Cross*Canada survey of susceptibility of Neisseria gonorrhoeae isolates to six antimicrobial agents J.R. DILLON, PH D. L. ElDus, MD; B.B. DIENA, DVM, PH D

The minimum inhibitory concentrations (MICs) of penicillin, ampicillin, tetracycline, erythromycin, spectinomycin and sulfadiazine were determined for 732 isolates of Neisseria gonorrhoeae collected in 1973-74. Comparison of the results of this survey with data from other Canadian studies showed that the percentage of isolates resistant to tetracycline had not changed since 1966, but that the percentage of erythromycin-resistant isolates had decreased. After an initial increase in 1966 the percentage of penicillin-resistant isolates stabilized. Spectinomycin-resistant Isolates were not found. Positive correlations were observed between the MICs of the antibiotics tested; the highest positive correlations were between penicillin and ampicillin and between penicillin and tetracycline. A positive correlation was also noted between penicillin resistance and increasing spectinomycin MICs. Finally, a significant seasonal variation in MICs was found, the trend being towards Increasing MICs during the summer. Les concentrations inhibitrices minimums (CIM) de Ia p6nicilline, de l'ampicilline,

de Ia t6tracycline, de l'6rythromycine, de Ia spectinomycine et de Ia sulfadiazine ont 6te determinees pour 732 souches de Neisseria gonorrhoeae isol6es en 1973-74. La comparaison des resultats de cette etude avec les donn6es d'autres etudes canadiennes a montr6 que le pourcentage des souches r6sistantes A Ia t6tracycline n'a pas chang6 depuis 1966, mais que le pourcentage des souches resistantes & l'erythromycine a diminu6. AprAs une augmentation initiale en 1966 le pourcentage des souches r6sistantes A Ia penicilline s'est stabilis6. Aucune souche resistante a Ia spectinomycine n'a et6 retrouv6e. Des corr6lations positives ont 6t6 observees entre les CIM des antibio. tiques test6s; les plus fortes correlations positives ont 6te entre Ia penicilline et l'ampicilline et entre Ia p6nicilline et Ia tetracycline. Une autre correlation positive a aussi 6t6 not6e entre Ia resistance A Ia p6nicilline et l'augmentation des CIM A Ia spectinomycine. Finalement, une variation saisonniAre significative des CIM a 6t6 retrouv6e, Ia tendance favorisant une augmentation des CIM durant l'et6.

From the bureau of bacteriology, Laboratory Centre for Disease Control, health protection branch, Health and Welfare Canada Reprint requests to: Dr. J.R. Dillon, Bureau of bacteriology, Laboratory Centre for Disease Control, health protection branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ont. KiA 0L2

The incidence of Neisseria gonorrhoeae infection has been increasing in Canada1 and in other parts of the world,2 as has resistance of the organism to some of the clinically important antibiotics used in gonorrhea therapy.3-8 In addition, positive corre-

lations have been found between the susceptibility of a given isolate to one antibiotic and the sensitivity of that isolate to other antibiotics.6'9-11 In Canada 47 860 cases of gonorrhea were reported in 1974, the largest number since 1946.12 Since the increase in antibiotic resistance has important implications in the treatment of gonococcal infection, a survey to monitor the trend in antibiotic susceptibility of N. gonorrhoeae isolated in Canada was undertaken in 197374. The survey and its results are described in this paper. Material and methods Receipt of gonococcal isolates

Cultures of N. gonorrhoeae isolates were sent once every 3 months from 10 of the provincial public health laboratories to the bureau of bacteriology, Laboratory Centre for Disease Control (LCDC), Ottawa; the cultures represented all gonococcal isolates received by the laboratories during the week prior to shipment. Of the 824 cultures sent in 1973-74, 92 were nonviable or grossly contaminated. Antibiotic sensitivity tests were carried out on the remaining 732; the numbers tested during the fall, winter, spring and summer quarters were 117, 164, 180 and 211 respectively.

CMA JOURNAL/AUGUST 12, 1978/VOL. 119 223

sion was made by transferring a large 46.0% of the isolates were sensitive Subculture, preservation inoculum from Columbia blood agar and 35.5% and 33.1% were resistand identification Cultures were generally shipped plates to S mL of trypticase soy ant to penicillin and ampicillin re(Oxoid). The turbidity of the spectively. A high correlation was by air in Amies transport medium in broth suspension was adjusted to a no. 1 found between the MICs of the two screw-capped tubes. Upon arrival MacFarland standard,14 correspond- agents. Ampicillin, however, was they were subcultured on Columbia ing to approximately 3 X 108 bacte- more active than penicillin in that blood agar (Oxoid) and were incu- ria per millilitre, and was further all of the isolates were inhibited by bated for 18 hours in a humid atdiluted 00-fold. 1 Antibiotic plates 0.5 p.g/mL of the former. mosphere at 370C in the presence were inoculated The distribution of tetracycline with cotton swabs of 5% carbon dioxide. Cultures MICs was unimodal; 70.8% of the that had been impregnated with the found to be contaminated were furisolates were sensitive and 10.2% bacterial suspension. The plates were ther subcultured, first on Thayerwere resistant to this antibiotic. incubated for 48 hours in a humid Martin medium and then on ColumWith erythromycin 17.2% of the atmosphere at 370C in the presence bia blood agar. Gonococcal suspenisolates were sensitive, 4.5% were of 5% carbon dioxide. The minimum sions were stored at -700C in heart resistant and the remainder, 78.3%, inhibitory concentration (MIC) was infusion broth (Difco) supplemented taken as concentration exhibited the intermediate at which sensitivity. with 10% glycerol. Prior to identification an aliquot the antibiotic completely inhibited With spectinomycin 25.7% of the were sensitive (MIC 5.0 of frozen culture was thawed and growth. The antibiotics investigated isolates .g/mL or less) and the rest exhibited and their MIC ranges are listed in subcultured on Columbia blood agar. intermediate susceptibility; none were Table I. Isolates were identified as N. gonorresistant to more than 15.0 p.g/mL. rhoeae by their gram stain and their Statistical analysis With sulfadiazine 45.8% of the isooxidase and sugar fermentation relates were sensitive (MIC range 2.0 MIC distributions by season were actions. Sugar fermentation tests analysed by means of the Kolmogoto 15.0 p.g/mL) and 29.3% were rewere carried out on cystine trypticase rov-Smirnov two-sample test.15 This sistant to 100 /Lg/mL or more. agar (Baltimore Biological Labora- test was also used to calculate the tories) containing 1% dextrose, malCorrelations between MICs tose and sucrose (on separate plates) significance of the interaction beThe Kolmogorov-Smirnov twotween the antibiotics. Regression incubated for 24 hours. analyses of the data were also con- sample test was used to determine correlations between the MICs of ducted. Antibiotic sensitivity testing penicillin, ampicillin, tetracycline and All antibiotic sensitivity tests were Results spectinomycin. High positive correlacarried out on chocolate agar (Oxoid tions were found between the MICs gonococcal agar base containing 1% Susceptibility of gonococcal isolates of penicillin and ampicillin. A posihemoglobin); 1 % Kellogg's13 defined Penicillin and ampicillin exhibited tive correlation was also found besupplement was subsequently added bimodal MIC distributions, with tween the MICs of penicillin and to the medium. Appropriate volumes peaks at 0.025 and 0.5 U or p.g/mL tetracycline, especially at the higher of antimicrobial stock solutions were respectively (Table I); 45.1 % and concentrations of these antibiotics; added to 200 mL of molten chocolate agar to give the desired final concentrations. The antibiotic concentrations tested were as follows: penicillin (Ayerst Laboratories) 0.025, 0.05, 0.1, 0.5, 0.75, 1.0 and 2.0 U/mL; ampicillin (Ayerst) 0.025, 0.05, 0.1, 0.5, 0.75, 1.0 and 2.0 .g/mL; erythromycin (Abbott Laboratories, Ltd.) 0.05, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 /Lg/mL; tetracycline hydrochloride (Lederle Products Department, Cyanamid of Canada Ltd.) 0.05, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 p.g/mL; spectinomycin (Upjohn Company of Canada) 5.0, 7.5, 10.0, 15.0, 20.0, 40.0 and 80.0 p.g/mL; and sulfadiazine (Abbott) 2.0, 5.0, 10.0, 15.0, 20.0, 50.0 and 100.0 ,xg/mL. Media were dispensed into 18.8 (R) 100 X 15-mm Petri plates in 25-mL itch Is .xpresud In units pr volumes. In testing the antibiotic sensitivities of the isolates a gonococcal suspen-

224 CMA JOURNAL/AUGUST 12, 1978/VOL. 119

LILS.K)

for example, the tetracycline MICs were significantly lower (P < 0.00 1) for penicillin-sensitive isolates than for isolates with intermediate penicillin sensitivity, and were significantly higher (P < 0.00 1) for penicillinresistant isolates (MIC 0.5 U/mL 4.0 2.0 .-

0

S

(2)

(2)

1.0 f -0@ (12) (8) LIJ

-

0.5 F

-J L)

L&J

I-

0

0

(123)

(7)

or more) than for isolates wiith inter- found if the range of penicillin MICs mediate penicillin sensitivity increased from sensitive to intermeThe relation between the MICs of diate sensitivity. However, the specpenicillin and spectinomycin was not tinomycin MICs increased signifias clear-cut as that between the MICs cantly (P < 0.001) when the peniof penicillin and tetracycline No in- cillin MICs increased from the intercrease in spectinomycin M ICs was mediate sensitivity range to the resistant range. Logarithmic linear regression analysis of the data (Figs. 0@ 0 * 1 and 2) illustrated that, with an (1) (91 )(1 ) (3) increase in the penicillin MICs, the * tetracycline MICs increased by a 0 000 * much greater factor than did the (10) (36)(6) (2) (3) S spectinomycin MICs. Seasonal differences in MICs

0

(22) 0

*(22)

0.25

Statistical analysis of the MIC distributions by season indicated that the isolates received during the summer were significantly more resistant (P range 0.02 to 0.001) to all the antibiotics tested than isolates collected during the fall, the winter or, in some instances, the spring. In addition, the MIC distributions of penicillin and ampicillin fluctuated less from season to season than the MIC distributions of tetracycline, spectinomycin and sulfadiazine.

00

(58) (5)

(37)

(26) (4)

0 (12)

0 (11)

(-)

0.125 1-

0.05

0

0

(76)

(5)

*

0

(12)

(3)

Discussion I

.025

.05

.10

I

.50 .75 1.0

The determination of MICs for

2.0

MIC PENICILLIN

FIG. 1-Logarithmic regression analysis of peniciflin and tetracycline minimum inhibitory concentrations (MICs). Log linear equation: log2 (MICtet) = -2.21 + 0.351 log2 (MIC..).

(48)

0

(6)

0

(20)

*

(79)

0@

(i4)(2)

* *

101..

(78)

S

(6)

7.5

(8)0)

* (2)

5

.20 cm MICs.

isolates of N. gonorrhoeae is important because increased resistance to penicillin has been correlated with increased frequency

of treatment

failure with this antibiotic.1.1' In addition, increasing MICs of penicillin have necessitated increased doses for efficient treatment of gonococcal diseases.16 Rates of treatment failure have also been positively correlated with increased MICs of ampicillin" and tetracycline.1' The penicillin MICs determined from our survey were compared with those from other Canadian studies (Table II). Interpretation of the data must be qualified because the methods used in each of the investigations differed. In earlier studies, carried out by Amies. in 1959 and by Snell, Norris and Strong21 in 1962, about 82% of the isolates were found to be sensitive to between 0.01 and 0.03 U/mL, and to 0.05 U/mL respectively of penicillin; no isolates were found to be resistant to 0.4 or 1.0 U/mL. In surveys carried out 7 years later Amiesw found that the proportion of penicillin-sensitive isolates had decreased to 38.8%, and that 27.4% of the isolates required

Table III-Tetracycline IWICs determined In vmrlousCmuadl.astudl.s

ute.

196P. 10*7319

1$7041.' 117$q4, our study

. of isolates Inhibited; .0.5 LO >1.0

53.5 08 708

30.1 11 21 19

10.3 10.2

spectinomycin MICs. Maness and References Sparling9 did not find a positive in- 1. GODDEN JO: International symposium teraction between the two antibiotics, on gonorrhea. Can Med Assoc J 109: 1043, 1973 but othersu have reported a positive correlation between penicillin and 2. KIRALY K, CAUSSE G: Gonorrhea spectinomycin MICs. Moreover, a worldwide problem, in International Kousa and colleagues27 stated that Symposium on Gonorrhea, cat no H47-5/ 1975, Health and Welfare Canmore treatment failures were obada, Ottawa, 1975, p 14 served with spectinomycin among patients with gonorrhea and lower sen- 3. HART G: Penicillin resistance of gonococci in South Vietnam. Med J Aust sitivity to penicillin. Because of these 2: 638, 1973 differing opinions the sensitivity patterns of gonococcal isolates for these 4. REYN A: Sensitivity of N. gonorrhoeae to antibiotics. Br J Vener Dis 37:145, antibiotics should be monitored. 1961 Seasonal differences in MICs were noted throughout this study. The 5. REYN A, BENTZON MW: A study of the relationships between the sensitivimost important such trend was toties of Neisseria gonorrhoeae to sowards increased resistance to all antidium penicillin G, four semi-synthetic biotics of isolates tested during the penicillins, spiramycin, and fusidic summer. In Greenland Olsen's found acid. Br J Vener Dis 44: 140, 1968 a higher frequency of penicillin re- 6. Idem: Relationships between the sensisistance during the summer but detivities in vitro of Neisseria gonortected no seasonal variation in tetrarhoeae to spiramycin, penicillin, strepcycline resistance. At present we tomycin, tetracycline, and erythromycin. Br J Vener Dis 45: 223, 1969 have no explanation for the seasonal variations in MICs. Follow-up 7. SCHROETER AL, LUCAS JB: Gonorrhea studies should be conducted to de- diagnosis and treatment. Obstet Gynecol 39: 274, 1973 termine whether they are permanent characteristics. 8. SPARLING PF: Antibiotic resistance in In conclusion, our study, in Neisseria gonorrhoeae. Med Gun North Am 56: 1133, 1972 comparison with other Canadian studies,19"4". has shown that the 9. MANESS MJ, SPARLING PF: Multiple sensitivity of isolates of N. gonorantibiotic resistance due to a single mutation in Neisseria gonorrhoeae. J rhoeae to penicillin did not decrease Infect Dis 128: 321, 1973 between 1966 and 1974. In addition, the percentage of isolates resistant to 10. PHILLIPS I, RIDLEY M, RIMMER D, et al: In-vitro activity of twelve antitetracycline remained the same, while bacterial agents against Neisseria the resistance to erythromycin degonorrhoeae. Lancet 1: 263, 1970 creased. No isolates were resistant to spectinomycin. Despite the apparent 11. POWELL JT, BOND JH: Multiple antibiotic resistance in clinical strains of stabilization of susceptibility to gonorrhoeae isolated in Neisseria penicillin and ampicillin, the reSouth Carolina. Antimicrob Agents cent emergence of plasmid-mediated Chemother 10: 639, 1976 f.-lactamase-producing N. gonorVenereal Disease in Canada 1974. rhoeae41 strongly suggests that sus- 12. Health and Welfare Canada, health ceptibility testing of isolates of N. protection branch, bureau of epidegonorrhoeae should be continued. miology, Ottawa, 1975, p 1 We thank the directors of the provincial 13. KELLOGG DS JR, PEACOCK WL JR, DEACON WE, et al: Neisseria gonorpublic health laboratories for their rhoeae. 1. Virulence genetically linked cooperation: Dr. E.J. Bowmer, Vanto clonal variation. J Bacteriol 85: couver; Dr. J.M.S. Dixon, Edmonton; 1274, 1963 Dr. H.E. Robertson, Regina; Dr. J.G. Fox, Winnipeg; Dr. Sandu Toma, To- 14. BLAIR JE, LENNETrE EH, TRUANT JP (eds): Manual of Clinical Microronto; Dr. S.S. Kasatiya, Ville de Laval, biology, American Society for MicroPQ; Dr. J.S. MacKay, Saint John; Dr. biology, Bethesda, Md, 1970, p 677 R.S. Martin, Halifax; Dr. John Craig, Charlottetown; and Dr. R.W. Butler, St. 15. SIEGEL 5: Nonparametric Statistics for John's. the Behavioral Sciences, McGraw, Thanks are also due to Dr. Paul New York, 1956, p 127 Varughese for supervising the technical 16. HOLMES KK, KARNEY WW, HARNISCH aspect of the survey, and to Mr. Yang JP, et al: Single-dose aqueous procaine Mao, disease statistics and operational penicillin G therapy for gonorrhea: planning, Laboratory Centre for Disthe use of probenecid and cause of ease Control, for his assistance in contreatment failure. J Infect Dis 127: ducting statistical analyses of the data. 455, 1973 228 CMA JOURNAL/AUGUST 12, 1978/VOL. 119

17. JAFFE HW, BIDDLE JW, THORNSBERRY

C, et al: National gonorrhea therapy monitoring study. In . vitro antibiotic susceptibility and its correlation with treatment results. N Engi J Med 294: 5, 1976 18. LEIGH DA, LE FRANC J, TURNBULL

AR: Sensitivity to penicillin of Neisseria ganorrhoeae. Relationship to the results of treatment. Br J Vener Dis 45: 151, 1969 19. GIVAN KF, KEm A: Antibiotic sensitivities of Neisseria gonorrhoeae in the Toronto area. Can Med Assoc J 111: 44, 1974 20. AMIES CR: Development of resistance of gonococci to penicillin: an eightyear study. Can Med Assoc J 96: 33, 1967 21. SNELL E, NoRRIs DA, STRONG J: Sus-

ceptibility of N. gonorrhaeae to antibiotics: a study of 200 consecutive strains isolated in Winnipeg in 1962. Can Med Assoc J 89: 601, 1963 22. AMIES CR: Sensitivity of Neisseria gonorrhoeae to penicillin and other antibiotics. Studies carried out in Toronto during the period 1961-1968. Br J Vener Dis 45: 216, 1969 23. ROBSON HG, SALIT TE: Susceptibility of Neisseria gonorrhoeae to seven antibiotics in vitro. Can Med Assoc J 107: 959, 1972 24. OLSEN GA: Consumption of antibiotics in Greenland, 1964-1970. IV. Changes in the sensitivity of N. gonorrhoeae to antibiotics. Br J Vener Dis 49: 33, 1973 25. SAVAGE GM: Spectinomycin (Tobricm), a new antibiotic for the treatment of gonorrhea. mt J Clin Pharmacol Biopharm 6: 143, 1972 26. Idem: Spectinomycin related to the chemotherapy of gonorrhea. Infection 1: 227, 1973 27. KousA M, LASSUS A, JXRvELXINEN L,

et al: Spectinomycin hydrochloride in the treatment of uncomplicated gonorrhea in males and females. Br J Vener Dis 50: 291, 1974 28. BROWN J, TABERT 0, HANNA JD, et

al: Treatment of gonorrheal urethritis with spectinomycin hydrochloride. Can Med Assoc J 110: 173, 1974 29. ASHFORD WA, GOLASH RG, HEMI4IING

VG: Penicillinase-producing Neisseria gonorrhoeae. Lancet 2: 657, 1976

30. BOWMER EJ: Penicillinase-producing Neisseria gonorrhoeae. Can Dis Wkly Rep 2: 189, 1976 31. ELWELL LP, ROBERTS M, MAYER LW,

et al: Plasmid-mediated beta-lactamase production in Neisseria gonorrhoeae. Antimicrob Agents Chemother 11: 528, 1977