate and reduced their mutagenicities to 31.4-67.9% at the dose of 10 mg/plate. However, it did not inhibit the mutagenicities of 2-amino-9H-pyrido[2,3-b]indole, 2-amino-3-methyl-9 H-pyrido[2,3-b]indole, benzo[a]pyrene,N-ethyl-N'-nitro-N-nitrosoguanidine and 1-nitropyrene. The extract itself showed no mutagenicity. The chemopreventive activity of M. loriformis was examined using azoxymethane (AOM)-induced aberrant crypt focus (ACF) formation in the colon of F344 rats. The extract at doses of 0.1-1.0 g/kg wt significantly inhibited ACF formation in the initiation stage (21-51%), although it was more effective at a lower dose. In the post-initiation stage, the extract also tended to inhibit ACF formation (12-27%) and significantly decreased the number of larger ACFs that have more than 3 aberrant crypts per focus. The extract inhibited the formation of O6-methylguanine and N7-methylguanine in the colonic mucosa and muscular layers but not or increased in the liver. These results indicate that M. loriformis extract has antimutagenic activity toward various known mutagens and that it inhibits AOM-induced ACF formation both in the initiation and post-initiation stages in the rat colon.