Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

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 (ϳ200 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.It has been suggested that most Escherichia coli O157:H7 infections in humans are food-borne illnesses and that dairy and beef cattle are reservoirs of E. coli O157:H7 (10). In addition, E. coli O157:H7 seems to persist in food processing because of its acid and heat tolerance (18). Contamination with E. coli O157:H7 may frequently occur at various stages of food processing and drive up the potential for human infection (7). Thus, the elimination of E. coli O157:H7 from the animal intestine might be effective for the prevention of the infection. In previous reports, dietary manipulations, such as a fasting followed by a reseeding or administration of Lactobacillus casei, induced the clearance of E. coli O157:H7 from animal gastrointestinal tracts (13,17,22).Previous reports also discussed the role of bacteriophage in reducing enteropathogenic bacteria in live animals and gastrointestinal models (2,4,12,19,23,25). It is known that the elevated levels of virulent phage in human feces correlate with diseased conditions (8). Thus, phage may play an important role in affecting pathogenic bacteria in intestinal environments.The emergence of infectious disease caused by drug-resistant bacteria requires alternatives to conventional antibiotics (1, 3, 6, 26). Phage therapy is one possible option, and it can provide an economical tool for controlling pathogens in the intestinal tract without affecting the viability of other normal flora (14, 15). Three virulent E. coli O157 antigen-specific phages, designated KH1, KH4, and KH5, have been analyzed in an attempt to control ...

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

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For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L). By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage. 15% of the Mu(L) population was completely resistant to PP01 infection. Mu(L) also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions.

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  10 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 933W phages. On the other hand, 1 mg/l chlorine inactivated the 933W phage significantly. r

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.

The previously isolated T-even type coliphage PP01, specifically infective to Escherichia coli O157:H7, uses the outer membrane protein OmpC as a receptor. The characterization of a spontaneous PP01-resistant strain indicated that it had lost ompC due to the deletion of a 14-kbp region upstream of and partially including ompC. Two host range mutants, able to infect an ompC null mutant, were isolated. Sequencing of gene 38, which codes for the receptor recognition protein Gp38, indicated three mutations in one mutant and two in the other. Both mutant proteins had a Gly208Arg, a Gly161Arg or Gly101His replacement, respectively, and the one mutant phage in addition a Trp189Arg replacement. These alterations suggest that the host range was mediated by a more positively charged Gp38.