Katsunori Mizoguchi

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

ABSTRACT The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h−1 and by 3 orders of magnitude at a lower dilution rate (0.327 h−1). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h−1 and persisted until the end of the experiment (∼200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.

Functional analysis of antibacterial activity of Bacillus amyloliquefaciens phage endolysin against Gram-negative bacteria

To analyze the antibacterial activity of Bacillus amyloliquefaciens phage endolysin, nine deletion derivatives of the endolysin were constructed. Each deletion mutant was overexpressed, purified and characterized. The catalytic domain was located on the N‐terminal region and the C‐terminus had an affinity with the bacterial envelope. The enzymatic activity remained in spite of the deletion of the C‐terminal 116‐amino acid region; however, the antibacterial activity was lost. These results indicate that antibacterial action requires both the C‐terminal cell‐binding and the N‐terminal enzymatic activities.

Antibacterial activity of Bacillus amyloliquefaciens phage endolysin without holin conjugation

To characterize the enzymatic activity and antibacterial activity of endolysin encoded by a Bacillus amyloliquefaciens phage, the open reading frame encoding endolysin was amplified by PCR and cloned into the expression plasmid pET21d(+). The resultant plasmid was used to transform Escherichia coli JM109(DE3). Production of endolysin in the cytosol facilitated cell lysis without coproduction of holin, which is considered to degrade or alter the cytoplasmic membrane. The phage endolysin was overexpressed and purified. Although the specific activity of the purified phage endolysin towards lyophilized Micrococcus luteus cells was 1/11 of the activity of chicken egg white lysozymes, the endolysin showed stronger antibacterial activity towards E. coli W3110, E. coli JM109(DE3) and Pseudomonas aeruginosa PAO1 than chicken egg white lysozymes. The antibacterial activity of the endolysin towards these three bacterial strains was marked when EDTA was added to the endolysin solution.

Seasonal change and fate of coliphages infected to Escherichia coli O157:H7 in a wastewater treatment plant.

Seasonal change of virulent phage infected to two E. coli O157:H7 strains (O:157-phage) in the influent of a domestic wastewater treatment plant in the central part of Japan and fate of O:157-phage in the plant were monitored almost monthly from March 2001 to February 2002. Coliphage infected to nonpathogenic E. coli O157:H7 ATCC43888 (43888-phage) was detected for 1 year. On the other hand, phage infected to pathogenic E. coli O157:H7 EDL933 (EDL-phage) was detected intermittently. Concentration of EDL-phage was almost one-tenth of that of 43888-phage. The progressive decrease in phage concentration with the treatment steps was observed. No phage was detected in the supernatant from the secondary settling tank and effluent. PCR amplification of the Stx 2 gene that encodes Shiga toxin (Stx) was observed when O:157-phage concentration in the influent was high x10(3) PFU/ml order. Concentration and percentage of suspended O:157-phage decreased with the progress of the wastewater treatment. 933W phage, which encodes Stx 2 gene, was more fragile and sensitive to chlorination than T4 phage. However, addition of 0.02 mg/l chlorine, in conformance with the required concentration of the plant, did not affect the viability of T4 and 933 W phages. On the other hand, 1mg/l chlorine inactivated the 933 W phage significantly.

Fate of Coliphage in a Wastewater Treatment Process

The fate of coliphage in a wastewater treatment plant in the central part of Japan was investigated from March to December 2001. A relative abundance of coliphage, 1000-10,000 PFU/ml determined with three different Escherichia coli strains, was detected in the influent. But, no remarkable seasonal change in the phage concentration in the influent was observed during the ten-month test period. Almost ten times higher coliphage concentration was detected by the F+ E. coli strain than by the other two F- strains. The RNA phage was more stable than the DNA phage against aerobic treatment using activated sludge. Most of the phages in the influent and primary settling tank were detected as suspended forms. Anaerobic-aerobic treatment enhanced adsorption of the phage by the solid particles. Almost no phage was detected in the effluent. Aerobic treatment using activated sludge and/or the addition of flocculants such as PAC was effective for the removal of coliphage, an index of enteric viral pollution.

Plastic deformation of Al13Fe4 particles in Al–Al13Fe4 by high-speed compression

Abstract Spray-formed Al–Fe alloys having undergone high-speed deformation were examined under a high-voltage electron microscope. Two types of specimens were examined; one containing fine Al 13 Fe 4 particles, and the other containing large particles. In the former specimen, deformation is found to proceed in three patterns, depending on specimen thickness and strain rate: (1) without deformation of the Al 13 Fe 4 ; (2) breaking of the Al 13 Fe 4 ; or (3) melting of the Al 13 Fe 4 . Local melting is found to alter some of the Al 13 Fe 4 particles, to impart five-fold symmetry in diffraction or an amorphous structure. In the latter specimen, introduction of glide dislocations enabled us to determine a shear system in the mc102 monoclinic c 2/ m crystal of Al 13 Fe 4 . On the bases of these observations, the mechanism of high-speed deformation is discussed while taking into account the highly stressed and/or heated states of Al 13 Fe 4 embedded in Al matrix.