Motomichi Takahashi

Molecular and Microbiological Characterization of Clostridium difficile Isolates from Single, Relapse, and Reinfection Cases

ABSTRACT In this study, we investigated the correlation between the microbiological characteristics of Clostridium difficile clinical isolates and the recurrence of C. difficile-associated disease (CDAD). Twenty C. difficile isolates recovered from 20 single infection cases and 53 isolates from 20 recurrent cases were analyzed by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping, and the cytotoxicity, antimicrobial susceptibility, and sporulation/germination rates of the isolates were examined. Recurrent cases were divided into relapse or reinfection cases by the results of C. difficile DNA typing. Among the 20 recurrent cases, 16 cases (80%) were identified to be relapse cases caused by the initial strain and the remaining 4 cases (20%) were identified to be reinfection cases caused by different strains. All 73 isolates were susceptible to both vancomycin and metronidazole, but resistance against clindamycin, ceftriaxone, erythromycin, and ciprofloxacin was found in 87.7%, 93.2%, 87.7%, and 100% of the isolates, respectively. No correlations between DNA typing group, cytotoxicity, and sporulation rate of isolates and infection status, i.e., single, relapse, or reinfection, were observed. However, the isolates recovered from relapse cases showed a significantly higher germination rate when incubated in medium lacking the germination stimulant sodium taurocholate. These results indicate that the germination ability of C. difficile may be a potential risk factor for the recurrence of CDAD.

Experimental infection of germ-free mice with hyper-toxigenic enterohaemorrhagic Escherichia coli O157:H7, strain 6.

A mouse enterohaemorrhagic Escherichia coli (EHEC) infection model was developed with a combination of germ-free (GF) mice and hyper-toxigenic EHEC (HT-EHEC) O157:H7 strain 6. The HT-EHEC strain 6 produced both Shiga-like toxin (SLT)-1 1.0 microg/mI and SLT-2 8.2 microg/ml in vitro. Eight-week-old germ-free mice were inoculated intragastrically with 5.0 x 10(7) cfu of HT-EHEC strain 6. All GF mice challenged with the HT-EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days after the challenge, culminating in death within 5 days. The HT-EHEC colonised well at a level of 10(8) - 10(9) cfu/g of faeces 5 days after the challenge. Both SLT-1 and SLT-2 were demonstrated in the faeces of the mice for 5 days after challenge. Histological examination of the colons of the infected mice showed congestion of the lamina propria mucosa, mild inflammatory cell infiltration and goblet cell depletion. In proximal tubules of the renal cortex, epithelial swelling with scattered necrotic cells was recognised. Endothelial swelling and mononuclear cell infiltration were also observed in the glomeruli. The brain showed acute neuronal necrosis in the cerebrum and slight loss of Purkinje cells in the cerebellum. Passive immunisation with anti-SLT antisera prolonged the life of the mice without anyneural symptoms. Microscopically, all tissue specimens from the passively immunised mice were not remarkable. These results indicate that the infection of GF mice with HT-EHEC is a useful animal model to study the pathogenicity of SLT-producing E. coliand the toxins.

Efficacy of Clostridium butyricum preparation concomitantly with Helicobacter pylori eradication therapy in relation to changes in the intestinal microbiota

Antibiotic associated diarrhea due to human intestinal microbiota abnormalities is a side effect of H. pylori eradication therapy. We examined intestinal microbiota changes during H. pylori eradication therapy and the preventive effect of CBM588 as a probiotic agent. Nineteen patients with gastro‐duodenal ulcer were randomly divided into three groups: group A (without probiotics), group B (with regular doses of CBM588) and group C (with double doses of CBM588). The incidence of diarrhea and soft stools during H. pylori eradication therapy was 43% in group A and 14% in group B, while none of the patients in group C reported diarrhea or soft stools. Both bacterial counts and detection rates of bifidobacteria and/or obligate anaerobe were decreased by eradication therapy. However, bacterial counts of obligate anaerobes in group C were significantly higher than in group A (P < 0.05). Additionally, during eradication therapy C. difficile toxin A was detected in both group A and group B but not in group C.

Induction of secretion of interleukin-8 from human gastric epithelial cells by heat-shock protein 60 homologue of Helicobacter pylori

Escherichia coli cells expressing fusion proteins consisting of beta-galactosidase and bacterial heat-shock protein (HSP) 60 of E. coli, Yersinia enterocolitica or Helicobacter pylori were constructed, and designated as HY1, HY2 or HY3, respectively. Fusion proteins prepared from HY2 and HY3 induced secretion of interleukin-8 (IL-8) from human gastric epithelial KATOIII cell cultures. On the other hand, the parent strain (E. coli pop2136), PEX (pop2136 transformed by vector) and fusion protein prepared from HY1 did not induce IL-8 secretion from KATOIII cells. Other human gastric (MKN45) and non-gastric cell lines (Int 407 and A549) did not secrete IL-8 following treatment with these proteins. These results indicate that H. pylori HSP60 induces IL-8 secretion from human gastric cells and the levels of IL-8 differ among the various gastric cell lines, suggesting that HSP60 might be an important virulence factor associated with chronic gastric inflammation following H. pylori infection in man.

Inhibition of the cytotoxic effect of Clostridium difficile in vitro by Clostridium butyricum MIYAIRI 588 strain

In contrast to most modern pharmaceuticals, probiotics are used in many parts of the world with little or no research data on the complex system of interactions that each strain may elicit in the human body. Research on probiotics has recently become more significant, as probiotics have begun to be prescribed by clinicians as an alternative for some gut infections, especially when antibiotics are contraindicated. This study attempted to elucidate the inhibitory interaction between the Japanese probiotic strain Clostridium butyricum MIYAIRI 588 (CBM588) and the hospital pathogen Clostridium difficile, which is responsible for a large proportion of antibiotic-associated diarrhoea and colitis. CBM588 has previously shown effectiveness against C. difficile in vivo, and here it was found that the toxicity of C. difficile in in vitro co-culture with CBM588 was greatly decreased or absent. This was dependent on the inoculation ratio and was not accounted for by the small degree of growth and mRNA inhibition observed. CBM588 and its cell-free supernatant also had no effect on toxin already secreted into the culture medium, and culture of the two strains separated by a semi-permeable membrane resulted in loss of the inhibition. Therefore, it was concluded that the detoxification probably occurred by the inhibition of toxin protein production and that this required close proximity or contact between the two species. The low-pH conditions caused by organic acid secretion were also observed to have inhibitory effects on C. difficile growth, metabolism and toxicity.

Effect of Bacterial Flora on Postimmunization Gastritis following Oral Vaccination of Mice with Helicobacter pylori Heat Shock Protein 60

ABSTRACT In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increased in all animals at 10 weeks p.i. Anti-H. pylori HSP60 immunoglobulin G was detected in serum at 3 weeks p.i. in mice vaccinated with either H. pylori HSP60 or GroEL. Significant increases in the gastritis scores were observed only in SPF mice immunized with H. pylori HSP60. These results indicate that oral vaccination with H. pylori HSP60 has partial protective effects on subsequent H. pylori infection but also induces postimmunization gastritis. However, GF mice immunized with H. pylori HSP60 did not suffer from severe gastritis. Therefore, the presence of bacterial flora appears to contribute to the induction of postimmunization gastritis.

Studies of the effect of Clostridium butyricum on Helicobacter pylori in several test models including gnotobiotic mice

The interaction between Clostridium butyricum and Helicobacter pylori was examined in vitro and in vivo. The culture supernate of C. butyricum MIYAIRI 588 inhibited the growth of H. pylori even when its pH was adjusted to 7.4. The bactericidal effect of butyric acid on H. pylori was stronger than that of lactic, acetic or hydrochloric acids. Flow cytometric analysis showed that pre-incubation of gastric epithelial (MKN45) cells with H. pylori and C. butyricum inhibited the adhesion of H. pylori to the cells. Persistent infection with H. pylori in the gastric mucosa of germ-free mice was observed for 5 weeks. Cure of persistent infection with H. pylori in the gnotobiotic mice was demonstrated following infection with C. butyricum. The probiotic agent, C. butyricum MIYAIRI 588 may have some beneficial effects on H. pylori infection.

Intestinal microbiota and secretory immunoglobulin A in feces of exclusively breast‐fed infants with blood‐streaked stools

Episodes of blood‐streaked stools are not uncommon in exclusively breast‐fed infants under 6 months of age. Such bleeding is thought to be associated with food protein‐induced proctocolitis, however the pathomechanism remains unclear. The aim of this study was to investigate intestinal microbiota and secretory immunoglobulin A in the feces of exclusively breast‐fed infants with blood‐streaked stools. Fecal specimens from 15 full‐term infants with blood‐streaked stools and 15 breast‐fed healthy infants were studied and the results compared. All infants had been delivered vaginally and exclusively breast‐fed. The fecal microbiota were investigated by phylogenetic analysis combined with culture methods for some bacterial species, and feces were assessed for the presence of fecal secretory immunoglobulin A by enzyme‐linked immunosorbent assay. Phylogenetic cluster analysis revealed four major clusters of fecal bacteria, cluster A being found only in healthy infants. The Bacteroides fragilis group was observed more frequently in controls than in patients (P < 0.05). In the controls, the predominant species belonging to the Enterobacteriaceae group was Escherichia coli, whereas in the patients it was Klebsiella (P < 0.05). Concentrations of secretory immunoglobulin A were high in one third of the healthy controls. In conclusion, the pathomechanism of rectal bleeding in exclusively breast‐fed infants may be related to differences in the composition of their intestinal flora.

Genetic relatedness between Japanese and European isolates of Clostridium difficile originating from piglets and their risk associated with human health

Clostridium difficile colonization in pig intestine has been a public health concern. We analyzed C. difficile prevalence among piglets in Japan to clarify their origin and extent of the associated risk by using molecular and microbiological methods for both swine and human clinical isolates and foreign isolates. C. difficile was isolated from 120 neonatal piglet fecal samples. Toxin gene profile, antimicrobial susceptibilities, PCR ribotype, and multiple-locus variable-number tandem-repeat analysis (MLVA) type of swine isolates were determined and compared with those of human clinical and foreign isolates. One-hundred C. difficile strains were isolated from 69 (57.5%) samples, and 61 isolates (61%) were toxin gene-positive. Some isolates were resistant to antimicrobials, contributing to antibiotic-associated diarrhea by C. difficile. These results suggest that C. difficile, prevalent among Japanese pigs, is a potential risk for antibiotic-associated diarrhea. Furthermore, PCR ribotype 078 (12 isolates), which has been linked to multiple outbreaks worldwide, was the third-most frequently isolated of the 14 PCR ribotypes identified. Moreover, MLVA revealed that all 12 PCR ribotype 078 isolates were genetically related to European PCR ribotype 078 strains found in both humans and pigs. To date, in Japan, many breeding pigs have been imported from European countries. The genetic relatedness of C. difficile isolates of Japanese swine origin to those of European origin suggests that they were introduced into Japan via imported pigs.

Safety assessment of the Clostridium butyricum MIYAIRI 588® probiotic strain including evaluation of antimicrobial sensitivity and presence of Clostridium toxin genes in vitro and teratogenicity in vivo

Probiotics are live microorganisms ingested for the purpose of conferring a health benefit on the host. Development of new probiotics includes the need for safety evaluations that should consider factors such as pathogenicity, infectivity, virulence factors, toxicity, and metabolic activity. Clostridium butyricum MIYAIRI 588® (CBM 588®), an anaerobic spore-forming bacterium, has been developed as a probiotic for use by humans and food animals. Safety studies of this probiotic strain have been conducted and include assessment of antimicrobial sensitivity, documentation of the lack of Clostridium toxin genes, and evaluation of CBM 588® on reproductive and developmental toxicity in a rodent model. With the exception of aminoglycosides, to which anaerobes are intrinsically resistant, CBM 588® showed sensitivity to all antibiotic classes important in human and animal therapeutics. In addition, analysis of the CBM 588® genome established the absence of genes for encoding for α, β, or ε toxins and botulin neurotoxins types A, B, E, or F. There were no deleterious reproductive and developmental effects observed in mice associated with the administration of CBM 588®. These data provide further support for the safety of CBM 588® for use as a probiotic in animals and humans.