SEXUALLY TRANSMITIED DISEASES

In vitro activities of sparfloxacin, ceftriaxone, penicillin,

tetracycline and doxycycline against Chlamydia trachomatis

and Neisseria gonorrhoeae

HAZEL TALBOT, BSC, RT, BARBARA ROMANOWSKI, MD, FRCP

H TALBOT, B ROMANOWSKI. In vitro activities of sparfloxacin, ceftriaxone , penicillin, tetracycline and doxycycline against Chlamydia trachomatis and Neisseria gonorrhoeae. Can J Infect Dis 1992;3(3): 114-117. In vitro spa rfloxacin was highly active against 223 penicillin-susceptible isolates of Neisseria gonorrhoeae with a 90% minimal inhibitmy concentration (MIC90) of 0.004 pg/mL. Resistant strains of N gonorrhoeae totalled 55: 32 were penicill inase-producing and 23 chromosomally resistant. The MIC90 for these isolates was 0 .004 pg/mL and 0.008 pg/mL. respectively. Chlamydia trachomatis was also very susceptible with an MIC50 of 0.063 pg/mL and a 50% minimal bactericidal concentration of 0 .032 pg/mL for 11 isolates .

Key Words: Chlamydia trachomatis. In vitro spwjloxacin. Neisseria gonorrhoeae

Activite in vitro de la sparfloxacine, de la ceftriaxone, de la penicilline, de la tetracycline et de la doxycycline centre Chlamydia trachomatis et Neisseria gonorrhoeae

RESUME: La sparfloxacine in vitro est fo rtement active contre 223 isola ls de Neisseriagonorrhoeae sensibles a la penicilline avec une concentration minirnale inhibitrice (CMl) de 90 % a 0 .004 ~tg/mL. Les sou ches resistantes de N gonorrhoeae sonl a u nombre de 55: 32 etaient productrices de penicillinase et 23 etaient chromosomiquement resistantes. La CMl90 etait de 0 .004 pg/ mL el a 0.008 ~tg/mL respectivement pour ces isolats. Chlamydia trachomatis etait a ussi Lres sensible a une CMI50 de 0,063 ~tg/mL et a une concentration minimale bactericide de 50 % a 0 .032 ~tg/mL pour 11 isolals.

SPARFLOXACIN IS ONE OF THE NEW FLUOROQUINOLONE

antibiotics. It is hoped that this agent will demon­strate both in vitro and in vivo activity against Neisseria gonorrhoeae and Chlamydia trachomatis . Quinolones such as norfloxacin . enoxacin, ofloxacin and cipro­floxacin have been documented to be effective in vitro

and in vivo against penicillin-susceptible and -resistant strains of N gonorrhoeae (1). However, these agen ts have been disappointing in their activities against C trachomatis , a major pathogen in both cervicitis and urethritis. With dramatic increases in the incidence of both chlamydia! infections and resistant strains of

Clinical Inves tigation Unit. Sexually Transmitted Disease Control. Alberta Health. Edmonton, A lberta Correspondence and reprints : Dr B Romanowski. Sexually Transmitted Disease Control. 4th F loor. 101 05-109 Street. Edmonton.

A lberta T5J 1MB. Telephone (403) 427-2830 This research was presented in part at the 3 rd International Symposium on New Quinolones, Vancouver. July 1990 Received for publication January 12. 1991. Accepted April 13. 1991

114 CAN J INFECT D1s VOL 3 No 3 M AY I Ju NE 1992

gonorrhea, new agents are required to facilitate management of these conditions .

In this study, the in vitro activity of sparfloxacin was compared with activities of ceftriaxone, penicillin, tetra­cycline and doxycycline against both N gonorrhoeae and C trachomatis.

PATIENTS AND METHODS The antimicrobial agents tested were sparOoxacin

(Parke-Davis, Michigan). ceftriaxone (Hoffmann -La Roche Ltd). penicillin G potassium (Wyeth Ltd). and tetracycline hydrochloride and doxycycline hydrochlo­ride (Ffizer Canada) . They were obtained in powder form and all except sparfloxacin were solubilized in sterile distilled water. Sparfloxacin was solubilized according to the manufacturer's directions in 95% ethanol, sterile distilled water and 1 N sodium hydroxide. Powders were stored dessicated at 4°C. Stock solutions of the drugs and test media containing serial twofold dilutions of the agent were prepared the day of use.

Clinical isolates of C trachomatis were obtained from 1988 to 1990 from patients attending the Edmonton Sexually Transmitted Disease Clinic with nongonococ­cal urethritis or mucopurulent cervicitis. Standard isolation methods were used (2 .3). Eleven isolates of C trachomatis were passed an average of 14 tin1es (range eight to 21) in cell culture medium (4) with 1 pg/mL cycloheximide and no antibiotics. All cell culture main­tenance was done in medium containing no antibiotics. In vitro sensitivity testing was done using McCoy cells grown in 96-well plates. Isolates retrieved from - 70°C were diluted to yield lxl03 to 5xl03 inclusions per well. Duplicate trays were inoculated and centrifuged at 1000 g for 1 hat 34°C. The inoculum was in1mediately removed and the monolayers overlayed with cell culture medium containing 1 pg/mL cycloheximide and serial twofold dilutions of the test antibiotic. Cultures were incubated for 48 hat 37°C in 0.3% carbon dioxide. and one tray was stained with Ouorescein-conjugated monoclonal antibody (Kallestad Canada) to determine the minimal inhibitory concentration (MIC) . The MIC was defined as the lowest concentration at which at least 99 .9% inhibition of inclusions occurred, ie. one or two inclusions were considered an endpoint in some cases.

The companion tray was used to determine the min­imal bactericidal concentration (MBC) as follows. The antibiotic medium was removed . the monolayer rinsed twice, and 0. 2 mL of cell culture medium was added . The tray was then frozen at - 70°C, thawed and passed to new monolayers vvith incubation in antibiotic-free medium. MBC endpoints were defined in the same manner as the MIC. Appropriate antibiotic-free control wells were included along with control organism type K strain UW-31/cx (ATCC VR 887). All tests were per­formed at least in duplicate .

Clinical isolates of N gonorrhoeae consisted of 223

CAN J INFECT D1s VOL 3 No 3 MAY/ JUNE 1992

In vitro activity of spartloxacin

penicillin-susceptible, 32 penicillinase-producing. and 23 chromosomally mediated resistant strains. The iso­lates of N gonon-hoeae were obtained in Edmonton from the Se),,,'Ually Transmitted Disease Clinic, Kasper Laboratories and the Provincial Laboratory of Public Health. All penicillin-sensitive isolates were obtained from 1988 through 1990. and penicillin-resistant iso­lates from 1984 through 1990. Standard methods were used to isolate and identify the organism (4,5) . All isolates were screened for penicillin resistance, and beta-lactamase production was assessed using the chromogenic cephalosporin technique (nitrocefin) (Cefinase; BBL Microbiology Systems, Maryland). An organism with chromosomally mediated resistance is one with an MIC of penicillin of 1 mg/ mL or more which does not produce beta-lactamase. N gonorrhoeae with chromosomally mediated resistance have resistance due to changes in the cell wall. whereas penicillinase­producing N gonorrhoeae have plasmid-directed penicillinase production.

In vitro sensitivity testing for N gonon-hoeae was done using agar dilution techniques (4-8) on GC medium base agar (Difeo Laboratories, Michigan) with 1 % Kellogg's defined supplement. A suspension of the organism adjusted to a 0.5 McFarland turbidity stand­ard in 0.7% casamino acids solution was inoculated with a Steer·s replicator thus delivering 105 colony­forming units to the plate containing serial twofold dilutions of the test antibiotic. Incubation was at 37°C in 7% carbon d ioxide for 18 to 24 h.

Control strains were WHO 111 , w1-10 v and WHO VII and were obtained from the Antimicrobials and Molecular Biology Division , Laboratory Center for Disease Con­trol, Ottawa, Ontario. Appropriate antibiotic-free plates were included. The MIC was defined as the concentra­tion inhibiting growth with a slight haze considered negative. All tests were performed at least in duplicate.

RESULTS Table 1 summarizes drug activities against the 11

isolates of C b·achomatis. MBC was usually lower than MIC by one dilution, reflecting tJ1e appearance of abnor­mal inclusions near the MIC which were not infectious. The abnormal inclusions made endpoints difficult to interpret. The MBC was therefore considered a better metJ1od of analyzing drug activities in fuis study. Spar­floxacin had an MBC50 value of 0.032 pg/mL and demonstrated activity comparable to tetracycline and doxycycline.

Table 2 outlines the MIC values for N gonon-hoeae. Against penicillin-susceptible (penicillin MIC less than 1 mg/ mL). penicillinase-producing and chromosomally mediated resistant strains of N gonon-hoeae the M!Cgo of sparfloxacin was 0.004, 0.004 and 0.008 pg/mL, respectively. All N gonon-hoeae isolates witJ1 chromosomally mediated resistance to penicillin had tetracycline MICs less than or equal to 2 pg/mL. The

115

TALBOT AND ROMANOWSKI

TABLE l In vitro activity against 11 isolates of Chlamydia trachomatis

Minimal inhibitory concentration (µg/ ml) Minimal bactericidal concentration (µg/ ml) Antimicrobia l agent Range 50% of isolates Range 50% of isolates Sparfloxacin 0.032 to greater than 0. 125 0.063 0.016 to 0.063 0.032

Ceftriaxone Greater than 32 Greater than 32 16 16

Penicillin Greater than 0.5 Greater than 0.5 0.063 to 0.5 0.125

Tetracycline 0. 125 to 0.25 0.125 0.032 to 0.063 0.063

Doxycycline 0.063 to 0.125 0.063 0.016 to 0.063 0.063 Minimal bactericidal concentration is the lowest concentrotion with at least 99. 9% inhibition of inclusions after passage of antibiotic-treated cultures in antibiotic-free medium. Minimal inhibitory concentration is the lowest concentration with at least 99. 9% inhibition of inclusions

TABLE 2 In vitro activity against 278 isolates of Neisseria gonorrhoeae

Antimicrobia l Penicillin MIC<l µg/ ml (n=223) PPNG (n=32) CMRNG (n=23) agent Range 50% 90% Range 50% 90% Range 50% 90%

Sparfloxacin $0.00025 to 0.004 0.002 0.004 0.001 to0. 125 0.004 0.004 0.002 to 0.008 0.004 0.008

Ceftriaxone $0.00025 to 0.008 0.002 0.004 0.002 to 0.016 0.008 0.016 0.004 to 0.032 0.016 0.016

Penicillin $0.032 to 0.5 0.063 0.25 32 to >256 >256 >256 l to 2 2 2

Tetracycline 0.125to 16' 0.25 0.5 0.5 to 16 2 2 0.5 to 2 2 2

Doxycycline 0.063 to 2 0.25 0.5 0.5 to 8 2 2 1 to 2 2 2 CMRNG N gonorrhoeae with chromosomally mediated resistance (MIC of penicillin ?.1 pg/ml, beta-lactamase negative); MIC Minimal inhibitory concentration; PPNG Penicillinase-producing N gonorrhoeae (beta-lactamase positive). ·one isolate was tetracycline resistant with a tetracycline MIC of 16 pg/ ml. All other isolates in the penicillin-sensitive category had M/Cs of tetracycline 52 ,,g/mL

MICso and M!Cgo values of sparfloxacin against all penicillin-resistant strains of N gonorrhoeae were lower than ceftriaxone by one or two dilutions . Sparfloxacin and ceftriaxone had identical MIC values against penicillin-susceptible isolates . Tetracycline demonstrated intermediate activity against both penicillinase-producing strains and chromosomally mediated strains.

DISCUSSION Sparfloxacin demonstrated excellent in vitro activity

against C trachomatis and penicillin -susceptible and -resistant strains of N gonorrhoeae. Similar results against C trachomatis were reported by Nagayama and Kitajima (9), Oh and Bowie (10). Wise and Andrews (11). Wong and Stamm (12), and Nakamura (13). Methods of C trachomatis antibiotic susceptibility testing h ave not been standardized. The method used in this stu dy has been used by others (14). Variations in methods for chlamydial in vitro testing have been discussed in other papers (15-17) .

In the present study, the test antibiotic was added immediately after centrifu gation and the MBC defined as the point of inhibition after one passage of the antibiotic-treated culture in antibiotic-free medium. Ninety-six-well plates were used instead of vials and f1uorescein-conjugated mon oclonal antibody stain in place of iodine. The present resu lts are s im ilar to those reported in other studies for the tetracyclines, ceftriaxone and penicillin (17-19) . Although not used clinically in the treatment of chlamydial infections , ceftriaxone and penicillin are important antibiotics in

116

the treatment of oth er sexually transmitted diseases and were included for control purposes. Both agen ts had some activity with MBCso valu es of 0 .125 and 16 mg/mL for penicillin and ceftriaxon e, respectively.

When difficulties arose determining the MIC, due to the appearance of abnormal inclusions. the MBC clarified the MIC. It was eviden t that a berrant inclu­sions were not infectious and therefore MBC values were generally lower than MIC values for each an­tibiotic. Using the definition of the endpoint as the lowest concen tration at which at least 99 .9% inhibition of inclusions occurred allowed the a u thors to assign definite endpoints (as opposed to the ·greater than· values used in a previous study [4] wh en inclusions persisted past th e obvious dropoff point). Again . in som e cases on e or two inclusions pers is ted past the 99.9% inhibition point and in these instances 'greater than' values would h ave been assign ed had absolute endpoints been defined. Defining th e endpoint as the complete absence of inclusions would not h ave facilitat­ed a con cise comparison of antibiotics. Th e bodies that pers isted may h ave been identifia ble althou gh aberrant inclu sions or antigenic m aterial (20) .

Recent research h as shown that if a sm all num ber of organisms in the population has antibiotic resistance, they may be detected when large inoculums are used (2 1). The inoculum in this study ranged from 1000 to 5000 inclusion-forming units per well . Speculatively. the latter inoculum m ay h ave been high enough that resistant organism s were manifest.

Sparfloxacin demonstrated excellent activity against

CAN J INFECT D IS VOL 3 No 3 M AY/JUNE 1992

all strains of N gon orrhoeae tested. For penicillin-sus­ceptible strains. th ese results were comparable to those of ceftriaxone but for resistant strains, sparfloxacin achieved results on e to two dilutions lower than ceftri­axone . Similar results have been reported by Gransden and King (22) as well as Kojima et al (23) and Nakamura (13).

Clinical trials of the new quinolone agents have so far resulted in varied ou tcomes. None of th e currently available quinolone agents is effective in a single dose for eradication of C trachomatis infection even though in vitro activity of some quinolones (ciprofloxacin and ofloxacin) is relatively high. Trials using ciprofloxacin.

ACKNOWLEDGEMENTS: This work was funded by a granl from Parke-Davis (US). We especial ly thank Kasper Laboratories and the Edmonton Provincial Laboratory of Public Health for donating N gonorrhoeae isolates. We also thank U1e pharmaceutical companies which kindly supplied the anU­biotic powders for this study.

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1987:ii: 1319-22. 2. Ripa KT. Mardh PA. Cu!Uvation of Chlamydia trachomatis

in cyclohexirnide-treated McCoy cells. J Clin Microbial 1977:6:328-3 1.

3. Smith TF. Brown SD. Weed LA. Diagnosis of Chlamydia trachomatis infections by cell cultures and serology. Lab Med 1982:13:92- 100.

4. Talbot H. Romanowski B. In vitro activiLies of lomefloxacin. tetracycline. pen icillin. spectinomycin and ceftriaxone against Neisseria gonorrhoeae and Chlamydia trachomatis. Antimicrob Agents Chemother l 989:33:2049-51.

5. Romanowski B . Hardy JS. Rafter MS. Dral,er J. Enoxacin in the U1erapy of anal and pha1-yngeal gonococcal infections. SexTransm Dis 1989:16:190- 1.

6. Barry AL. Agar dilution techniques. In: Barry AL. ed. The Antimicrobic Susceptibi li ty Tesl: Princip les and Practices . Ph iladelphia: Lea and Febiger. 1976:76-90.

7. Dillon JR. Laboratory Methods for N gonoTThoeae (MIC. Auxotype. Screening for PPNG). Ottawa: Health and Welfare Canada. 1983.

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9. agayarna A. Kilajimas S. Nakoa T. In vilro acUviUes of sparfloxacin and other new quinolones against Chlamydia trachomatis. 3 rd lnlernational Symposium on lhe ew Quinolones. 1990:283. (Absl)

10. Oh H. Bowie WR. In vilro activity of sparnoxacin and olher quinolones against Chlamydia trachomatis. 3rd lnternaUonal Symposium on the New Quinolones. 1990:287. (Absl)

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CAN J INFECT D 1s VOL 3 No 3 MAY/JUNE 1992

In vitro activity of sparfloxacin

ofloxacin, fleroxacin and norfloxacin for seven to 10

days demonstrated failure rates of Oto 70% (24). Fur­ther research is needed on this group of antimicrobial agents before a recommendation can be made for their general use in the treatment of chlamydia! infections.

Based on the findings of the present study, the future of sparfloxacin is promising. The inciden ce of resistant N gonorrhoeae is increasing and sparfloxacin shows excellent in vitro activity against these strains. In vitro results against C trachomatis are comparable to those of the tetracyclines. Ultimately, the place of sparfloxacin in vivo will depend upon tolerability and pharmacokinetic characteristics.

12. Wong K, Slarnm WE. Antimicrobial activity of sparnoxacin (Cl -978. Al-4140) vs Chlamydia trachomatis (CT) in cell culture. 3rd International Symposium on the New Quinolones, 1990:284. (Abst)

13. Nalmmura S. ln vitro and in vivo antibacterial activities of AT-4140. a new broad-spectrum quinolone. Antimicrob Agents Chemother 1989:33: 1167-73.

14. Segreti J. Kessler HA, Kapell KS. Trenholme GM. In vitro activities of A-56268 (TE-031) and four other antibiotics against Chlamydia trachomatis . Antimicrob Agents Ch emother 1987:31: 100-1.

15. Mardh PA. Paavonen J. Puolakkainen M. Chlamydia. New York: Plenum Medical Book Company. 1989:103-5.

16. 01iel JD. Ch emotherapy. In: Oriel D, Ridgway G. Schachter J. Taylor -Robinson D. Ward M. eds . Ch lamydia! Infections. Cambridge: Camb1·idge Universi ty Press. 1986:513-23 .

17. Slarnrn WE. Chlamydia trachomatis: Antibiotic sensitivity and chemotherapy. In: Reeve P. ed. Chlamydia! Infections. Berlin: Springer -Verlag. 1987:34-44.

18. Eh rel JME. Judson FN. Susceptibility testing of Chlamydia trachomatis: From eggs to monoclonal antibodies . Antim icrob Agents Chemother 1988:32: 1295-9 .

19. Hammerschlag MR. Gleyzer A. In vitro activity of a group of broad-spectrum cephalosporins and other bela-lactam antibiotics against Chlamydia trachomatis. Antimicrob Agents Ch emoth er 1983:23:493-4.

20. How SJ. Hobson D. Hari. A. Quayle E. A comparison of U1e in-vilro activity of antimicrobials against Chlamydia trachomatis. Antimicrob Agents Chemoth er 1985: 15:399-404.

2 1. Jones RB. Van Der Pol B. Martin DH. Shepard K. Partial characterization of Chlamydia trachomatis isolates res istant lo multiple antibiotics. J Infect Dis 1990: 162: 1309- 15.

22. Grandsen WR. King A. Phillips I. A comparative study of lhe in vilro acUvity of sparnoxacin against Neisseria gonorrhoeae. 3 rd InlernaLional Symposium on the New Quinolones. 1990:46. (Absl)

23 . Kojima T. Inoue M. Milsuhash i S. In vitro activity of AT-4140 against clinical bacterial isolates. Antimicrob Agents Chemolher 1989:33: 1980-8.

24 . Toomey KE. Barnes RC. Trealmenl of Chlamydia trachomatis genital infections. Rev Infect Dis 1990: l 2(Suppl 6):S645-55.

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