In vitro activities of sparfloxacin , ceftriaxone , penicillin , tetracycline and doxycycline against Chlamydia trachomatis and Neisseria gonorrhoeae

In vitro sparfloxacin was highly active against 223 penicillin-susceptible isolates of Neisseria gonorrhoeae with a 90% minimal inhibitory concentration (MIC90) of 0.004 μg/mL. Resistant strains of N gonorrhoeae totalled 55; 32 were penicillinase-producing and 23 chromosomally resistant. The MIC90 for these isolates was 0.004 μg/mL and 0.008 μg/mL, respectively. Chlamydia trachomatis was also very susceptible with an MIC50 of 0.063 μg/mL and a 50% minimal bactericidal concentration of 0.032 μg/mL for 11 isolates.

S PARFLOXACIN IS ONE OF THE NEW FLUOROQUINOLONE antibiotics.It is hoped that this agent will demonstrate both in vitro and in vivo activity against N eisseria 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 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 manufacture r'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 lxl0 3 to 5xl0 3 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 In vitro activity of spartloxacin penicillin-susceptible, 32 penicillinase-producing. a nd 23 chromosomally mediated resistant strains.The isolates of N gonon-hoeae were obtained in Edmonton from the Se),,,'Ually Transm itted Disease Clinic, Kasper Laboratories and the Provincial Laboratory of Public Health.All pen icillin-sensitive isolates were obtained from 1988 throu gh 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 10 5 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 appear ance 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 (pen icillin 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

DISCUSSION
Sparfloxacin demonstrated excellent in vitro activity against C trachomatis and penicillin -susceptible and -resistant strains of N gonorrhoeae.Sim ilar results against C trachomatis were reported by Nagayama and Kitajima (9), Oh and Bowie (10).Wise and Andrews (11).Wong and Stamm (12), and Nakam ura (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 u sed in stead of vials and f1uorescein-conjugated mon oclonal antibody stain in place of iodine.The present resu lts are s im ila r to those reported in other studies for the tetracyclines, ceftriaxone and penicillin (17)(18)(19) .Although not used clinically in the treatment of chlamydial infections , ceftriaxone and penicillin are important antibiotics in the treatm ent 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, resp ectively.
When difficulties arose determining the MIC, due to the appearance of a bnormal inclus ions.the MBC clarified the MIC.It was eviden t that a b errant inclusions were not infectious and therefore MBC values were generally lower than MIC values for each antibiotic.Using the definition of the endp oint 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• valu es used in a previous study [4 ] wh en inclusions persisted past t h e obvious dropoff point).Again . in som e cases on e or two inclusions p ers is ted past the 99.9% inhibition point and in these instances 'greater than' values would h ave been assign ed h ad absolute endpoints been defined.Defining t h e endpoint as the com p lete a bsen ce of inclus ions would n ot h ave facilitated a con cise compa rison of antibiotics.Th e bodies that pers isted may h ave b een identifia ble althou gh aberrant inclu s ions or antigenic m aterial (20) .
Recent research h as s hown that if a s m all num ber of organisms in the popula tion has antibiotic resistance, they may be detected when large inoculums a re used (2 1).Th e inoculum in this study ran ged from 1000 to 5000 inclusion-forming units p er well.Speculatively. the latter inoculum m ay h ave b een high enough that resistant organism s were manifest.
Sparfloxacin demonstrated excellent activity against 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.
ofloxacin, fleroxacin a nd norfloxacin fo r seven to 10 days demonstrated failure rates of Oto 70% (24).Further research is needed on this group of a ntimicrobial agents before a recommendation can b e 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 a re comparable to those of the tetracyclines.Ultimately, the place of sparfloxacin in vivo will depend upon tolerability and pharmacokinetic characteristics.

In vitro activity against 278 isolates of Neisseria gonorrhoeae
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 o f 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 p enicillinase-producing strains and chromosomally mediated strains.