PubMed · 8726773
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A J DiMarino, W W Bond. 1996. Flexible gastrointestinal endoscopic reprocessing.. https://doi.org/10.1016/s0016-5107(96)70301-2
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Although Shigella is a major foodborne pathogen, its growth in foods has received little attention. Growth of S. flexneri 5348 inoculated into commercially available sterile foods (canned broths, meat, fish, UHT milk, baby foods) was studied at 10 to 37 degrees C. S. flexneri was enumerated by surface-plating on Tryptic Soy Agar and growth curves were fitted by means of the Gompertz equation. Observed growth kinetics values and values calculated using a previously developed response surface model compared favorably for growth at 19 to 37 degrees C, but not at < 19 degrees C. To refine the model, additional data were collected for growth at 10 to 19 degrees C. A total of 844 tests in BHI broth, representing 197 variable combinations of temperature (10-37 degrees C), pH (5.0-7.5), NaCl (0.5-5.0%) and NaNO2 (0-1000 ppm) was used for the revised model. The revised model, developed in BHI, gave significantly better agreement of calculated growth kinetics values with those observed in foods at 10 to 19 degrees C.
The effects of Lactobacillus strains isolated from intestinal tracts for starter cultures of fermented sausage on the growth rate and enterotoxin production of Staphylococcus aureus were studied at two fermentation temperatures of 20 degrees C and 35 degrees C. Initial inoculated populations in the sausage batter were approx. 10(4) cfu/g for S. aureus and 10(7) cfu/g for the Lactobacillus strain as a starter culture. Samples of sausage were taken during fermentation and analyzed for pH and microbial populations. In control lots without inoculation of Lactobacillus strains, staphylococcal enterotoxin was detected during fermentation at each temperature. Of three intestinal Lactobacillus strains, L. rhamnosus FERM P-15120 and L. paracasei subsp. paracasei FERM P-15121 inhibited the growth and enterotoxin production of S. aureus in sausages during fermentation at both temperatures, although L. acidophilus FERM P-15119 could not satisfactorily suppress them. The effect of the two selected strains in meat fermentation (i.e., fermentation time, acid production, inhibition of S. aureus) was the same as that of a commercial L. sake starter culture for fermented sausage. These results suggest the intestinal Lactobacillus strains selected in this study could be utilized as a starter culture to produce new fermented meat products that are microbiologically safe.
The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents. AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents. Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG). Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants. BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells. We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine. When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG. The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine. The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8. All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG. Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG. This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity. The next most common residues found at position 140 were arginine (7%) and asparagine (7%). Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively). These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.