No abstract

The combination of polymyxin B and rifampin resulted in an additive effect against all 12 clinical isolates of Serratia marcescens examined, including multiple-drug-resistant isolates.Very recently, Perez Urena et al. (5) demonstrated that the combination of rifampin and polymyxin B was synergistically effective against all strains of Proteus vulgaris, P. mirabilis, and Pseudomonas aeruginosa examined. As we had previously encountered several clinical isolates of Serratia marcescens (bacteriocin type 18) that proved resistant against all currently available antibacterial drugs, including gentamicin, tobramycin, nalidixic acid, and cotrimoxazole, it was of interest to determine whether the above combination of antibiotics was similarly effective. Furthermore, we wished to determine whether amikacin (BB-K8), a semisynthetic derivative of kanamycin (6), was effective against these problem isolates, since it had previously been shown that this latter drug, at a concentration of 6.25 ,ug/ml or less, inhibited 48 of 50 isolates of S. marcescens (2). (ii) Media. Brain heart infusion agar and broth, tryptic soy broth, and Mueller-Hinton agar and broth were purchased from Difco Laboratories, Detroit, Mich. The organisms were maintained on brain heart infusion agar slants at 4 C, and in a mixture of 1 volume of brain heart infusion broth plus 1 volume of heat-inactivated bovine serum (Behringwerke, Marburg, Germany) at -65 C.(iii) Antibiotics. Rifampin (lot no. 9124) was a gift from Ciba-Geigy AG, Wehr/Baden, Germany; stock solutions (2,000 zg/ml) were prepared in 20 vol% of aqueous methanol, membrane filter sterilized, and stored at 4 C in the dark. Amikacin (BB-K8) was obtained through the courtesy of Bristol Laboratories, Syracuse, N.Y. (lot no. 74F 1805); polymyxin B was a gift from Pfizer GmbH, Karlsruhe, Germany (no lot number given). Aqueous stock solutions of the latter two drugs (2,000 gg/ml) were filter sterilized and stored in small aliquots at -65 C. Antibiotic "high content" disks were procured from Difco.(iv) Antibiotic susceptibility tests. Disk susceptibility tests were carried out in accordance with the technique of Bauer et al. (1). Minimal inhibitory concentrations of rifampin, polymyxin B, and amikacin were determined in a microtiter system (Cooke Engineering Co., Alexandria, Va.). The antibiotic master dilutions had been prepared conventionally in Mueller-Hinton broth and were delivered with dropper pipettes (0.05 ml). Bacterial inocula (0.05 ml) were adjusted to yield a final number of 1.5 x 106 colony-forming units/ml at 0 time (10). The combined activity of polymyxin B and rifampin likewise was examined in the microtiter system; U-shaped wells received 0.05 ml of each respective 4x antibiotic dilution or Mueller-Hinton broth (control) and 2 x 0.05 ml of the adjusted bacterial inoculum. Thus,

Chelation of fresh human serum with 0.01 M MgCl, (Mg) plus 0.01 M ethylene glycol tetraacetic acid failed to abrogate the bactericidal activity against "delayed serum-sensitive" strains of Serratia marcescens, whereas previously "promptly serum-sensitive" strains of S. marcescens and control strain Escherichia coli C were killed after an extended period of incubation. The addition of 0.01 M ethylenediaminetetraacetate to fresh human serum neutralized bactericidal activity against S. marcescens of either serum sensitivity category.

A multiple drug-resistant strain of Serratia marcescens (bacteriocin type 18) was isolated from three clinical patients. The isolates were found to carry a conjugally nontransferable, nonmobilizeable resistance plasmid (R-plasmid) with resistance-(r-)determinants against ten antimicrobial drugs: ampicillin, carbenicillin, chloramphenicol, gentamicin, kanamycin, neomycin, streptomycin, tobramycin, triple sulfonamides, cotrimoxazole, and – possibly – nalidixic acid, as determined with exposure to ‘curing’agents (ethidium bromide, acridine orange, and sodium dodecyl sulfate) and by the high rate of spontaneous loss of r-determinants. Dye-buoyant density centrifugation allowed recovery of R-plasmid DNA that measured roughly 24 μm in contour length; after ‘curing’ with concomitant loss of 9 r-determinants, the contour length of the R-plasmid DNA of one isolate (No. SE 154) had decreased to roughly 15 μm, and none was detected in the sole variant of the isolate that spontaneously had lost 11 r-determinants.

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-L-lysine, poly-L-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamineor protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10-3 and 3 x 10-4 for lambda DNA; between 10-6 and 3 x 10-8 for T7 DNA; and between 3 x 10-6 and 10-7 for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.