Transcriptional Downregulation of a Type III Secretion System under Reducing Conditions in Bordetella pertussis.

Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA-) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA- medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA- medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCE The type III secretion system (T3SS) of Bordetella pertussis forms a needlelike structure that protrudes from the bacterial cell surface. B. pertussis uses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.

Acanthamoeba castellanii supports extracellular survival of Helicobacter pylori in co-culture.

This study aimed to demonstrate whether Helicobacter pylori is able to survive in co-culture with a protozoan, Acanthamoeba castellanii, in order to further investigate a possible aqueous environmental mode of transmission. Numbers of H. pylori in co-culture with A castellanii were assessed by colony forming unit (CFU) assay and cell morphology was observed by electron microscopy. Viable and intact H. pylori in co-culture were detected and the number of H. pylori in co-culture with A. castellanii was significantly higher than in bacterial single culture. It was also shown that co-culture of H. pylori with A. castellanii physically separated by a filter membrane negated this survival effect, suggesting that adherence of H. pylori to A. castellanii affects its survival. Scanning electron microscopy revealed helical forms of H. pylori in co-culture with A. castellanii, but not in single culture. These results imply that mutual interaction between H. pylori and A. castellanii in the environment is critical for survival of H. pylori. In addition, the H. pylori gene expression profile was found to differ between single and co-cultured cells using RNA-sequence analysis.

Silviavirus phage ɸMR003 displays a broad host range against methicillin-resistant Staphylococcus aureus of human origin.

The emergence of life-threatening methicillin-resistant Staphylococcus aureus (MRSA) has led to increased interest in the use of bacteriophages as an alternative therapy to antibiotics. The success of phage therapy is greatly dependent on the selected phage possessing a wide host range. This study describes phage ɸMR003 isolated from sewage influent at a municipal wastewater treatment plant in Tokyo, Japan. ɸMR003 could infect 97% of 104 healthcare- and community-associated MRSA strains tested, compared with 73% for phage ɸSA012, which has a broad host range against bovine mastitis S. aureus. Genome analysis revealed that ɸMR003 belongs to the genus Silviavirus which has not been studied extensively. ɸMR003 recognizes and binds to wall teichoic acid (WTA) of S. aureus during infection. In silico comparisons of the genomes of ɸMR003 and ɸSA012 revealed that ORF117 and ORF119 of ɸMR003 are homologous to the putative receptor-binding proteins ORF103 and ORF105 of ɸSA012, with amino acid similarities of 75% and 72%, respectively. ORF104, which is an N-acetylglucosaminidase found in the ɸMR003 tail, may facilitate phage's infection onto the WTA-null S. aureus RN4220. The differences in tail and baseplate proteins may be key contributing factors to the different host specificities of ɸMR003 and ɸSA012. ɸMR003 showed strong adsorptivity, but not infectivity, against S. aureus SA003, which may be influenced by the bacterium's restriction modification system. This study expands our knowledge of the genomic diversity and host specificity of Silviavirus, which is a potential phage therapy candidate for MRSA infections.

Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion.

Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pyloriIMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System.

UNLABELLED: Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg(+) mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCE: Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis.

Community-Acquired Methicillin-Resistant Staphylococcus aureus Strain Positive for the Panton-Valentine Leucocidin Gene in a Middle-Aged Patient With Multiple Septic Pulmonary Emboli.

A 59-year-old man suffered from fever and chest pain for three days following an accidental bite to a lip ulcer. His lower lip showed swelling and tenderness, and chest computed tomography showed multiple bilateral nodules. He was diagnosed with septic pulmonary embolism and a lip abscess, and blood, sputum, and lip abscess cultures confirmed the presence of methicillin-resistant Staphylococcus aureus (MRSA). Despite the initiation of vancomycin, he rapidly developed respiratory failure and septic shock, necessitating intubation and noradrenaline support. Gentamicin was added on the seventh day of admission due to an insufficient effect, and vancomycin was switched to linezolid on the 14th day of admission. However, his respiratory failure persisted as bilateral pneumothorax developed. Blood culture was negative on the 14th day after admission, but the patient died on the 15th day after admission. The MRSA isolate was tested for the presence of the Panton-Valentine leukocidin (PVL) gene in conjunction with the USA300 strain. The prevalence of community-acquired (CA)-MRSA in the USA300 clone is increasing but still low in Japan, and this type of infection is commonly observed in people of all ages; this case is the first instance reported in Japan of a middle-aged patient with septic pulmonary embolism. Given the anticipated global increase in CA-MRSA infection caused by the USA300 clone and the emergence of USA300 with altered pathogenicity, it may be crucial to suspect PVL-positive CA-MRSA infections even in middle-aged or elderly patients presenting with septic pulmonary embolism as community infections.

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which B. pertussis causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in B. pertussis, and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of fim3 and bsp22, which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes - including fim3 and bsp22 - were expressed in the Bvg(+) phase during which the BvgAS two-component system was activated. Although fim3 and bsp22 were downregulated in a (p)ppGpp-deficient mutant, normal expression of fhaB, cyaA and ptxA persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences B. pertussis pathogenicity.

Molecular and microbiological characterization of Clostridium difficile isolates from single, relapse, and reinfection cases.

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.

Comparative analysis of gastric bacterial microbiota in Mongolian gerbils after long-term infection with Helicobacter pylori.

Quantitative (qt) real time PCR using 16SrDNA primers is useful for determination of the bacterial composition of the gastric microbiota in Mongolian gerbils. The aim of this study was to determine the change in the gastric microbiota after long-term infection with Helicobacter pylori. One year after inoculation with H. pylori, five gerbils were determined as H. pylori-positive and 6 gerbils H. pylori-negative by culture and real time qt PCR methods. The gastric microbiota of each group of gerbils was also compared with that of 6 gerbils uninfected with H. pylori. DNA from the Atopobium cluster, Bifidobacterium spp., Clostridium coccoides group, Clostridium leptum subgroup, Enterococcus spp. and Lactobacillus spp. were detected in the gastric mucus of both infected and uninfected gerbils. In contrast, Eubacterium cylindroides group and Prevotella spp. were detected only in H. pylori-negative gerbils. The numbers of C. leptum subgroup, C. coccoides group and Bifidobacterium spp. in gastric mucus of H. pylori-negative Mongolian gerbils were significantly lower than those in non-infected gerbils. The results obtained suggest that the composition of gastric indigenous microbiota in Mongolian gerbils may be disturbed by long-term infection with H. pylori, and that these changes may in fact inhibit H. pylori infection.

Effect of the steroid receptor antagonist RU486 (mifepristone) on an IFNγ-induced persistent Chlamydophila pneumoniae infection model in epithelial HEp-2 cells.

We have previously demonstrated that the steroid receptor antagonist mifepristone (RU486) causes growth inhibition of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells. In the present study, we assessed the efficacy of treatment with RU486 against persistent C. pneumoniae infection in interferon (IFN)γ-treated HEp-2 cells. Assessment of bacterial growth modification, the number of infectious progenies, the formation of inclusions, and the expressions of the C. pneumoniae genes 16S rRNA and hsp60 were investigated in cells with or without IFNγ stimulation in the presence of RU486, using an inclusion-forming unit (IFU) assay, fluorescence microscopic analysis, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that RU486 treatment produced growth inhibition and an absence of C. pneumoniae gene expression in normal HEp-2 cells and that this treatment failed to inhibit C. pneumoniae growth in HEp-2 cells stimulated with IFNγ. These results indicate that treatment with RU486 had a limited effect on C. pneumoniae growth only during the active developmental stage of the bacteria, suggesting that the bacterial target molecule of RU486 is not expressed sufficiently during persistent infection in which there is an aberrant developmental cycle. Thus, our findings provide valuable insight into the complicated chlamydial biological processes involved in the recurrent cycling between normal and persistent infections.

Bcr4 Is a Chaperone for the Inner Rod Protein in the Bordetella Type III Secretion System.

Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required to stabilize some T3SS components or localize them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pulldown assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pulldown assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we show that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS. IMPORTANCE The type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We show that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.

Modification of the immune response by bacteriophages alters methicillin-resistant Staphylococcus aureus infection.

There is an urgent need to develop phage therapies for multidrug-resistant bacterial infections. However, although bacteria have been shown to be susceptible to phage therapy, phage therapy is not sufficient in some cases. PhiMR003 is a methicillin-resistant Staphylococcus aureus phage previously isolated from sewage influent, and it has demonstrated high lytic activity and a broad host range to MRSA clinical isolates in vitro. To investigate the potential of phiMR003 for the treatment of MRSA infection, the effects of phiMR003 on immune responses in vivo were analysed using phiMR003-susceptible MRSA strains in a mouse wound infection model. Additionally, we assessed whether phiMR003 could affect the immune response to infection with a nonsusceptible MRSA strain. Interestingly, wounds infected with both susceptible and nonsusceptible MRSA strains treated with phiMR003 demonstrated decreased bacterial load, reduced inflammation and accelerated wound closure. Moreover, the infiltration of inflammatory cells in infected tissue was altered by phiMR003. While the effects of phiMR003 on inflammation and bacterial load disappeared with heat inactivation of phiMR003. Transcripts of proinflammatory cytokines induced by lipopolysaccharide were reduced in mouse peritoneal macrophages. These results show that the immune modulation occurring as a response to the phage itself improves the clinical outcomes of phage therapy.

Biological properties of Staphylococcus virus ΦSA012 for phage therapy.

Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

Ciliates promote the transfer of the gene encoding the extended-spectrum ß-lactamase CTX-M-27 between Escherichia coli strains.

OBJECTIVES: The mechanism by which Escherichia coli acquires multidrug resistance genes from other bacteria in the natural environment or livestock is still unclear. The ability of ciliates to promote the transfer of genes encoding extended-spectrum ß-lactamases (ESBLs) between the CTX-M-27 donor and clinically isolated recipient E. coli strains was investigated. METHODS: Equal amounts (∼10(9) cfu) of donor cefotaxime-resistant E. coli and recipient ciprofloxacin-resistant E. coli strains were mixed together in the presence or absence of 10(5) ciliates in Page's amoeba saline for 24 h, in the presence or absence of certain drugs (cytochalasin D, cycloheximide and latrunculin B). RESULTS: Gene transfer frequency in the presence of ciliates was estimated at ∼10(-6); in the absence of ciliates it was ∼10(-10). Protein synthesis (cycloheximide) or phagocytosis (cytochalasin D or latrunculin B) inhibitors significantly reduced the frequency of gene transfer. CONCLUSIONS: Ciliates promote the transfer of genes encoding ESBLs between E. coli strains, implying that the presence of ciliates may provide a significant impact on emerging multidrug-resistant bacteria.

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori.

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air-liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2-3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.

Dual interleukin-17A/F deficiency protects against acute and chronic response to cigarette smoke exposure in mice.

IL-17A and IL-17F are both involved in the pathogenesis of neutrophilic inflammation observed in COPD and severe asthma. To explore this, mice deficient in both Il17a and Il17f and wild type (WT) mice were exposed to cigarette smoke or environmental air for 5 to 28 days and changes in inflammatory cells in bronchoalveolar lavage (BAL) fluid were determined. We also measured the mRNA expression of keratinocyte derived chemokine (Kc), macrophage inflammatory protein-2 (Mip2), granulocyte-macrophage colony stimulating factor (Gmcsf) and matrix metalloproteinase-9 (Mmp9 ) in lung tissue after 8 days, and lung morphometric changes after 24 weeks of exposure to cigarette smoke compared to air-exposed control animals. Macrophage counts in BAL fluid initially peaked at day 8 and again on day 28, while neutrophil counts peaked between day 8 and 12 in WT mice. Mice dual deficient with Il17a and 1l17f showed similar kinetics with macrophages and neutrophils, but cell numbers at day 8 and mRNA expression of Kc, Gmcsf and Mmp9 were significantly reduced. Furthermore, airspaces in WT mice became larger after cigarette smoke exposure for 24 weeks, whereas this was not seen dual Il17a and 1l17f deficient mice. Combined Il17a and Il17f deficiency resulted in significant attenuation of neutrophilic inflammatory response and protection against structural lung changes after long term cigarette smoke exposure compared with WT mice. Dual IL-17A/F signalling plays an important role in pro-inflammatory responses associated with histological changes induced by cigarette smoke exposure.

Analysis of the microflora in the stomach of Mongolian gerbils infected with Helicobacter pylori.

BACKGROUND AND AIMS: Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences. The presence of some gastric flora with a suppressive effect on H. pylori suggests inhibitory microflora against H. pylori infection. The aim of the present study was to analyze the microflora in the stomach of Mongolian gerbils with H. pylori infection. METHODS: H. pylori ureA was detected by polymerase chain reaction (PCR) in the fecal samples of infected Mongolian gerbils. H. pylori was isolated from the gastric mucosa of the gerbils by microaerophilic cultivation. Gastric microflora were isolated by aerobic and anaerobic culture, and the identification of gastric bacterial species was performed by API20E and API20A. RESULTS: Oral administration of H. pylori TK1402 induced colonization and gastric inflammation of the stomach of the Mongolian gerbils. According to the frequency of detection of H. pylori ureA in fecal samples, the gerbils were divided into three groups (frequently detected, moderately detected and infrequently detected). According to the analysis of the gastric microflora in the frequently and infrequently detected groups, Lactobacillus spp. and Eubacterium limosum were isolated from the former and latter group, respectively. CONCLUSION: Some gastric flora, such as Lactobacillus spp., may inhibit colonization by H. pylori.

Assessment of in vitro biofilm formation by Helicobacter pylori.

BACKGROUND AND AIMS: Biofilms are surface-bound communities of bacterial cells that are implicated in their survival. As with various bacteria studied to date, Helicobacter pylori can have an alternate lifestyle as a biofilm. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. However, the mechanisms of its biofilm development remain unclear. We analyzed the basic characteristics of the biofilm-forming ability in strain TK1402. METHODS: In order to characterize the biofilm-forming ability of the H. pylori strains, auto-aggregation, motility and hydrophobicity, which are important factors in biofilm formation by other bacteria, were analyzed. Further, we tested whether cell growth participated in biofilm formation in strain TK1402. RESULTS: There were no significant differences in the auto-aggregation, motility and hydrophobicity of strain TK1402 compared with the other strains. On the other hand, pre-culture of this strain for 24-48 h resulted in decreased biofilm formation. CONCLUSION: TK1402 is a strong biofilm-forming strain of H. pylori in Brucella broth supplemented with 7% fetal calf serum. It is possible that biofilm-forming cell growth is a principal factor in biofilm development.

In vitro antibacterial activity of Phx-3 against Helicobacter pylori.

Phx-3, one of the phenoxazine derivatives, is reported to have inhibitory effect on Mycobacterium species and Chlamydia pneumoniae but not Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes. The bactericidal activities of Phx-3 against Helicobacter pylori strains have not been assessed. Then, we measured minimum inhibitory concentration of Phx-3 for Helicobacter strains and assessed the morphological and biochemical effects of Phx-3 on H. pylori. In present study, it has shown that H. pylori strains including clarithromycin resistant strain and Helicobacter musterae were killed effectively by the treatment with Phx-3. Furthermore, severe morphological changes such as membrane blebbing and formation of hollows in H. pylori were detected. In addition, induction of heat shock protein 60 was observed. Taken together, Phx-3 has antibacterial activity against Helicobacter pylori.

Effect of probiotic bacterial strains of Lactobacillus, Bifidobacterium, and Enterococcus on enteroaggregative Escherichia coli.

The effects of nine probiotic strains of Lactobacillus, Bifidobacterium, and Enterococcus on the growth, adhesion activity, and biofilm formation of enteroaggregative Escherichia coli (EAggEC) were examined. The culture supernatant of the E. faecium strain, with or without pH adjustment to a neutral pH, had a strong bactericidal effect on EAggEC, including induction of membrane damage and cell lysis. Supernatants of the L. casei ss. casei and L. casei ss. rhamnosus strains also had a bactericidal effect on EAggEC, but this activity was abolished by pH adjustment to a neutral pH. No inhibitory effect of the culture supernatants of Bifidobacterium or E. faecalis strains was detected. Adhesion of EAggEC to intestinal epithelial cells was not inhibited by the bacterial strains tested. Two strains of L. casei enhanced EAggEC biofilm formation, which was characterized by increased bacterial proliferation. These results suggest that the three different bacterial species; Lactobacillus, Bifidobacterium, and Enterococcus, have different effects on EAggEC, and that further analysis is required for the practical use of these bacteria as probiotics against EAggEC infection.