Effects of Ethidium Bromide on Growth and on Loss of the Penicillinase Plasmid of Staphylococcus aureus

Abstract

Ethidium bromide (EB) was more efficient than ethyl violet or rifampin as a curing agent for the penicillinase plasmids of Staphylococcus aureus strains. The effects of EB on growth and on the loss of the penicillinase plasmid of PS 81 were studied in detail. The growth rates of PS 81 and an EB-cured derivative were identical in broth, but the cured derivative had a shorter lag in the presence of added 6 x 10-6 M EB. The shortened lag was due to prior exposure to EB as the cured derivative and an EB-treated but uncured strain of PS 81 gave identical growth lag and growth rates in the presence of EB. The curing of PS 81 by EB occurs in three phases. After a 4 to 5 hr lag, there is a 100-fold increase in the number of penicillinase-negative cells, and the proportion of cured cells continues to rise until 10 to 12 hr. Thereafter, the population becomes refractory to further curing, and the proportion of penicillinase-negative cells remains constant at about 20% of the total. Penicillinase-positive survivors of EB treatment showed increased EB resistance and were cured at lower rates upon subsequent EB treatment. Isolated colonies of the parental strain PS 81 were heterogeneous in their EB sensitivity. Thus, EB does not competitively favor spontaneously cured penicillinase-negative cells but appears to act in a manner analogous to acridine orange on the plasmids of enteric bacteria.Plasmid elimination. The method of Bouanchaud, Scavizzi, and Chabbert (4) was used for plasmid elimi-1200 on August 1, 2020 by guest

Ethidium Bromide-Resistant Mutant of Bacillus subtilis

“…Sporulation of strain EB8 was blocked when grown in the presence of EB (10 jig/ml) on either AK agar or in x 2 Shaeffer sporulation medium. After 16 h of growth at 37 C with vigorous shaking in x 2 Shaeffer medium containing 10 Ag of EB/ml, microscope examination revealed no spores or intermediate stages. However, the cells were highly motile and their morphology appeared normal.…”

“…Ethidium bromide (EB) (2, 7-diamino-9phenylphenanthridium-1-ethyl bromide) has been used for a variety of biological purposes. EB induces petite mutations in yeast (19), it has been used to eliminate penicillinase plasmids and resistance transfer factors from bacterial cells (2,16), it is an inhibitor of nucleic acid biosynthesis (20), and it functions as a mutagen in bacterial cells (13).…”

Plasmid curing in bacteria

“…Curing was usually observed at a high frequency, and the results obtained were more reproducible than with acridine dyes. Rubin and Rosenbaum [19] also successfully cured peni.cillinase plasmids in Staphylococcus aureus strains using ethidium bromide.…”

Nature of the Genetic Determinant Controlling Exfoliative Toxin Production in Staphylococcus aureus

“…Various physical and chemical agents can be used to enhance the frequency of plasmid elimination over that of spontaneous loss, but the frequencies of elimination will vary according to the strain and conditions used (16). The increased loss or curing of genetic markers after growth of some strains in curing agents or at elevated temperatures is indicative of plasmid location of the markers, but only if it can be shown that treatment did not select for cells from which plasmid markers were lost spontaneously (17). Possible selective effects can be ruled out (i) by determining the rate and time of appearance of negative variants during treatment of the wild-type culture, and (ii) by showing that growth at the elevated temperature or in the presence of the curing agent does not competitively favor plasmid-negative cells over plasmid-positive cells (9,17).…”

“…Ethidium bromide (EB) has proven to be a potent agent for eliminating penicillinase plasmids (3,14,17). Rubin and Rosenblum (17) observed that the curing frequencies for staphylococcal penicillinase plasmids were consistently greater with EB than with other curing agents.…”