Masanari Ikedo

Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

Selective enrichment with a resuscitation step for isolation of freeze-injured Escherichia coli O157:H7 from foods.

ABSTRACT We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detectE. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modifiedE. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coliO157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.

Burkholderia uboniae Sp. Nov., l‐Arabinose‐Assimilating but Different from Burkholderia thailandensis and Burkholderia vietnamiensis

A polar multitrichous Gram‐negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA‐DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, biochemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA‐DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone‐8(Q8). G+C content of DNA is 69.71%.

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing.

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2– by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione‐catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione‐catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coll Menadione‐catalyzed LC was sensitive to antibiotic action. A decrease in menadione‐catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione‐catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells.

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 × 1.2 μm in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 × 3.0 μm and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)‐containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM‐containing medium by both light‐microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40‐fold within 24 hr after inoculation, whereas in the four other less or non‐infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.

An Ultrastructural Study of HeLa Cell Invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium‐attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair‐like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane‐bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane‐bound vacuoles.

Effects of Biocidal Treatments to Inhibit the Growth of Legionellae and Other Microorganisms in Cooling Towers

The effects of biocidal treatments for cooling towers were examined through the use of chemicals and ultraviolet irradiation to inhibit the growth of legionellae and other microorganisms. In the water of cooling towers without continuous biocidal treatments, heterotrophic bacteria and bacterivorous protozoan first appeared, and then legionellae increased up to 104 CFU/100 ml. When a UV sterilizer was connected to the cooling tower, the legionellae count was 1/10 or 1/100 of that in the nontreated tower water. In the water of towers supplemented continuously with the biocidal chemicals, legionellae were not found during a 4‐month period. The biocidal treatments tested were proved to suppress the increase of legionellae in cooling‐tower water, and thus are useful in preventing the outbreak of legionellosis due to inhalation of contaminated aerosol from the cooling tower system.

Ecological Studies of Legionella Species

The occurrence and viable counts of Legionella pneumophila in acid‐treated water samples of 62 cooling towers on the main island of Japan were determined by inoculating them onto plates of Wadowsky‐Yee‐Okuda (WYO) agar medium. WYO plate cultures of 39 (63%) of the samples yielded L. pneumophila with viable counts ranging from 10 to 104 colony‐forming units per 100 ml.

Rapid, sensitive and simple detection method for koi herpesvirus using loop‐mediated isothermal amplification

New methods were developed for the detection of koi herpesvirus (KHV, CyHV‐3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI‐5 PCR amplicon (SphI‐5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real‐time based on the increase in turbidity, with magnesium pyrophosphate as the by‐product. The reactions were carried out under isothermal conditions at 65°C for 60 min. The detection limit of both LAMP was six copies, equal to the modified SphI‐5 PCR. No cross‐reactivity with other fish pathogenic viruses and bacteria was observed. SphI‐5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI‐5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.

Evaluation of a Novel Automated Chemiluminescent Assay System for Antimicrobial Susceptibility Testing

ABSTRACT The newly developed Rapid Lumi Eiken/IS60 (RL/IS60) system automatically determines MICs by detecting chemiluminescence produced in the reaction of a chemiluminescent probe and oxygen metabolites from living microorganisms. The present study evaluated this system for accuracy in antimicrobial susceptibility testing. Chemiluminescence intensities after 4 h of cultivation of clinically important strains were plotted against various concentrations of antimicrobial agents, which resulted in curves reflecting the levels of susceptibility. Sixty-percent inhibitory concentrations based on the susceptibility curves agreed with MICs determined by the reference microdilution method. When the MICs of antimicrobial agents for four quality control (QC) strains (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) were determined by the RL/IS60 system, most (91.1%) of them were within the QC limits proposed by the National Committee for Clinical Laboratory Standards. The system was further assessed for a total of 162 clinical isolates, including E. coli, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Morganella morganii, P. aeruginosa, Haemophilus influenzae, S. aureus, coagulase-negative staphylococci, Enterococcus faecalis, Enterococcus faecium, and Streptococcus pneumoniae. Overall, there was 90.6% agreement between the RL/IS60 system and the reference microdilution method. Our results suggest that the RL/IS60 system provides rapid and reliable MICs of a variety of antimicrobial agents for clinical isolates as well as QC strains.