Yoko Okubo

Antimicrobial activity of nutmeg against Escherichia coli O157.

We examined the difference between Escherichia coli O157 and non-pathogenic E. coli in their tolerance to spices. Various spices (5 g each) were homogenized at 25 degrees C for 10 min with 5 ml of 70% ethyl alcohol, and the supernatant solutions obtained by centrifugation were used as spice extracts. When the E. coli strains were incubated with each spice extract at concentrations of 0.01% and 0.1%, a noteworthy difference was observed between the O157 and non-pathogenic strains in their tolerance to nutmeg. The populations of the non-pathogenic strains could not be reduced, but those of the O157 strains were remarkably reduced. Antibacterial activity by the nutmeg extract was also found against the enteropathogenic E. coli O111, but not against enterotoxigenic (O6 and O148) and enteroinvasive (O29 and O124) E. coli. When we examined the antibacterial effect of volatile oils in nutmeg on the O157 and non-pathogenic E. coli strains, all O157 strains tested were found to be more sensitive to beta-pinene than non-pathogenic E. coli strains.

Subunit interaction of monomeric alanine racemases from four Shigella species in catalytic reaction

Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer). To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor. The enzymes were eluted on gel filtration as a monomer of about 39,000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine. An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer. The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60,000 and 76,000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively. D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium. The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes. The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands. These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction. Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.

Acid tolerance and gad mRNA levels of Escherichia coli O157:H7 grown in foods

We examined the acid tolerance and gad mRNA levels of Escherichia coli O157:H7 (three strains) and nonpathogenic E. coli (strains K12, W1485, and B) grown in foods. The E. coli cells (approximately 30,000 cells) were inoculated on the surface of 10 g of solid food samples (asparagus, broccoli, carrot, celery, cucumber, eggplant, ginger, green pepper, onion, potato, radish, tomato and beef) and in 10 ml of cows milk, cultured statically at 10-25 degrees C for 1-14 days, and subjected to an acid challenge at 37 degrees C for 1 h in LB medium (pH 3.0). When grown at 20 and 25 degrees C in all foods, except for tomato and ginger, the strains showed a stationary-phase specific acid tolerance. The acid tolerance of the O157 strains changed depending on the types of foods (3-10% survival), but was clearly lower than that of the cells grown in EC medium (more than 90% survival). Tomato and ginger induced relatively high acid tolerances (10-30% survival) in the O157 strains irrespective of the growth phase, probably because of their acidity. No remarkable difference was observed in the acid tolerance between the O157 and nonpathogenic strains grown in all foods. When grown at 10 and 15 degrees C in the foods and EC medium, none of the strains showed the stationary-phase specific acid tolerance. In beef, broccoli, celery, potato and radish, the acid tolerance showed a tendency to decrease with the prolonged cultivation time. In other foods, the acid tolerance was almost constant (about 0.1% survival) irrespective of the growth stage. The mRNA level of glutamate decarboxylase genes (gadA and gadB) correlated to the acid tolerance level when the E. coli cells were grown at 25 degrees C, but was very low even in the stationary phase when the E. coli cells were grown at 15 degrees C or below.

Effect of ethyl alcohol on growth and intracellular alanine racemase of psychrotrophs.

The psychrotrophic alanine racemase from Pseudomonas fluorescens, a typical psychrotroph, is less resistant to organic solvents than the enzymes from thermophilic and mesophilic bacteria (Okubo et al., J. Home Econ. Jpn., 46: 1135-1140, 1995). To further elucidate this difference, we examined the effect of ethyl alcohol on the growth and intracellular alanine racemase activity of three typical psychrotrophs-P. fluorescens, Bacillus psychrosaccharolyticus and B. psychrophilus-in comparison with two mesophiles, Escherichia coli and B. subtilis. Although all the bacteria grew to the early stationary phase when cultivated at 22 degrees C for 36 h in the absence of ethyl alcohol, the growth of the psychrotrophs was more effectively suppressed by the addition of 3 and 5% ethyl alcohol to the medium than that of the mesophiles. The intracellular alanine racemase activity of the psychrotrophs was also more markedly reduced in the presence of ethyl alcohol than that of the mesophiles. When bacterial cells of each strain grown at 22 degrees C for 36 h in the absence of alcohol were suspended in 0-5 % ethyl alcohol solution and incubated at 30 degrees C for 1 h, both the survival ratio and intracellular alanine racemase activity of the psychrotrophs were lower than those of the mesophiles. Thus, ethyl alcohol effectively reduced both the growth of the psychrotrophs and their intracellular alanine racemase activity. Low concentrations of various other alcohols also repressed the growth of the psychrotrophs at 10 degrees C.

Evaluation of an alanine racemase gene as an indicator for the detection of various Escherichia coli: Reactivity of the gene fragment with various E. coli O157:H7 isolates

We determined whether various Escherichia coli O157:H7 isolates could be detected using a probe, an alanine racemase gene (dadX) fragment, specific to E. coli. One hundred strains of E. coli O157:H7 were isolated from fecal samples of healthy cattles and human patients with diarrhea and from a leg of beef. Most isolates (95 strains) produced both verocytotoxin 1 (VT1, Shiga-like toxin I) and verocytotoxin 2 (VT2, Shiga-like toxin II), whereas four isolates produced only VT2. Although neither VT1 nor VT2 was detected in one isolate under the conditions used, the VT2 gene was detected. When all of the isolates were analyzed by a DNA hybridization method involving the use of the probe, positive signals were obtained from all isolates. The DNA hybridization method used for detection of E. coli is considered to detect various E. coli O157:H7 isolates which differ in the types of verocytotoxin they produce.

A new sterility test for cow's milk using alanine racemase gene as the index

Oligonucleotide primers designed from consensus sequences of alanine racemase genes were used for a sterility test of cows milk by polymerase chain reaction (PCR). Commercial cows milk in two 250 ml packages was separately centrifuged at 5000 x g for 10 min, bacterial cells in each precipitate were cultivated at 30 and 55 degrees C for 5 h in Luria-Bertani medium, and the cells from each culture were mixed and used for the PCR after being treated with 0.1 N NaOH at 60 degrees C for 10 min. When we performed the PCR using DNAs from various bacteria and eukaryotes as the templates, a unique PCR product of about 390 bp was amplified only from the bacteria. The sensitivity of the PCR method was such that an initial inoculum of 1 CFU of Bacillus stearothermophilus, Escherichia coli, and Pseudomonas fluorescens per 250 ml of cows milk could be detected. When we analyzed 14 types of commercial cows milk, all samples which were positive by the standard sterility test at 30 or 55 degrees C were also found positive by the PCR method.

Alanine racemase gene fragments as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods

Abstract Alanine racemase gene fragments containing non-conserved regions of the gene were evaluated as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods. The gene fragments were amplified from genomic DNA of each bacterium by polymerase chain reaction with degenerate oligonucleotide primers, and labeled with digoxigenin as probes for detecting each bacterium. Foods and bacteria were treated at 25°C for 10 min in 0.1 N NaOH containing 0.5% SDS before being directly spotted onto nylon membranes for DNA hybridization. When the specificities of the probes were analyzed using a total of 86 strains (23 genera and 65 species) of bacteria including 29 Bacillus strains (20 species), each probe was specific for the respective target bacteria. A variety of foods inoculated with B. stearothermophilus or B. psychrosaccharolyticus were positive as determined by hybridization with the respective probe, whereas uninoculated foods were negative. The alanine racemase gene fragments could be used as specific probes for detecting B. stearothermophilus and B. psychrosaccharolyticus in foods.