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Antimicrobial Agents and Chemotherapy, April 2005, p. 1584-1586, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1584-1586.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In Vitro Effects of Spectinomycin and Ceftriaxone Alone or in Combination with Other Antibiotics against Chlamydia trachomatis

Shuxian Shang,1 Longqing Xia,1 Mingying Zhong,1 Jinping Zhang,1 Jianbin Zhao,1 Xiangdong Gong,1 David Mabey,2 and Qianqiu Wang1*

National Center for STD and Leprosy Control, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China,1 Clinical Research Unit, London School of Hygiene and Tropical Medicine, London, England2

Received 17 July 2004/ Returned for modification 20 July 2004/ Accepted 3 December 2004


   ABSTRACT
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The in vitro effects of spectinomycin and ceftriaxone, alone or in combination with erythromycin, ofloxacin, and doxycycline, against Chlamydia trachomatis were investigated by the checkerboard method and compared by Ridit (reference identical unit) analysis. A combination of spectinomycin with erythromycin or doxycycline was found to be more effective than that of ceftriaxone.


   TEXT
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Patients with genital chlamydial infection are often coinfected with Neisseria gonorrhoeae. Many studies suggested the coinfection rate is between 20 and ~40% (17). Among the antimicrobials routinely used in the treatment of genital gonococcal infection, there are rarely effective agents that are also effective in a single dose against Chlamydia trachomatis (6, 7). The Centers for Disease Control and Prevention (CDC) recommends that treatment of gonococcal infection should be accompanied by treatment for genital chlamydial infection (2-5). Although the cost-effectiveness of this dual therapy has been evaluated (9, 14), little is known about the in vitro effects of interactions between the drugs used. Our aim was to investigate the in vitro effects of spectinomycin and ceftriaxone, which are commonly used in the treatment of gonococcal infection, alone or in combination with erythromycin, ofloxacin, or doxycycline, respectively, against C. trachomatis by a microdilution checkerboard assay.

A total of 10 C. trachomatis strains were obtained as fresh isolates from endocervical or urethral swabs from patients with urogenital symptoms at our sexually transmitted disease clinic. The isolates were typed by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein of C. trachomatis by the method of Frost et al. (13). They consisted of serovars E (n = 4) and F (n = 6). Two reference strains, E-BOUR (ATCC VR-348B) and L3 (CDC), were used as quality controls. Antimicrobial agents were all obtained as powders from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China, and solubilized according to the manufacturer's instructions.

MICs were determined by the method described previously (8, 18, 19). Each test was performed in triplicate. Briefly, chlamydia strains (4 x 104 inclusion-forming units/ml) were inoculated onto confluent monolayers of McCoy cells (1 x 105 to ~2 x 105 cells/well) in 96-well microtiter plates (Linbro, Flow Laboratories) with growth medium (1% fetal calf serum in RPMI 1640). After the inoculate had been centrifuged at 1,250 x g for 1 h at 30°C, the supernatant was replaced with maintenance medium (growth medium supplemented with 1 µg of cycloheximide per ml and 0.35% glucose) containing serially twofold-diluted antimicrobials. After incubation at 35°C for 48 h, the cultures in plates were fixed with acetone, stained with a fluorescein isothiocyanate (FITC)-labeled rabbit antibody specific to elementary bodies of C. trachomatis (Virostat, Portland, Maine), and observed under a fluorescence microscope. The MIC was defined as the lowest drug concentration at which no inclusion body was seen.

The in vitro effect of antimicrobial combination was evaluated by a two-dimensional (8-by-8) checkerboard microdilution technique. The procedures were similar to the determination of the MIC. The concentrations tested ranged from 4 to 1/16 times the MICs. The fractional inhibitory concentration (FIC) index for combinations was calculated as follows (11):

(A) is the concentration of drug A in a well which is the lowest inhibitory concentration for C. trachomatis in its row or column. (MICA) is the MIC of drug A for C. trachomatis. FICA, the fractional inhibitory concentration of drug A, is derived from dividing (A) by (MICA). (B), (MICB), and FICB are defined in the same fashion for drug A. The interaction is defined as synergistic if the FIC index is ≤0.5, antagonistic if >4.0, and indifferent if between 0.5 and 4.0. Each isolate for every combination was tested in triplicate. For a given isolate with a given combination, if synergism (or indifference or antagonism) was observed in two or three runs of the triplicate, synergism (or indifference or antagonism) was reported.

The effects of different combinations were compared by reference identical unit (Ridit) analysis. Ridit, introduced by Bross in 1958, is a sensitive statistical method to analyze ordinal data. The Ridit score does not require a normal distribution and preserves differences present in the (ordinal) data. Ridit analysis illustrates the relative position of the score of a particular group against an identified distribution of a reference group (20). The analysis was carried out by the calculation function of Excel Table.

In each batch of microdilution tests, the MICs of the five antimicrobial agents for strains VR-348B and L3 all fell within the ranges previously observed by other investigators using the same method (10, 15, 22). The ranges of MICs of spectinomycin, ceftriaxone, erythromycin, ofloxacin, and doxycycline were 128 to 256, 10 to 20, 0.06 to 0.5, 0.25 to 1, and 0.015 to ~0.125 µg/ml, respectively, in our study.

The in vitro effects and comparisons of combinations are shown in Tables 1 and 2. Indifference was observed in most of the combinations. When the combination of spectinomycin with erythromycin, ofloxacin, or doxycycline was compared to that with ceftriaxone, respectively, both combinations of spectinomycin with erythromycin (P = 0.014) and doxycycline (P = 0.005) were more synergistic than the combinations of ceftriaxone with the correspondent antibiotics. The combinations of ofloxacin with spectinomycin and ceftriaxone were of no significant difference (P = 0.32).


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  		TABLE 1. Comparison of the three combinations with spectinomycin or ceftriaxone

 

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  		TABLE 2. Comparison between spectinomycin and ceftriaxone in the three combinations

 
Our results are similar to those observed with other bacteria in previous studies. Burrows et al. (1) observed that the combination of erythromycin with spectinomycin was synergistic or additive against 27 of 33 Pasteurella haemolytica isolates. Flatz et al. (12) found synergism in penicillin-resistant pneumococci for the combination of ceftriaxone and levofloxacin, with a FIC value of 0.25. Mayer et al. (16) found synergism in 4 of 18 clinical isolates of Pseudomonas spp. for the combination of ceftriaxone with ofloxacin. Shishkov et al. (21) showed that spectinomycin-tetracycline was a most effective combination against Mycoplasma gallisepticum. Yet, there is an issue in our study which may be worthwhile to mention here. The interpretation of the FIC index was described to have different definitions, which may lead to inconsistent conclusions (23). The ineffectiveness of combinations of ceftriaxone in our study may be more clear (data are not shown) if the definition for antagonism is, traditionally, a FIC index of no less than 2 (11).

Most of the synergism between spectinomycin and doxycycline or spectinomycin and erythromycin occurred at a sub-MIC or even much lower concentration of spectinomycin (e.g., 32 µg/ml). The average reduction of MICs was two- or fourfold. Though the synergism was very weak, our study still recommended spectinomycin rather than ceftriaxone for this dual therapy because of the significant difference between the combinations of spectinomycin and ceftriaxone. In other words, when ceftriaxone is prescribed in the therapy, the interval between or the sequence of the administration of ceftriaxone and erythromycin or doxycycline may be prolonged or shifted to avoid possible antagonism and to achieve the best effect from combination therapy. In addition, in vivo study is required to confirm the results and the clinical significance of the conclusion in this study. Besides, more effective combinations need to be explored for this dual therapy.


   ACKNOWLEDGMENTS
 
We thank A. Eley, D. T. A. Te Dorsthorst, Shunzhang Ye, and Changgeng Shao for guidance with the experimental methods and for review of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Center for STD and Leprosy control, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China. Phone: 86-25-85478045. Fax: 86-25-85421323. E-mail: wangqianqiu{at}china.com. Back


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Antimicrobial Agents and Chemotherapy, April 2005, p. 1584-1586, Vol. 49, No. 4
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.4.1584-1586.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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