Saturday EVIDENCE FOR TWO DISTINCT TYPES OF PENICILLINASE-PRODUCING NEISSERIA GONORRHή PETER L. PERINE CLYDE THORNSBERRY WILLIAM SCHALLA JAMES BIDDLE MARTIN S. SIEGEL KWEI-HAY WONG SUMNER E. THOMPSON Center for Disease Control, Atlanta,

Since their

Summary

Georgia 30333,

recognition early

in

U.S.A.

1976,

penicillinase (&bgr;-lactamase)-producing Neisseria gonorrhœœ (P.P.N.G.) have been isolated in more than 15 countries. Most strains isolated in or epilinked with the Far East are relatively tetracycline in vitro, are phenotypically or wild-type proline-dependent auxotypes, and carry a plasmid with a molecular weight of 5800 000 (5.8×106) daltons coding for &bgr;-lactamase production. In contrast, P.P.N.G. epidemiologically linked with West Africa are

demiologically resistant

to

susceptible to tetracycline, require arginine for growth, and their gene coding for &bgr;-lactamase synthesis is contained in a smaller 3·2×106 dalton plasmid. Moremore

43% of the Far Eastern strains, but none of those from West Africa, have an additional 24·5×106 dalton conjugative plasmid which transfers the &bgr;-lactamase R factor(s) to other gonococci. The presence of this conjugative plasmid may explain the relatively high prevalence of P.P.N.G. in certain areas of the Far East. over,

Introduction

of

Neisseria penicillinase-producing first infection were reported in gonorrhcece (P.P.N.G.) U.S.A.l.2 in 1976. Since and England3’ patients in the then 14 other countries in Europe, Africa, North America, New Zealand, and the Far East have reported cases to the World Health Organisation (G. Antal, personal communication). In many instances infections caused by P.P.N.G. were directly linked with sexual contacts in the Far East or West Africa. Some of the biological properties of representative strains of P.P.N.G. isolated in differCASES

parts of the world indicate that there tinct geographic types ofp.p.N.G.

ent

are two

dis-

Methods 94

P.P.N.G.

cultures

were

tested. The cultures

were

by one of us (P.L.P.) from the Philippines during gonorrhoea in September, 1976; the rest were sent eau

a

collected

survey of

to the Burof Laboratories, Center for Disease Control, U.S.A., from

12

November 1977

individual laboratories, State and national health departments, and the W.H.O. Collaborating Centre for Reference and Research in Gonococci, Denmark. Epidemiological data, including age, sex, site, and probable source of P.P.N.G. infection, were available for most of the isolates tested. All cultures were confirmed as being N. gonorrhaeae by colony morphology, gram stain, oxidase test, and standard sugar utilisation patterns and as p-lactamase-producing organisms by the chromogenic cephalosporin test. Antimicrobial susceptibility tests were performed on each P.P.N.G. isolate by an agardilution method, with a Steers’ replicator used as described elsewhere.6 The inoculum contained 103-104 organisms. Antibiotics tested were penicillin, amoxycillin, cefuroxime, tetra-

cycline, chloramphenicol, erythromycin, and spectinomycin. Nutritional growth requirements (auxotyping) of each isolate were determined by the method of LaScolea and Young.7 Each isolate was tested for auxotrophy on at least two different lots of basal medium with the same inoculum and replicator system that was used for the antimicrobial susceptibility tests. Gonococcal plasmids were identified and characterised by the agarose-gel electrophoresis method of Meyers et al.* The molecular weight of closed, circular gonococcal plasmid D.N.A. was estimated by comparing its migration with plasmid D.N.A. preparations of known molecular weights. Results

The

biological properties of P.P.N.G. from various listed in the accompanying table by country

sources are

of isolation. Several of the European isolates were obtained from patients whose infection was acquired in the Far East and have been previously reported9e.g., three of the seven cases in Holland were a cluster representing the first, second, and third generation transfer of P. P. N. G. from a Filipino air steward. 10

Plasmid Analysis The molecular weight of 5800 000 (5.8x106 daltons) of the p-lactamase-coding plasmid" from P.P.N.G. isolated in the Far East or from patients who acquired their infection in this area of the world was always greater than the molecular weight of the plasmid (3-2 x 106 daltons) present in the P.P.N.G. strains from patients whose infections were epidemiologically linked with West Africa (Ghana and Ivory Coast). All P.P.N.G. isolates, irrespective of geographic origin, possessed a 2.6 x106 dalton plasmid of no known function. In addition, 43% of Far Eastern strains contained a plasmid of 24.5 x 106 daltons, which was not found in any of the strains associated with West Africa or England.

AuxotypicAnalysis Most

P.P.N.G.

pically wild-type

from the Far East or

were

required proline 8046

for

either

phenotygrowth. In con-

994 BIOLOGICAL CHARACTERISTICS OF P.P.N.G

I

*

Number of strains.

* *

pro=proline, arg=arginine,

trast, all of the West African strains

I

I

required arginine

P.P.N.G.

Antibiotic Susceptibility

Testing Tetracycline was the only antibiotic tested which showed significantly different susceptibility patterns against P.p.N.G. from different sources. Strains epidemiologically linked to the Far East or containing the 5.8 x 106 dalton plasmid were significantly more resistant (p < 0-05 by a ttest with unequal variances) in vitro to tetracycline than the isolates that were epidemiologically linked to West Africa or containing the 3.2 x 106 dalton plasmid. For example, all 10 West African isolates were susceptible to 1-0 pLg/ml of tetracycline or less, whereas 16 of 60 (26%) Far Eastern isolates were resistant to tetracycline at this concentration. All of the P.P.N.G. were susceptible to cefuroxime (minimum inhibitory concentration (M.LC.