monoclonal antibody test (Du Pont Co., Wilmington, Del.).

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1992, p. 277-282 66-484/92/2277-6$2./ Copyright 1992, American Society for Microbiology Vol. 36, No. 2 In Vitro Antibiotic Susceptibilities of Neisseria gonorrhoeae Isolates in the Philippines T. E. CLENDENNEN 111,1* C. S. HAMES,2 E. S. KEES,' F. C. PRICE,' W. J. RUEPPEL,' A. B. ANDRADA,3 G. E. ESPINOSA,3 G. KABRERRA,4 AND F. S. WIGNALL' Navy Environmental and Preventive Medicine Unit Six, Pearl Harbor, Hawaii 9686-541, and U.S. Naval Hospital, Subic Bay,2 Olongapo City Health Department, Zambales,3 and Subic City Social Hygiene Clinic, Zambales,4 Philippines Received 7 May 1991/Accepted 13 November 1991 Antibiotic susceptibility surveillance testing was performed on clinical isolates of Neisseria gonorrhoeae collected in September 1989 in the Philippines. -Lactamase was produced by 77 (55%) of 14 isolates. In vitro MIC testing revealed significant resistance to penicillin (MIC for 9% of isolates [MIC], >64,ug/ml), tetracycline (MIC9, 4 jig/ml), and cefmetazole (MIC9, 8,ug/mI). Spectinomycin resistance was rare (1 of 117), but the MIC9 was 32,ug/mI. Isolates were susceptible to fluoroquinolones and cephalosporins at the time of this survey, as evidenced by the MIC9s of ciprofloxacin (.25,ug/ml), norfloxacin (2.,ug/ml), ofloxacin (.625,Ig/ml), cefpodoxime (2.,ug/ml), cefotaxime (1.,ig/ml), ceftazidime (.25,Ig/mI), ceftizoxime (.25,ug/ml), and ceftriaxone (.6,ug/ml). To date, ceftriaxone resistance has not emerged, despite the widespread use of this antibiotic in the Philippines. Changes in the antibiotic resistance patterns of Neisseria gonorrhoeae isolates in the Philippines have heralded the emergence of resistant isolates in the United States over the last decade (7, 16). This geographic area continues to serve as a test ground for the emergence of antibiotic-resistant N. gonorrhoeae isolates because of antibiotic pressure consequent to the continuous use of inadequately dosed prophylactic antibiotics by those who are repeatedly infected and high endemic rates of gonococcal infections in prostitutes who live adjacent to military bases. Penicillin-resistant N. gonorrhoeae isolates were first reported in the Philippines in 1965 to 1966 (7, 16). As antibiotic therapy was altered in response to the demonstration of resistance to this agent and later to others, these changes were followed by the emergence of isolates resistant to the newer therapeutic regimens (1, 2, 4-6, 14). Unfortunately, it also appears that once antibiotic resistance develops, it persists in the local reservoir, despite the substitution of unrelated antimicrobial agents in new antibiotic regimens (1). The present study was undertaken to reassess the antibiotic susceptibility of N. gonorrhoeae isolates in the Philippines and, in particular, to examine the possibility of emerging ceftriaxone resistance, since this antibiotic is currently used to treat all gonococcal infections in the area from which these isolittes were collected. MATERIALS AND METHODS Gonococcal isolates. Isolates of N. gonorrhoeae were obtained from men attending military sick call with symptoms of a sexually transmitted disease and from female bar hostesses attending public health screening clinics. Initial isolations were made on modified Thayer-Martin agar (BBL Microbiology Systems, Cockeysville, Md.). Suspect colonies were identified by colony morphology, Gram staining, oxidase activity (SpotTest oxidase reagent; Difco Laboratories, Detroit, Mich.), and reaction in the Gonochek II * Corresponding author. 277 monoclonal antibody test (Du Pont Co., Wilmington, Del.). Overnight subcultures were placed in cryoprotective medium (Trypticase soy broth [BBL] with 2% glycerol [Mallinckrodt, Inc., Paris, Ky.]) and frozen in liquid nitrogen until tested. Thawed specimens were plated on chocolate agar prepared from GC agar base (BBL), 1% bovine hemoglobin (BBL), and 1% IsoVitaleX (BBL). Pure colonies reisolated on chocolate agar were tested as 18-h growth in second subcultures. j-lactamase testing.,b-lactamase production was tested by use of nitrocefin disks (Cefinase; BBL) with Haemophilus influenzae ATCC 1211 as a negative control. TABLE 1. Antibiotic" In vitro susceptibilities of N. gonorrhoeae isolates in the Philippines MIC (ug/rml) No. of isolates 9% 5% Range tested Penicillin G (,L neg) 16. 2..2->64. 61 Penicillin G (PL pos) >64. 16..6->64. 77 Azlocillin (PL neg) >2..5.3->2. 48 Azlocillin (1L pos) >2. >2..3->2. 6 Erythromycin 2..5.6->4. 135 Tetracycline (PL neg) >4. 2..6-32. 6 Tetracycline (,L pos) 4. 1..6->4. 75 Spectinomycin 32. 32. 16.->128. 117 Trospectinomycin 16. 8. <2.->128. 8 Cefoxitin 4. 1..6->8. 134 Cefmetazole (,L neg) >8. 8..6->8. 59 Cefmetazole (,L pos) 8. 2..5->8. 75 Cefuroxime 2..125.15->8. 135 Cefpodoxime 2..3.2->4. 134 Cefotaxime 1..3.2->8. 134 Ceftazidime.25.6.15->4. 124 Ceftizoxime.25.8 <.1->4. 137 Ceftriaxone.6.8 <.1->1. 134 Norfloxacin 2..6.8->4. 134 Ofloxacin.625.38.5->5. 139 Ciprofloxacin.25.4 <.1->2. 135 " PL neg,,-lactamase negative;,l pos,,b-lactamase positive. Downloaded from http://aac.asm.org/ on April 26, 218 by guest

278 CLENDENNEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. ( c (D ) CU ) ) - 8-7n- 5- - 42.6 4- --= 2 1---_ 49 1.3 <OR=.6.12TO 1 >OR= 2 Minimum Inhibitory Concentration (ug/ml) FIG. 1. Relationship of,b-lactamase carriage to in vitro penicillin susceptibility in 61,3-lactamase-negative ( M ) and 77,B-lactamasepositive ( M ) isolates. Antimicrobial agents. Standard antimicrobial agent reference powders were provided as follows: penicillin G, Marsham Pharmaceuticals, Cherry Hill, N.J.; tetracycline hydrochloride, Lederle Laboratories, Wayne, N.J.; spectinomycin hydrochloride, trospectinomycin hydrochloride, cefmetazole sodium, and cefpodoxime proxetil, The Upjohn Co., Kalamazoo, Mich.; azlocillin sodium and ciprofloxacin hydrochloride, Miles Pharmaceuticals, West Haven, Conn.; cefoxitin sodium and norfloxacin sodium, Merck Sharp & Dohme, West Point, Pa.; cefuroxime sodium and ceftazidime, Glaxo, Inc., Research Triangle Park, N.C.; cefotaxime sodium, Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N.J.; ceftizoxime sodium, Smith, Kline & French, Philadelphia, Pa.; ceftriaxone, Roche Laboratories, Nutley, N.J.; erythromycin base, Abbott Pharmaceuticals, Inc., North Chicago, Ill.; and ofloxacin, Imperial Pharmaceuticals, Manila, Philippines. 1 9-8--- c 7-.co cn CD 6- O 5- cm co ) 4- X- 3-2- I 1- - <OC)R =2 4 >OH =8 Minimum Inhibitory Concentration (ug/mi) FIG. 3. Relationship of 13-lactamase carriage to in vitro cefmetazole susceptibility in 59 3-lactamase-negative ( 1 ) and 75 3-lactamase-positive ( 1 ) isolates. Antibiotic stock solutions were prepared in reagent-grade water and frozen at -7 C. Serial twofold dilutions of the antibiotics were prepared in reagent-grade water on the day of use. Antibiotic susceptibility testing. Antibiotic susceptibility testing was conducted as previously described (15). Quality control organisms inoculated on each plate were Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922. Subcultures were incubated in a humidified atmosphere of 5% CO2 for 24 h at 35 C. MICs were read as the lowest concentration of antibiotic that inhibited growth (11). Statistical analysis. The effect of P-lactamase production on susceptibility to each drug was examined by chi-square analysis with Epilnfo Version 5. (Centers for Disease Control Epidemiology Program Office, Atlanta, Ga.). Significance was defined as P c.5. Data are presented sep Downloaded from http://aac.asm.org/ on April 26, 218 by guest ac c) CY) D C bu - 5-- 4-, 3-,- 2-1 - -- 1.4 1 1- <OR=.6.12 TO 1 >OR= 2 Minimum Inhibitory Concentration (ug/mi) FIG. 2. Relationship of,b-lactamase carriage to in vitro azlocillin susceptibility in 48,-lactamase-negative ( M ) and 6,-lactamasepositive ( M ) isolates..) ce CO ) 3-2- 1- - 3=.25.5 TO 1 > OR = 2 Minimum Inhibitory Concentration (ug/ml) FIG. 4. Relationship of,b-lactamase carriage to in vitro tetracycline susceptibility in 6,-lactamase-negative ( M ) and 75 P-lactamase-positive ( M ) isolates. 3

VOL. 36, 1992 N. GONORRHOEAE ISOLATES FROM THE PHILIPPINES 279 1-97.6 9 U1) 'o ) U1) CD Q ro U) ) co ) 2-1 -_- -------------_- -. ------74-1----.9 O- < OR =32 64 > OR =128 Minimum Inhibitory Concentration (ug/m) n=1 17 8-- 6--_. 5-- 4- FIG. 5. <OR=.5 1 TO 4 > OR =8 Minimum Inhibitory Concentration (ug/mi) n=135 Distribution of erythromycin susceptibility. rately for drugs for which 1-lactamase production was found to have a significant effect on susceptibility. RESULTS One hundred forty isolates were confirmed to be N. gonorrhoeae. Although no single drug was evaluated against all isolates (Table 1), 3-lactamase was detected in 77 of 14 (55%) isolates and conferred resistance to penicillin (MIC, >2 jg/ml) in 76 of 77 (98%) (13). Thirty-two of 61 (52%) 3-lactamase-negative isolates also were penicillin resistant (P <.1) (Fig. 1). Seventy-one percent (43 of 6) of,3-lactamase-positive and 18% (9 of 48) of P-lactamasenegative isolates were resistant to azlocillin (MIC, >2,ug/ml) n U) U) 8--- - - _ 1W- 91.5 9- -- 7-1-_.. _ S///////////m...-.--- _ -.-_.... 1.3 1.3 O-,,,,,,,,,,,,,I I,,,,,,,.... C,,,,I,,I,I <OR= 32 64 >OR = 128 Minimum Inhibitory Concentration (ug/ml) n=8 FIG. 7. Distribution of trospectinomycin susceptibility. (P <.1) (Fig. 2). Sixty-one percent (36 of 59) of 3-lactamase-negative and 17.3% (13 of 75) of 3-lactamase-positive isolates were resistant to cefmetazole (MIC, >8,ug/ml) (P <.1) (Fig. 3). Thirty-two percent (24 of 75) of 3-lactamasepositive and 66% (4 of 6) of P-lactamase-negative isolates were resistant to tetracycline (MIC,.2 jig/ml) (P <.1) (Fig. 4) (8). A few isolates were resistant to erythromycin, spectinomycin, trospectinomycin, cefoxitin, and cefuroxime, but the association with P-lactamase production was not significant (Fig. 5 to 9) (8, 9, 13). A single isolate was resistant to ciprofloxacin (MIC,.4,ug/ml) and was,-lactamase negative (Fig. 1) (13). None of the isolates tested was found to be resistant to cefotaxime, cefpodoxime, ceftazi- Downloaded from http://aac.asm.org/ on April 26, 218 by guest ) U3) ao 5-_ 4-- --/////-_- -- 3 --. - U) FIG. 6. Distribution of spectinomycin susceptibility. <OR=2 4 >OR=8 FIG. 8. Distribution of cefoxitin susceptibility.

28 CLENDENNEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. 6- *D 5- ) 4- ) ( 3 U) t 6 5 CY) a4- ) - 1- /// 2.2 /// // // <OR= 1 2 >OR =4 Minimum Inhibitory Concentration (ug/ml) n=135 FIG. 9. Distribution of cefuroxime susceptibility. dime, ceftizoxime, ceftriaxone, norfloxacin, or ofloxacin (Fig. 11 to 17) (8, 13). 5 () D c L ) 1-3 9= 8- _ 5-~ 4-~ DISCUSSION This survey revealed that established resistance to penicillin and other P-lactamase-susceptible antibiotics, tetracyclines, and erythromycin has continued at a high prevalance. The MICs for 9% of isolates (MIC9s) of penicillin G (>64,ug/ml), tetracycline (4 [±g/ml), and erythromycin (>4,ug/ml) were consistent with those reported in previous studies carried out in the Philippines (1, 2, 4, 1, 14). Azlocillin, a ureidopenicillin, presented no advantage over penicillin G, since it too was susceptible to P-lactamase (12). The r-lac- 7-3- 2- - <OR= 1 2 >OR =4 Minimum Inhibitory Concentration (ug/mo n=135 FIG. 1. Distribution of ciprofloxacin susceptibility. ' --:: -- :--:-:- -: 1.5. <OR =8 16 TO 32 > OR = 64 FIG. 11. Distribution of cefotaxime susceptibility. tamase-stable broad-spectrum cephalosporins were highly active against all or most of the strains and should each be effective at the usual preferred dose. The fluoroquinolones tested were effective against all or most of the strains in vitro and may represent a viable treatment option in the Philippines. Although no fluoroquinolone antibiotics have been officially recommended in the Philippines since the discontinuation of rosoxacin use by health department clinics several years prior to this study, the MIC9 of norfloxacin is now 16-fold higher than the MIC98 reported in 1982 data (3). The uncontrolled availability of antibiotics has previously been cited as a confounding factor in the selection of a therapeutic regimen in and near Subic Bay and probably () Co ) ) ) cn ) CD 1-97.e 9/ 8 _////// 4-~ 7-6- 5-3- 2-1---._ -///_._. /////////// 3. o- //////S,e//,,,,,.o < OR =2 4TO8 > OR = 16 FIG. 12. Distribution of cefpodoxime susceptibility. Downloaded from http://aac.asm.org/ on April 26, 218 by guest

VOL. 36, 1992 N. GONORRHOEAE ISOLATES FROM THE PHILIPPINES 281 ) OD ) ) - 166 1-1. 9-8- 7-6- 5-4- 3-2- 1-.. <OR =8 16 > OR =32 Minimum Inhibitory Concentration (ug/m) n=124 FIG. 13. Distribution of ceftazidime susceptibility. contributes significantly to the development and persistence of resistant strains (6). Also of interest is the presence of a P-lactamase plasmid in 55% of all strains and chromosomally mediated resistance to penicillin in 57% of the 1-lactamasenegative strains as well. This fact probably reflects the ongoing practice by sexually active persons of continuously taking subtherapeutic doses of oral penicillins. Spectinomycin and the investigational drug trospectinomycin were active against greater than 9% of strains, but the overall level of resistance of susceptible strains is increasing. Spectinomycin (MICgo, 32,ug/ml) is probably useful in the treatment of those for whom ceftriaxone is contraindicated. The prevalence of resistance to spectinomycin has declined from 22% is ) a 3 D - 1 1. 9-8- 7-5- 4-3- 2-1- <OR =8 16 TO 32 > OR = 64 Minimum Inhibitory Concentration (ug/ml n=137 FIG. 14. Distribution of ceftizoxime susceptibility. < OR =.25 > OR =.5 Minimum Inhibitory Concentration (ug/ml) n=134 FIG. 15. Distribution of ceftriaxone susceptibility. ) () in 1988 to less than 1% in late 1989, but the MIC9 and M'C5 have continued to rise since the discontinuation of spectinomycin therapy in public health clinics (1). Trospectinomycin (MIC9, 16,ug/ml) was about twice as active as spectinomycin, but class resistance resulting from the abuse of spectinomycin probably limits the application of this drug in the control of gonorrhea in the Philippines. Class resistance and lack of Food and Drug Administration approval may preclude the use of trospectinomycin in the clinical setting. While broad-spectrum cephalosporins and fluoroquinolones remain viable alternatives for the treatment of gonorrhea in the Philippines, rising MICs and sporadic resistance suggest increased antibiotic resistance in the future. Continued systematic monitoring of in vitro suscep- D - 2 D <OR =4 8 > OR = 16 FIG. 16. Distribution of norfloxacin susceptibility. Downloaded from http://aac.asm.org/ on April 26, 218 by guest

282 CLENDENNEN ET AL. am CO) s U) (D ) 1-96.4 9-. // 8-6...,., W////////.....,,,,,......~~~~~., _ 7 -...X////X... -., _ 1-.,,... --------.......... 6//////// 3.6 -. <4R2 4 >OR=8 Minimum Inhibitory Concentration (ug/mi) n=139 FIG. 17. Distribution of ofloxacin susceptibility. tibility patterns coupled with rigorous clinical follow-up of reported treatment failures in gonorrhea cases in the Philippines will be necessary to inhibit the spread of future ceftriaxone-resistant strains from the busy port area of Subic Bay. ACKNOWLEDGMENTS We thank S. T. Bernardo and E. A. Aquino for expert technical assistance in collecting and characterizing the isolates, R. K. Hanson for assistance in carrying out statistical analyses, and E. Donegan for reviewing the manuscript. REFERENCES 1. Berg, S. W., and W.. Harrison. 1981. Spectinomycin as primary treatment of gonorrhoea in areas of high prevalence of penicillinase-producing Neisseria gonorrhoeae. Sex. Transm. Dis. 8:38-39. 2. Berg, S. W., M. E. Kilpatrick, W.. Harrison, and J. A. McCutchan. 1979. Cefoxitin as a single-dose treatment for urethritis caused by penicillinase-producing Neisseria gonorrhoeae. N. EngI. J. Med. 31:59-511. 3. Crider, S. R., S. D. Colby, L. K. Miller, W.. Harrison, S. B. J. Kerbs, and S. W. Berg. 1984. Treatment of penicillin-resistant Neisseria gonorrhoeae with oral norfloxacin. N. Engl. J. Med. 311:137-14. ANTIMICROB. AGENTS CHEMOTHER. 4. Crider, S. R., M. E. Kilpatrick, W.. Harrison, S. B. J. Kerbs, and S. W. Berg. 1984. A comparison of penicillin G plus a f-lactamase inhibitor (sulbactam) with spectinomycin for treatment of urethritis caused by penicillinase-producing Neisseria gonorrhoeae. Sex. Transm. Dis. 11:314-317. 5. Harrison, W. O., R. R. Hooper, P. J. Wiesner, A. F. Campbell, W. W. Karney, G. H. Reynolds,. G. Jones, and K. K. Holmes. 1979. A trial of minocycline given after exposure to prevent gonorrhea. N. Engl. J. Med. 3:174-178. 6. Harrison, W. O., F. S. Wignall, S. B. J. Kerbs, and S. W. Berg. 1984. Oral rosoxacin for treatment of penicillin-resistant gonorrhoea. Lancet i:566. 7. Holmes, K. K., D. W. Johnson, S. Stewart, and P. A. Kvale. 1968. Treatment of "penicillin-resistant" gonorrhea in military personnel in S. E. Asia: a cooperative evaluation of tetracycline and of penicillin plus probenecid in 1263 men. Mil. Med. 133:642-646. 8. Jones, R. N., T. L. Gavan, C. Thornsberry, P. C. Fuchs, E. H. Gerlach, J. S. Knapp, P. Murray, and J. A. Washington II. 1989. Standardization of disk diffusion and agar dilution susceptibility tests for Neisseria gonorrhoeae: interpretive criteria and quality control guidelines for ceftriaxone, penicillin, spectinomycin, and tetracycline. J. Clin. Microbiol. 27:2758-2766. 9. Jones, R. N., E. H. Gerlach, F. P. Koontz, P. R. Murray, M. A. Pfaller, J. A. Washington, M. E. Erwin, and C. C. Knapp. 1991. Interpretive criteria and quality control guidelines for Neisseria gonorrhoeae susceptibility test standardization for cefotetan. J. Clin. Microbiol. 29:363-366. 1. Joyce, M. P., B. B. Aying, G. H. Vaughan, D. S. Herip, C. G. Hayes, G. Espinosa, A. Andrada,. P. Daily, and L. W. Laughlin. 1988. Drug resistance patterns of gonococcal isolates in the Philippines, abstr. C-42, p. 339. Abstr. 88th Annu. Meet. Am. Soc. Microbiol. 1988. 11. Knapp, J. S. 1988. Laboratory methods for the detection and phenotypic characterization of Neisseria gonorrhoeae strains resistant to antimicrobial agents. Sex. Transm. Dis. 15:225-233. 12. Kucers, A., and N. M. Bennett (ed.). 1987. The use of antibiotics, 4th ed., p. 228. J. B. Lippincott Co., Philadelphia. 13. National Committee for Clinical Laboratory Standards. 199. Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 14. Sanchez, P. L., F. S. Wignall, T. R. Zajdowicz, S. Kerbs, S. W. Berg, and W.. Harrison. 1983. One gram of cefoxitin cures uncomplicated gonococcal urethritis caused by penicillinaseproducing Neisseria gonorrhoeae (ppng). Sex. Transm. Dis. 1:135-137. 15. Schwarcz, S. K., J. M. Zenilman, D. Schnell, J. S. Knapp, E. W. Hook III, S. Thompson, F. N. Judson, and K. K. Holmes. 199. National surveillance of antimicrobial resistance in Neisseria gonorrhoeae. J. Am. Med. Assoc. 264:1413-1417. 16. Seigel, M. S., S. E. Thompson, and P. L. Perine. 1977. Penicillinase-producing Neisseria gonorrhoeae. Sex. Transm. Dis. 4:32-33. Downloaded from http://aac.asm.org/ on April 26, 218 by guest