Focused light introduction into transmission electron microscope via parabolic mirror.

We developed a novel light optics system installed in a scanning transmission electron microscope (STEM) to introduce a focused light accurately adjusted at the electron beam irradiation position using a parabolic mirror. With a parabolic mirror covering both the upper and lower sides of the sample, the position and focus of the light beam can be evaluated by imaging the angular distribution of the transmitted light. By comparing the light image and the electron micrograph, the irradiation positions of the electron beam and the laser beam can be accurately adjusted to each other. The size of the focused light was confirmed to be within a few microns from the light Ronchigram, which is consistent with the simulated light spot size. The spot size and position alignment were further confirmed by laser-ablating only a targeted polystyrene particle without damaging the surrounding particles. When using a halogen lamp as the light source, this system allows investigating optical spectra in comparison with cathodoluminescence (CL) spectra at exactly the same location.

The comparison of the intensity of human intestinal spirochetes between Brachyspira pilosicoli and Brachyspira aalborgi infections.

The agglutination titers of Brachyspira pilosicoli (B. pilosicoli) and Brachyspira aalborgi (B. aalborgi) were examined in colitis patients with human intestinal spirochetes. Among three cases of colitis patients, the titer of B. pilosicoli was extremely high in two cases while the titer of B. aalborgi was extremely high in one case. These three cases had symptoms of colitis, such as watery diarrhea, and we diagnosed the case as Brachyspira- related colitis. These findings suggest that the agglutination titers of Brachyspira may be useful in cases of Brachyspira- related colitis. Severe symptoms, such as abdominal pain and diarrhea, were observed in cases with high antibody titer of B. aalborgi, as well as B. pilosicoli, indicating that B. aalborgi could also cause symptomatic colitis.

Simultaneous isolation of two species, Brachyspira pilosicoli and Brachyspira aalborgi, from a patient with ulcerative colitis.

We succeeded in the simultaneous isolation of Brachyspira (B.) aalborgi and B. pilosicoli from a patient with ulcerative colitis. B. pilosicoli grew quickly and formed colonies within 7 days, while the growth of B. aalborgi was very slow and took over 21 days. Simultaneous isolation of B. pilosicoli and B. aalborgi from a common specimen is generally recognized to be difficult, mainly due to differences in their growth requirements and the growth rates. However, we succeeded in isolating both species from a patient with ulcerative colitis and this is first evidence. The present results suggest that ulcerative colitis may be caused by simultaneous infection with B. pilosicoli and B. aalborgi.

A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Drug-susceptibility of isolates of Brachyspira hyodysenteriae isolated from colonic mucosal specimens of pigs collected from slaughter houses in Japan in 2009.

Twenty nine isolates identified as Brachyspira hyodysenteriae were most susceptible to carbadox and metronidazole, whereas they were resistant to macrolides. The isolates showed intermediate susceptibility to tiamulin, lincomycin, penicillin G, ampicillin, chloramphenicol, tetracycline, enrofloxacin and valnemulin, with MIC50 values ranging from 0.39 to 3.13.

Human intestinal spirochaetosis in two ulcerative colitis patients.

A histological examination of colonic biopsies of the longitudinal and irregularly-shaped ulcerative lesions of a 37-year-old man and 61-year-old man with ulcerative colitis showed so-called "fringe formation," a typical finding of Brachyspira infection. The antibody titer to Brachyspira aalborgi showed marked elevation in both cases, and the patients were each treated with 1,000 mg of metronidazole for 14 days. Colonoscopy performed after treatment showed an improvement in the ulcerative lesions in both patients. These results indicate the possibility that intestinal spirochaetosis infection should be considered as an infectious complication in patients with ulcerative colitis receiving long-term steroid therapy.

Human intestinal spirochetosis in an immunocompromised host: evaluation of eradication therapy by endoscopy, histopathology and bacteriology.

Human intestinal spirochetosis (HIS) is a colorectal infectious disease caused by Brachyspira species. We describe HIS in an immunocompromised, 62-year-old Japanese man who presented at Jichi Medical University Hospital with symptoms of diarrhea and bloody stool. He had rheumatoid arthritis that had been treated with immunosuppressive drugs for 10 years. Colonoscopy revealed multiple erythematous spots in the cecum and colon. A histopathological examination identified intestinal colonization by spirochetes, and Brachyspira pilosicoli was isolated from biopsy specimens, indicating a diagnosis of HIS. Metronidazole eradicated the spirochetes, the intestinal mucosa recovered to normal, and the clinical symptoms disappeared. This case suggests that it is important to keep in mind HIS in the differential diagnosis of immunocompromised patients with chronic diarrhea and bloody stool.

Human intestinal spirochaetosis in northern Japan.

A histological diagnosis of human intestinal spirochaetosis (HIS) was made in 114 patients during the period 1994-2007. All patients lived in three prefectures in the northern part of Honshu, Japan. Most patients were elderly and male. Twenty-nine patients complained of abdominal pain, bloody stools, diarrhoea or bowel symptoms, but most patients showed no direct symptoms of bowel disease, and occult faecal blood detected at medical check-up was the main reason for colonoscopic examination. There were no homosexual patients and no immunosuppressed patients. HIS was evenly distributed throughout the whole colorectum. PCR analysis of Brachyspira aalborgi and Brachyspira pilosicoli revealed that more patients were infected with B. aalborgi. Follow-up PCR studies confirmed that infestation with B. aalborgi could be repeatedly detected over a 6 year period. This study, involving over 100 patients, identified the characteristic features of HIS in northern Japan. The results suggest that these spirochaetes may be harmless commensals that cause no obvious pathological alterations in infected individuals.

Imprint cytology detects floating Brachyspira in human intestinal spirochetosis.

Human intestinal spirochetosis is a colorectal infectious disease caused by 2 Brachyspira species. Its diagnosis is established by histology, culture, and polymerase chain reaction, but the value of cytologic examination in routine practice remains unclear. In this study, imprint cytology of biopsy specimens was examined for cytologic features specific to human intestinal spirochetosis. Specimens were obtained from 65 colorectal regions (1-3 regions from each case) in 25 ultrastructurally and/or genetically confirmed human intestinal spirochetosis cases (20 with Brachyspira aalborgi, 3 with B pilosicoli, 2 with both genotypes). In cytologic specimens, spirochetes tended to be floating freely within the mucus and intestinal fluid, whereas the "fringe formation" of spirochetes typically observed in histologic specimens was indistinct in cytologic specimens. Spirochetes were identified in 58 regions (89.2%) and 23 cases (92.0%) by cytology, against in 50 regions (76.9%) and 22 cases (88.0%) by histology (no significant differences). In 6 of 8 regions exhibiting positive cytology and negative histology, B pilosicoli was present within the mucus. Hence, B pilosicoli may tend to float in the mucus. In conclusion, cytologic examination would be useful for the routine identification of human intestinal spirochetosis, especially if B pilosicoli is involved. Further, we suggest the existence of differences in biological behavior between these spirochetes.

Human intestinal spirochetosis accompanied by human immunodeficiency virus infection: a case report.

We present a middle-aged, heterosexual Japanese man with mixed infections including human intestinal spirochetosis, which led us to the detection of human immunodeficiency virus (HIV) infection. The patient had syphilis without related physical or neurological findings. An examination for the serum antibody for HIV performed 9 years previously was negative. In a complete medical checkup at the present time, human intestinal spirochetosis and unspecified entamebic cysts were suggested by histological examination of colonic biopsy material and parasitic examination of the intestinal fluid, respectively. Moreover, a serological test for the antibody for HIV was positive. In specimens obtained by colonoscopy, Brachyspira aalborgi was diagnosed by ultrastructural study and the polymerase chain reaction method for bacterial 16S ribosomal deoxyribonucleic acid. Although HIV infection remains at low prevalence in Japan, we recommend examination for HIV infection in patients with human intestinal spirochetosis, especially when other co-infections are apparent.

Application of real time PCR for diagnosis of Swine Dysentery.

Evaluation of a genetic diagnostic technique using real time PCR of Swine Dysentery (SD) was performed using nox primers. Culture, ordinary PCR and real time PCR were compared in this experiment. Sixty-seven specimens from pigs with clinical signs of SD brought to a slaughterhouse in Shibaura, Tokyo, were used. B. hyodysenteriae was isolated from 49 of the pigs, was detected by ordinary PCR in 49 of the pigs and was detected by real time PCR in 54 of the pigs. Furthermore, we were able to determine the numbers of B. hyodysenteriae cells in all positive specimens by real time PCR. The rapid diagnostic technique established in this experiment was useful for detection of B. hyodysenteriae because it was more effective than ordinary PCR and culture.

Identification and characterization of a novel multidrug resistance operon, mdtRP (yusOP), of Bacillus subtilis.

Using comparative genome sequencing analysis, we identified a novel mutation in Bacillus subtilis that confers a low level of resistance to fusidic acid. This mutation was located in the mdtR (formerly yusO) gene, which encodes a MarR-type transcriptional regulator, and conferred a low level of resistance to several antibiotics, including novobiocin, streptomycin, and actinomycin D. Transformation experiments showed that this mdtR mutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream gene mdtP (formerly yusP), which encodes a multidrug efflux transporter, is cotranscribed with mdtR as an operon. Disruption of the mdtP gene completely abolished the multidrug resistance phenotype observed in the mdtR mutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to the mdtRP promoter, thus leading to repression of its transcription. Moreover, gel mobility shift analysis indicated that an Arg83 --> Lys or Ala67 --> Thr substitution in MdtR significantly reduces binding affinity to DNA, resulting in derepression of mdtRP transcription. Low concentrations of fusidic acid induced the expression of mdtP, although the level of mdtP expression was much lower than that in the mdtR disruptant. These findings indicate that the MdtR protein is a repressor of the mdtRP operon and that the MdtP protein functions as a multidrug efflux transporter in B. subtilis.

Participation in aflatoxin biosynthesis by a reductase enzyme encoded by vrdA gene outside the aflatoxin gene cluster.

Three reactions from hydroxyversicolorone to versicolorone, from versiconal hemiacetal acetate to versiconol acetate, and from versiconal to versiconol are involved in a metabolic grid in aflatoxin biosynthesis. This work demonstrated that the same reductase of Aspergillus parasiticus catalyzes the three reactions. The gene (named vrdA) encoding the reductase was cloned, and its sequence did not show homology to any regions in aflatoxin gene cluster. Its cDNA encoding a 38,566Da protein was separated by three introns in the genome. Deletion of the vrdA gene in A. parasiticus caused a significant decrease in enzyme activity, but did not affect aflatoxin productivity of the fungi. Although the vrdA gene was expressed in culture conditions conducive to aflatoxin production, it was expressed even in the aflR deletion mutant. These results suggest that the vrdA is not an aflatoxin biosynthesis gene, although it actually participates in aflatoxin biosynthesis in cells.

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae.

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.

Inactivation of KsgA, a 16S rRNA methyltransferase, causes vigorous emergence of mutants with high-level kasugamycin resistance.

The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10(-6). More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA(+) strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tsp(r) and Rif(r)), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic--failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.

Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus.

The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene's function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G(1) (AFG(1)). LC-MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG(1). We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG(1) from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A.parasiticus strain significantly enhanced the AFG(1) formation from OMST, whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG(1), which required NADPH or NADH, indicating that NADA is a precursor of AFG(1); in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme required for G-aflatoxin biosynthesis from OMST, and that it catalyzes the reaction from NADA to AFG(1), the last step in G-aflatoxin biosynthesis.

Comparison of the hemagglutination of formalinized American channel catfish (Ictalurus punctatus) erythrocytes between formalinized and live Aeromonas hydrophila isolated from the catfish rearing in Kasumigaura lake in Japan.

Formalinized Aeromonas (A.) hydrophila agglutinated loosely with the formalinized American channel catfish erythrocytes (FACCE), while live A. hydrophila agglutinated tightly with the FACCE. There was a significant difference on the number of attaching bacterial cells to the FACCE (p<0.01) (n=40 erythrocytes) between formalinized and live A. hydrophila. The other bacteria such as Salmonella (S.) Typhimurium ST-5, Escherichia (E.) coli V-517 and Staphylococcus (S.) hyicus ATCC1249 used in this experiment did not attach the FACCE.

Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria.

To investigate the distribution of lactic acid bacteria (LAB) inhabiting canine intestines, a total of 374 gram-positive LAB and bifidobacteria (BF) isolated from large intestinal contents in 36 dogs were classified and identified by phenotypic and genetic analyses. Based on cell morphological sizes, these isolates were divided into seven biotypes containing the genera Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus. The LAB and BF isolates were classified into 38 chemotypes based on SDS-PAGE protein profile analysis of whole cells. Furthermore, partial 16S rDNA sequencing analysis demonstrated the presence of 24 bacterial species in the 38 chemotypes from 36 dogs. The identified species consisted of ten species belonging to the genus Lactobacillus (78.8%), seven species to the genus Bifidobacterium (6.8%), five species to the genus Enterococcus (11.6%), one species of Streptococcus bovis (2.0%), and one species of Pediococcus acidilactici (0.8%). In particular, the most predominant species in canine intestines were L. reuteri, L. animalis, and L. johnsonii and were found in the high frequency of occurrence of 77.8, 80.6, and 86.1%, respectively. Besides these, Enterococcus faecalis, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, and Streptococcus bovis were also isolated in the present study. The sequences of the isolates also showed high levels of similarity to those of the reference strains registered previously in the DDBJ and the similarity was above 97.2%. Their partial 16S rRNA genes were registered in the DDBJ.

The organic acid profiles in the feces of pigs at Brachyspira hyodysenteriae- or B. pilosicoli-positive farms.

We observed a significant difference in the organic acid profile of diarrheal feces between pigs infected with and free from pathogenic spirochetes. Diarrhea and loose feces were collected from growing pigs, held at 15 different commercial farms. A total of 106 samples were measured for organic acid concentration by HPLC and were checked for the presence of B. hyodysenteriae and B. pilosicoli by PCR. B. hyodysenteriae was detected in 3 samples collected from one farm. B. pilosicoli was detected in 5 samples collected from another farm. Lower concentrations of iso-butyrate and iso-valerate were likely associated with development of pathogenic spirochete infection.