In vitro assays of the efficacy of antimicrobial agents in controlling Chlamydia trachomatis propagation

Abstract

The antimicrobial susceptibility of a low-laboratory-passage, slow-growing, genital Chlamydia trachomatis strain was studied by five different procedures with the use of McCoy cells pretreated with 5-iodo-2-deoxyuridine. The effects of antimicrobial agents when added to cultures on day 0 or day 2 after inoculation with C. trachomatis and the effects of washing and reincubating treated cultures in antimicrobial-free media were investigated. Tetracycline and erythromycin inhibited C. trachomatis growth at concentrations attainable in human serum, although their actions were reversible and significantly higher concentrations were needed to "cure" 48-h infected cultures. On a weight basis, spectinomycin was relatively ineffective in inhibiting C. trachomatis growth. The minimal inhibitory concentration of penicillin measured by our assay procedures was higher than that reported by other investigators. The five assay procedures used in this study were reproducible, and our results indicate that we can obtain more pertinent information about the efficacy of an antimicrobial agent in controlling C. trachomatis growth by using a combination of these assays than by simple minimal inhibitory concentration determinations, as had been previously described by other investigators. In addition, we failed to demonstrate changes in tetracycline susceptibility,of C. trachomatis isolates from two patients who had received tetracycline therapy.In vitro studies on the antimicrobial susceptibility of Chiamydia trachomatis in cell culture systems have been described (1-3, 8, 11). These studies often utilized fast-growing laboratoryadapted C. trachomatis strains, usually assayed inhibitory effects of drugs by the concurrent addition of drugs and C. trachomatis, and did not assay for the noninfectious reticulate body formn of C. trachomatis.In this study we examined the effects of tetracycline, erythromycin, spectinomycin, and penicillin on C. trachomatis in a 5-iodo-2-deoxyuridine (IUdR)-treated McCoy cell system. The bacteriostatic and bactericidal effects of these antimicrobial agents were measured by five different assay procedures. We attempted to parallel the in vivo situation by (i) using a nonlymphogranuloma venereum C. trachomatis strain that is slow growing, in low laboratory passage, and of a serotype common to United States isolates, (ii) treating already-infected cultures since, in vivo, antimicrobial agents must act on preestablished infections, and (iii) removing antimicrobial agents and reincubating cultures after treatment because antimicrobial agents are metabolically cleared in vivo and it is possible that organisms could survive in the reticulate body form and develop to the infective stage after removal of the antimicrobial agents. In addition to the one test strain used in all experiments, we also tested the tetracycline susceptibility of post-tetracycline-treatment isolates from two individuals. MATERIALS AND METHODS Celis. McCoy cells (mouse heteroploid cells) (4)were carried in antibiotic-free medium for at leas...

In vitro activity of clavulanic acid, amoxicillin, and ticarcillin against Chlamydia trachomatis

“…For most antimicrobial agents, MICs are well defined and the number of inclusions in cell cultures declines dramatically from numbers near those in controls without antimicrobial agents to nil over only a few dilutions (6,10,14,15). In contrast, with penicillins there are occasional inclusions over a wide range of concentrations (6,10,14,15). This probably accounts for the large discrepancies in published reports of MICs of penicillins (4,6,9,10,(13)(14)(15)(16).…”

“…In contrast, with penicillins there are occasional inclusions over a wide range of concentrations (6,10,14,15). This probably accounts for the large discrepancies in published reports of MICs of penicillins (4,6,9,10,(13)(14)(15)(16). In vivo, single-dose penicillin treatment does not usually eradicate C. trachomatis (7, 10), but multiple-dose penicillin treatment often results in negative follow-up cultures for weeks or longer (2,5,7,12).…”

“…Antimicrobial agents were reconstituted and diluted with 0.4 M sucrose phosphate the day of use (6,15). The final concentrations evaluated for ticarcillin were 960, 480, 30, 7.5, 1.875, and 0.469 ,uggml; for amoxicillin they were 64, 16, 4, 1, and 0.25 ,ug/ml; for clavulanic acid they were 32, 16, 4, 1, and 0.25 ,ug/ml; for the combination of ticarcillin and clavulanic acid in a 15:1 ratio they were 480 and 32, 30 and 2, 7.5 and 0.5, 1.875 and 0.125, and 0.469 and 0.031 p.g/ml;…”

“…1.8-g) vials by using a methodology previously described by me and others (6,15). Briefly summarized, C. trachomatis inocula yielding 20 to 30 inclusion forming units per 400 power field were centrifuged onto monolayers at 2,300 x g for 1 h, the antimicrobial agents were then added, and the cultures were incubated.…”

Lack of in vitro activity of cefoxitin, cefamandole, cefuroxime, and piperacillin against Chlamydia trachomatis

“…They were dissolved in 2-sucrose phosphate, and serial twofold dilutions were prepared. The experiments were performed in triplicate as described previously (3,9). Briefly, 5-iodo-2-deoxyuridine-treated McCoy cells maintained in antimicrobial-free medium were infected with an inoculum of C. trachomatis, which produced three to five inclusions per 400x field.…”