Comparative In Vitro Susceptibility of a Tetracycline-Resistant Chlamydia trachomatis Strain Isolated in Toulouse (France)

Background and Objectives: We recently reported the first isolation of a tetracycline-resistant Chlamydia trachomatis strain in Toulouse from a woman treated with tetracycline. To characterize this isolate, its in vitro susceptibility was compared with those of 34 other C. trachomatis isolates recovered in Toulouse.

Study Design: The susceptibilities of C. trachomatis strains were determined in terms of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using McCoy cells in 96-well microdilution plates, with an inoculum of 5.10³ to 1.10⁴ inclusion-forming units/ml. The antimicrobial agents tested were tetracycline, azithromycin, erythromycin, ofloxacin, and pristinamycin.

Cited Here... No difference was observed between the MICs and MBCs except for the tetracycline. Tetracycline-resistant strain MIC and MBC were >64 μg/ml, although <1% of the bacterial population showed resistance. For the other isolates, the MIC of tetracycline was ≤0.25 μg/ml. The antibiotics other than tetracycline were active in vitro against all strains.

Conclusions: These results show that the tetracycline resistance observed in Toulouse differs from the "heterotypic resistance" described previously in the United States in multiresistant C. trachomatis isolates. They confirm that the resistance we observed may be a new phenomenon.

GENITAL INFECTIONS due to Chlamydia trachomatis are the most common bacterial sexually transmitted diseases recognized throughout the world.¹ Conventionally, uncomplicated C. trachomatis infection is treated with antibiotics, usually tetracyclines or macrolides, that result apparently in very high cure rates.^2,3 Treatment failure is attributed usually to reinfection. We recently reported the first isolation of a tetracycline-resistant C. trachomatis strain in Toulouse from an asymptomatic woman treated with tetracycline.⁴ The patient denied having any sexual contact since the beginning of treatment and reported excellent compliance. She was retreated with pristinamycin and cured. Pristinamycin is a natural antibiotic that belongs to the family streptogramins, is composed of several compounds, and shares the antimicrobial activity spectra of macrolides and lincosamides. This drug has been shown to have high in vitro activity against genital pathogens, including C. trachomatis.⁵ To characterize the tetracycline-resistant isolate, its in vitro susceptibility to tetracycline, pristinamycin, and other conventional antimicrobial agents was compared with those of 34 other C. trachomatis strains recovered in Toulouse.

Methods

C. trachomatis Strains

The Toulouse tetracycline-resistant isolate and 34 clinical strains isolated at the Laboratory of Microbiology of the Purpan Hospital of Toulouse (1988 to 1995) were used. The 34 clinical strains were isolated from male urethras and from cervixes from patients before antibiotic treatment. Culture confirmation of clinical strains was performed by fluorescent antibody staining of cycloheximide-treated McCoy cells, with a monoclonal antibody specific to C. trachomatis (Syva Co.). The organisms were stored at -70°C until use.

Antimicrobial Agents

Antimicrobial agents used for susceptibility tests included tetracycline, ofloxacin (Roussel Laboratories, Paris, France), erythromycin (Abbott Laboratories, Rungis, France,), azithromycin, doxycycline (Pfizer Laboratories, Orsay, France), and pristinamycin (Rhone-Poulenc-Rorer Laboratories, Paris, France). All drug solutions were prepared and stored according to the manufacturers' instructions.

Antimicrobial Susceptibility Testing

The antimicrobial susceptibility of C. trachomatis strain was determined in terms of minimal inhibitory concentration(MIC) and minimal bactericidal concentration (MBC) using the dilution method.⁵ All clinical isolates and a laboratory strain of C. trachomatis (LGV-II ATCC VR-902) were passaged extensively in the laboratory and at least twice in antibiotic-free maintenance medium (Eagle's Minimum Essential Medium with 10% fetal bovine serum, 1% glutamine, and 5 g glucose/l). Monolayers of antibiotic-free McCoy cells were grown in 96-well microdilution plates seeded at a concentration of 2.0 × 10⁵ cells/ml. After 48 hours of incubation, the C. trachomatis strains were inoculated-5.10³ to 1.10⁴ inclusion-forming units per ml (IFU/ml). Monolayers were then centrifuged for 60 minutes at 1,500g, aspirated, and overlaid with appropriate serial dilutions of the antibiotic being tested. All dilutions were made with the above mentioned antibiotic-free maintenance medium containing 0.5 μg cycloheximide/ml.

Plates were prepared in duplicate, and antibiotic-free positive and negative controls were included on each plate. LGV-II ATCC VR-902 was included as quality standard in each MIC and MBC run. Cultures were incubated for 48 hours at 35 °C and then fixed and stained for counting of inclusions using a fluorescein-conjugated monoclonal antibody to Chlamydia genus-specific antigen (Ortho Diagnostic Systems). The MIC was defined as the lowest concentration of each antibiotic that completely inhibited inclusion formation. MBC was defined as the lowest antibiotic concentration required to inhibit the development of a single inclusion after 48 hours of incubation as described for MIC determination, followed by three washes, 48 hours of incubation in antibiotic-free medium, and one passage on cell monolayers grown in antibiotic-free medium.⁶

Growth in Tetracycline

McCoy cell monolayers in shell vials were inoculated with tetracycline-resistant isolate, and medium containing 8 μg/ml tetracycline was added. After 48 hours the isolate was titered and passaged in the same tetracycline medium as far as it was possible.

Results

The MICs and MBCs of antibiotics against the C. trachomatis strains isolated in Toulouse are listed in Table 1. No difference was observed between the in vitro susceptibility of the resistant isolate and the 34 other C. trachomatis strains, except for the tetracycline. The tetracycline-resistant isolate formed inclusions at concentrations of tetracycline and doxycycline of more than 64 μg/ml, whereas the other strains had MICs of tetracycline ≤0.25 and MBCs, ≤4 μg/ml. According to the break points of resistance established by the Comité Français de l'Antibiogramme,⁷ all strains were susceptible to azithromycin, erythromycin, ofloxacin, and pristinamycin.

Table 1

The tetracycline-resistant isolate did not grow well in McCoy cells. A high number of passages were required to achieve a yield sufficient for susceptibility testing. Attempts to expand the resistant organisms in medium containing 8 μg/ml tetracycline were unsuccessful. With each passage, the number of inclusion forming units per milliliter remained stable or decreased until after 3 to 11 passages, none were detectable.

Discussion

In our study, no difference was observed between the MICs and MBCs for the tetracycline-resistant isolate and 34 C. trachomatis strains isolated in Toulouse except for the tetracycline. Azithromycin, erythromycin, ofloxacin, and pristinamycin were active against all strains. Although the Toulouse resistant isolate formed inclusions at high concentrations of tetracycline, there was a decrease in the number and in the size of chlamydial inclusions for MICs ≥ 0.125 μg/ml.⁴ This suggested that only a small portion (<1%) of the bacterial population was resistant to tetracycline. When the resistant organisms were passaged on 8 μg/ml tetracycline medium, the number of inclusion forming units per milliliter remained stable or decreased. This heterogenous expression of resistance occurs in methicillin-resistant staphylococci. The phenomenon depends on the strain's genetic background but is not yet understood.⁸

Relative resistance of C. trachomatis to erythromycin and tetracycline resistance have been reported previously.^9,10 "Heterotypic" tetracycline-resistance of C. trachomatis was described in the United States with a high inoculum (>5.10⁴ IFU/ml) but did not appear to be limited to isolates from patients for whom treatment apparently failed.^10,11 Moreover, the heterotypic resistance was associated to erythromycin, sulfamethoxazole, and clindamycin resistance. The in vitro susceptibility of the Toulouse tetracycline-resistant isolate to erythromycin and azithromycin confirms that the resistance we observed with a standard inoculum from a patient for whom treatment apparently failed may be a new phenomenon.

C. trachomatis is an obligate intracellular organism and, like other species of Chlamydia, is capable of causing chronic unrecognized infections.¹² Conversely, persistent infection after appropriate therapy for C. trachomatis seems to be rare.^12,13 However, chlamydiae may persist in small numbers in a "latent" state and go undetected by cultural methods, as suggested by the detection of chlamydial deoxyribonucleic acids at a high percentage in women with postinfectious tubal infertility even after antibiotic treatment.¹⁴

The Toulouse tetracycline-resistant isolate, and particularly fully resistant organisms, did not grow well. A limited metabolic capacity has also been reported in organisms that remained in viable but culture-negative states during persistent infections.¹² In the case we reported, the short interval between the end of tetracycline therapy and the isolation of chlamydia together with the excellent compliance and the absence of sexual contact reported by the patient⁴ raised the possibility of a persistent infection. If the isolation of this tetracycline-resistant C. trachomatis strain in an asymptomatic woman was the result of persisting organism and not of new infection, the implications for the management of chlamydial infection could be considerable. In any case, the role of the tetracycline resistance has to be elucidated, and further studies are warranted.