Midori Ogawa

Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co‐culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto‐unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.

A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria

For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely low compared with the PI synthase activity of yeast. When CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate were incubated with the cell wall components of Mycobacterium smegmatis, both phosphatidylinositol phosphate (PIP) and PI were formed, as identified by fast atom bombardment-mass spectrometry and thin-layer chromatography. PI was formed from PIP by incubation with the cell wall components. Thus, mycobacterial PI was synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via PIP, which was dephosphorylated to PI. The gene-encoding PIP synthase from four species of mycobacteria was cloned and expressed in Escherichia coli, and PIP synthase activity was confirmed. A very low, but significant level of free [(3)H]inositol was incorporated into PI in mycobacterial cell wall preparations, but not in recombinant E. coli cell homogenates. This activity could be explained by the presence of two minor PI metabolic pathways: PI/inositol exchange reaction and phosphorylation of inositol by ATP prior to entering the PIP synthase pathway.

Inhibitory Effect of Capsular Antigen of Vibrio vulnificus on Bactericidal Activity of Human Serum

Opaque (Op) and translucent (Tr) colonial variants were isolated from Vibrio vulnificus strains. Op‐type variants were more resistant than the isogenic Tr‐type variants, but the survival rate of the Op‐type variants varied with the strains. Antisera were prepared by immunizing rabbit with whole cells of Op and Tr variants of some strains, in which the difference of the sensitivity between Op and Tr cells was remarkable. Then agglutination tests with their living and heat‐killed cells were carried out. The results suggested the presence of capsular antigen in Op cells and its absence in Tr cells, with the exception of the existence of a slight amount of capsular material in Tr variants of strain L‐180. The thin capsular layer of Tr cells of strain L‐180 was also demonstrated electron microscopically, but the layer was thinner than that of the isogenic Op cells. Results of determination of sugar content in the extracted capsular fraction also showed that Op to Tr transformation was due to loss of capsular antigen of the cells. These results confirmed the morphological studies previously reported which suggested the prevention of host defense system by the capsular material of the vibrio.

Gargling with povidone-iodine reduces the transport of bacteria during oral intubation

PurposeNosocomial pneumonia remains a common complication in patients undergoing endotracheal intubation. This study examined the transport of bacteria into the trachea during endotracheal intubation, and evaluated the effects of gargling with povidone-iodine on bacterial contamination of the tip of the intubation tube.MethodsIn the gargling group, patients gargled with 25 mL of povidone-iodine (2.5 mg· mL−1). In the control group, patients gargled with 25 mL of tap water. Before tracheal intubation, microorganisms were obtained from the posterior wall of the patient’s pharynx using sterile cotton swabs. After anesthesia, all patients were extubated and bacteria contaminating the tip of the tracheal tube were sampled and cultured.ResultsBefore orotracheal intubation, all 19 patients who gargled with tap water (control group) had bacterial colonization on the posterior walls of the pharynx. This group included five patients who had methicillin-resistant staphylococcus aureus (MRSA) in their nasal cavity preoperatively and MRSA was also detected in the pharynx of four patients. Bacterial colonization was observed in all 19 patients who gargled with povidone-iodine (gargling group) and four patients carried MRSA in their nasal cavity, although no MRSA was detected in the pharynx. In the control group, all the patients had bacterial colonization at the tip of the tube after extubation. Additionally, MRSA was detected in two of the four patients. In the gargling group, povidone-iodine eradicated general bacteria and MRSA colonies in the pharynx before intubation and at the tip of the tube after extubation.ConclusionGargling with povidone-iodine before oral intubation reduces the transport of bacteria into the trachea.RésuméObjectifLa pneumonie nosocomiale est une complication encore fréquente à la suite d’une intubation endotrachéale. Nous avons vérifié le transport des bactéries à l’intérieur de la trachée pendant l’intubation endotrachéale et évaluons les effets du gargarisme avec povidone iodé sur la contamination bactérienne de la pointe du tube d’intubation.MéthodeLes patients du groupe de gargarisme ont utilisé 25 mL de povidone iodé (2,5 mg· mL−1). Les patients témoins se sont gargarisés avec 25 mL d’eau du robinet. Avant l’intubation trachéale, les microorganismes ont été prélevés sur la paroi postérieure du pharynx au moyen de coton-tiges stériles. Après l’anesthésie, tous les patients ont été extubés et les bactéries de la pointe du tube trachéal ont été prélevées et mises en culture.RésultatsAvant l’intubation orotrachéale, on a détecté des bactéries sur les parois postérieures du pharynx chez les 19 patients témoins. Ce groupe comprenait cinq patients avec staphylocoque aureus résistant à la méthicilline (SARM) dans la cavité nasale avant l’opération. Le SARM a aussi été détecté dans le pharynx de quatre patients. Il y avait une colonisation bactérienne chez les 19 patients qui ont utilisé le mélange povidone iodé. On a retrouvé le SARM dans la cavité nasale de quatre patients, mais non dans le pharynx. Tous les patients témoins présentaient une colonisation bactérienne à la pointe du tube après l’extubation. De plus, le SARM a été détecté chez deux des quatre patients. Par contre, la povidone iodé a éliminé les bactéries en général et les colonies de SARM dans le pharynx avant l’intubation et à la pointe du tube après l’extubation.ConclusionLe gargarisme avec povidone iodé avant l’intubation orale réduit le transport bactérien dans la trachée.

Morphological Variety of Intracellular Microcolonies of Legionella Species in Vero Cells

Intracellular microcolonies of six Legionella species growing in Vero cells showed distinctly varied morphologies. The varieties were observed by light microscopy of Gimenez‐stained, Legionella‐infected Vero cells and by electron microscopy (EM). Legionella pneumophila Philadelphia‐1 formed needle‐shaped crystal‐like microcolonies. Legionella bozemanii WIGA formed microcolonies like wool balls containing filamentous cells. In EM, these organisms proliferated in endosomes, which were adjacent to swollen rough endoplasmic reticula. Legionella oakridgensis OR‐10 showed serpentine chains. Many mitochondria were observed around the microcolonies. Legionella jordanis BL‐540 formed spherical moss‐like microcolonies which were or were not surrounded by endoplasmic membranes. Legionella feeleii WO‐44C spread throughout the cytoplasm without making clusters. Legionella dumoffii Tex‐KL made big clusters that spread in the cytoplasm, a portion of which was outside the endosome membranes. These different morphologies imply diversity in modes of intracellular multiplication of Legionella spp.

Small and Rough Colony Pseudomonas aeruginosa with Elevated Biofilm Formation Ability Isolated in Hospitalized Patients

Pseudomonas aeruginosa is a key pathogen of nosocomial infection, and causes persistent infection in patients with specific diseases like cystic fibrosis (CF). It has been reported that patients affected with CF discharge, at a high frequency, small colony variants with high adherence ability. In routine laboratory testing, we found atypical small and rough type (SR) colony variants of P. aeruginosa. The SRs and the counterpart wild type (WT) colonies showed similar biochemical features, antimicrobial susceptibilities, pulsed‐field gel electrophoresis (PFGE) profiles, serotypes, and twitching motilities. The biofilm formation abilities of all the SR colonies, however, were extremely elevated as compared to those of the counterpart WT colonies. The frequency of SR‐positive patients was 3.1% of the P. aeruginosa‐positive inpatients (5/160), and that of the SR isolates was 0.6% of the P. aeruginosa strains (6/970) isolated in our laboratory over a period of 6 months. The SR‐positive patients did not have any common disease or particular antibiotics treatment. The PFGE profiles showed that the SRs and the counterpart WTs were identical to each other, and also that three of the five SR/WT pairs were clonally similar. The three pairs were recovered from the feces, urine, and endotracheal secretion, respectively, of three patients hospitalized in two distinct wards. The results suggest that P. aeruginosa spontaneously produced highly adherent SR colonies in hospitalized patients, and these colonies may tend to spread in a hospital.

Identification of Causative Pathogens in Eyes with Bacterial Conjunctivitis by Bacterial Cell Count and Microbiota Analysis

PURPOSE To determine the causative pathogens in eyes with bacterial conjunctivitis. DESIGN Evaluation of diagnostic test or technology. PARTICIPANTS Thirteen eyes diagnosed clinically with bacterial conjunctivitis and 12 eyes with normal conjunctival sac were studied. METHODS The bacterial cell numbers were counted in the samples stained by ethidium bromide (EtBr). The microbiota was determined by the clone library method using polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) gene with universal primers. In addition, examinations of smears and cultures of samples were performed. MAIN OUTCOME MEASURES Bacterial cell numbers determined by the EtBr staining method and microbiota analysis based on 16S rRNA gene of samples from eyes with bacterial conjunctivitis. RESULTS The bacterial cell numbers in the eyes with bacterial conjunctivitis were significantly higher than those in the normal conjunctival sacs. Ten of 13 samples from the eyes with bacterial conjunctivitis had positive PCR results. The remaining 3 samples and all 12 samples from the normal conjunctiva had negative PCR results. In 5 of the 10 PCR-positive samples, the predominant species accounted for 84.5% or more of each clone library. In the remaining 5 samples, the predominant and the second dominant species accounted for 27.4% to 56.3% and 19.0% to 26.8%, respectively, of each clone library. The number of detected species in the clone libraries was between 8 and 20 (average ± standard deviation, 7.5 ± 5.8). Bacteria were detected in 8 of the 10 bacterial conjunctivitis samples and in 5 of the 12 normal samples in the cultures. The number of species detected by cultures was 1 in the eyes with bacterial conjunctivitis and between 1 and 3 (mean ± standard deviation, 1.6 ± 0.9) in the normal conjunctiva. CONCLUSIONS These results showed that the bacterial cell number in a sample is a good method of determining bacterial conjunctivitis. The microbiota analysis detected a diverse group of microbiota in the eyes with bacterial conjunctivitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The combination of bacterial cell count and microbiota analysis is a good method for identifying the causative pathogens in cases of monomicrobial and polymicrobial conjunctivitis.

Isolation and characterization of a filamentous phage, Vf33, specific for Vibrio parahaemolyticus.

Phage Vf33, a filamentous phage about 1,400 nm long and 7 nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292 g/cm3. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80 C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single‐stranded circular DNA 8.4 kb in size. The viral genome was converted to a double‐stranded replicative form in the host cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.

Ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in Bacteria and Archaea, which contain inositol phospholipid

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.

Shiga toxin 1 and 2 induce apoptosis in the amniotic cell line WISH.

Objective: The aim of this study was to evaluate the toxicity of Shiga toxin (Stx) 1 and 2 on amniotic cells in vitro. Methods: WISH cells, which were derived from human amniotic cells, and Vero cells were cultured with or without Stxs. After 24 hours of culture, cell viability was measured by Cell Counting Kit-8, and extracted DNA was electrophoresed on a 1% agarose gel. The morphologic changes were observed by Papanicolaou staining, and the apoptotic index (percentage of apoptotic nuclei per total nuclei) was calculated. Quantification of apoptotic cells was also measured by an enzyme-linked immunosorbent assay. Results: The viability of WISH cells decreased in proportion to the concentrations of Stxs. Cellular ladder formation was observed by DNA electrophoresis of Stx-treated WISH cells, and the typical morphologic changes were observed by Papanicolaou staining. The proportion of apoptotic cell increased in response to Stxs. Conclusions: Stxs injured WISH cells directly and induced apoptosis in vitro. WISH cells were as sensitive as Vero cells to Stxs and cell death occurred by apoptosis.