PubMed · 4248662
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D M Bear, M Turck, R G Petersdorf. 1970. Ampicillin.. https://pubmed.ncbi.nlm.nih.gov/4248662/
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Changes in human granulocytic ehrlichiosis (HGE)-specific major outer membrane protein (p44 kD) were assayed by Western blot analysis in HL-60 cells in vitro infected by the HGE agent. Time course study demonstrated that the expression of p44 preceded the rise in cell infection as determined by the presence of intracellular morulae. To test whether the expression of p44 may be suitable for evaluating the effects of antibiotics in vitro, three recent isolates of the HGE agent were exposed to doxycycline and ampicillin during culture with HL-60 cells. Loss of infection concurrent with disappearance of the 44 kD protein was found with doxycycline treatment. In contrast, ampicillin treatment had no discernible effects on infection or 44 kD expression. There was excellent agreement between infection, as measured by morulae, and 44 kD expression (coefficient of correlation r = 0.97, p < 0.01). Following treatment with doxycycline, the 44 kD protein disappeared with an estimated t1/2 of approximately 24-30 h, which was considerably shorter than a t1/2 of >60 h calculated for loss of morulae. Measurement of p44 expression may be a more rapid and simple assay to determine antibiotic susceptibility of the HGE agent in cell culture. Furthermore, it may be used to indicate the presence of infection before morulae are apparent.
BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer. RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure. CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.
Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.