Technique for Determining the Bactericidal Effect of Drug Combinations

Abstract

Two paper strips, each containing different antimicrobial agents, were placed on plates on Mueller-Hinton agar to permit antibiotic to enter the agar. A filter membrane was placed on this plate, and the microorganisms were planted on the membrane. After 6 h of incubation at 37 C, the membrane was transferred to antibiotic-free Mueller-Hinton agar containing triphenyltetrazolium hydrochloride and incubated for 18 h at 37 C. Specific growth patterns were indicative of additive (indifferent), synergistic, or antagonistic effects of the drug combination used. Trimethoprim and sulfamethoxazole proved to act synergistically against 85% of Escherichia coli, 86% of Klebsiella, and 89% of Proteus mirabilis strains tested. A few strains resistant to either drug were susceptible to their combination. The technique was useful against organisms with widely differing susceptibilities to the two antimicrobial agents tested.A number of methods have been devised to establish the bactericidal effect of antibacterial agents against pathogenic organisms (8,9,11,13). The replica transfer method was introduced by Lederberg and Lederberg (10). Elek et al. (6,7) and Streitfeld (14) applied this method using a velvet replicator for the study of antibiotic combinations. Chabbert and Waterworth (4) applied the same principle using a cellophane membrane for the study of synergistic effects of antibiotics. They used filter paper strips impregnated with antibiotics placed at right angles to diffuse the drugs into the agar.In this paper we report the usefulness of a modified cellophane technique in which the cellophane has been replaced by a filter membrane. The technique was tested with the association of trimethoprim plus sulfamethoxazole. MATERIALS AND METHODSOne hundred strains each of Escherichia coli, Proteus mirabilis, and Klebsiella, isolated from specimens submitted to this laboratory, were tested for susceptibility to trimethoprim and sulfamethoxazole singly or in combination.Media and reagents. Media used were as follows: (i) Mueller-Hinton agar (MHA) (BBL) with 0.25% dextrose and 0.04% triphenyltetrazolium hydrochloride (TTC) (Biological Products, N.J.) (after the agar was autoclaved and cooled at 52 C [in a 52 C water bath ], 1 ml of a 4% solution of TTC in 20% alcohol was added to 100 ml of MHA with 0.25% dextrose); Trypticase soy broth (TSB) (BBL). Filter membranes #PHWPO9025, 9 cm in diameter (Millipore Corp., Bedford, Mass.), and filter paper grade #740E (Schleicher and Schuell, Inc. Keene, N.H.) were used. Trimethoprim and sulfamethoxazole were from Hoffman-La Roche, Inc., N.J. Trimethoprim (10 mg) was dissolved in 100 ml of boiling water containing 0.2 ml of hydrochloric acid p.a., and sulfamethoxazole (200 mg) was dissolved in 98 ml of boiling water containing 2 ml of a 4% sodium hydroxide solution.Antibiotic-impregnated plates were prepared in the following manner. Strips 4 cm long and 0.9 cm wide were cut from the filter paper (water absorption per strip was 0.29 ml). One strip was soaked in the trimethoprim solution (pr...

In vitro susceptibilities of suspected periodontopathic anaerobes as determined by membrane transfer assay

“…Because antimicrobial agents did not carry over with the thin cellophane membrane, bactericidal rather than inhibitory effects could be measured. The modification of the original technique proposed by Lorian and Fodor (9) (10). b Yoon and Newman (24).…”

“…The actual density of bacteria per area found in subgingival plaque is uncertain, but the density of lawn present on the membrane likely represents a more robust density than the recommended 105 bacteria used in the agar dilution method. Indeed, the rationale for using bacterial densities in the 105 range in conventional in vitro assays is that these levels are considered significant in wound sepsis and bacteremia (8) Drug carryover is often of major concern, particularly with agar diffusion and broth dilution methods, in which the bactericidal rather than the inhibitory effects of antimicrobial agents are tested (4,9). To circumvent this difficulty, Chabbert and Waterworth (4) devised a cellophane transfer technique in which the bacterial lawn could be removed from contact with the antimicrobial agent after a predefined exposure time (usually 6 to 18 h).…”

“…prepared from a 4% stock solution in 80% ethanol. TTC was used as an indicator of cell viability (9). Agar medium was prepared fresh weekly.…”

In vitro susceptibility of mutans streptococci to antimicrobial substances as determined by a membrane transfer assay

“…This observation suggests that these agents, when applied at the MBC or above, are immediate in action, and therefore, carry-over of the chemotherapeutic agent is not a confounding factor when results of this assay are interpreted. Moreover, the use of a membrane which restricts carry-over of accumulated agents when the membrane is transferred from contact with the agent-containing disks to a fresh agar plate was done intentionally to minimize carry-over (7).…”

“…Membranes were then transferred to fresh TS plates containing 0.04% triphenyl tetrazolium chloride dye (TTC; Sigma Chemical Co., St. Louis, Mo.). TTC was added to the medium to denote cell viability (2,7). After the plates were incubated for 24 h, the MBC was determined by observing the lowest dilution lethal to the bacteria in the area over which the disk was applied (nonreduced TTC is colorless) (2).…”