placebo-controlled trial with particular reference to the role of Campylobacter pylori. Gut 29:1386-1391.

30. Borsch, G., U. Mai, and K. M. Muller. 1988. Monotherapy or polychemotherapy in the treatment of Campylobacter pylori- related gastroduodenal disease. Scan. J. Gastroenterol. 23(S 142): 101-106.

31. Tytgat, G. N. J., E. Rauws, and W. Lan- genberg. 1986. The role of colloidal bis- muth subcitrate in gastric ulcer and gaslritis. Scand. J. Gastroenterol. 21(S 122):22-29.

32. Drumm, B. et al. 1988. Treatment of Campylobacter pylori-associated antral gastritis in children with bismuth sub- salicylate and ampicillin. J. Pediatr. 113:908-912.

33. Marshall, B. J. et al. 1988. Prospective double-blind trial of duodenal ulcer re- lapse after eradication of Campylobac- ter pylori. Lancet ii: 1437-1442.

34. Borody, T. J. et al. 1989. Recurrence of duodenal ulcer and Campylobacter py- lori infection after eradication. Med. J. Aust. 151:431--435.

35. Borsch, G., U. Mai, and W. Opferkuch. 1988. Oral triple therapy effectively eradicates Campylobacter pylori in man: a pilot study. Gastroenterology 94:A44.

36. Rauws, E. A. J. and G. N. J. Tytgat, 1990. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet i:1233--1235.

37. George, L. L. et al. 1990. Cure of duo- denal ulcer after eradication of Helico- bacterpylori. Med. J. Aust. 153: 145-149.

Editorial

Antimicrobial Susceptibility Testing of Neisseria meningitidis

Fred C. Tenover, Ph.D. Nosocomial Pathogens Laboratory Branch Hospital Infections Program National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA 30333

Neisseria meningitidis is a bacterial pathogen capable of invading the lungs, bloodstream, and central nervous sys- tem of humans and is one of the leading causes of bacterial meningitis in the United States, Although most meningo- coccal disease in the United States is sporadic, outbreaks of meningococcal meningitis still occur, often with high rates of morbidity and mortality (1, 2).

Until recently, meningococci were considered uniformly susceptible to penicillin. Typically, minimal inhibi- tory concentrations (MICs) of penicillin for meningococci were between 0.008 and 0.03 p.g/ml. Therefore, susceptibil- ity testing, except for determining the effectiveness of sulfonamides (3) or ri- fampin (4) when used for prophylaxis of persons exposed to patients with known meningococcal disease, was not undertaken in most laboratories.

In 1983, Dillon and coworkers re- ported the isolation of a beta-lactamase-

producing strain ofN. meningitidis (5). This organism was resistant to penicil- lin because of enzymatic inactivation of the drug and demonstrated MICs of penicillin of 256 p.g/ml. Beta-lactamase- producing isolates from South Africa were reported in 1988 by Botha (6). There was concern that beta-lactamase- producing meningococci would be- come more prevalent globally, but that has not occurred.

In 1987, S,-iez-Nieto et al. reported the isolation from patients in Spain of meningococci that were beta-lactamase negative but had penicillin MICs of 0.1 to 0.4 lag/ml (7). The following year, meningococci with MICs of penicillin up to 1.28 ~tg/ml were reported from the United Kingdom (8) and strains with MICs from 0.25 to >256 vg/ml were reported from South Africa (6). In 1992, meningococcal strains with MICs of penicillin of 0.12 to 0.25 lag/ml were documented in the United States (9; Tenover, F.C., unpublished observa- tions). These strains came from cities on the Eastern seaboard of the United States and from California. Thus, micro- biologists need to recognize that menin- gococcal isolates with decreased susceptibility to penicillin may be re- covered from patients anywhere in the United States. However, it is probably premature for most microbiology labo- ratories to initiate routine susceptibility

testing of meningococcal isolates. N. meningitidis is in that category of

pathogens for which the Antimicrobial Susceptibility Testing Subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) has not approved standardized antimicrobial susceptibility testing methods. MIC breakpoints that distinguish antimicro- bial-susceptible from antimicrobial-re- sistant organisms are lacking, as are the appropriate quality control strains and testing parameters to ensure the accu- racy of the results. Thus, important ground work remains to be completed before clinical laboratories begin to screen meningococci on a routine basis. Some of the problems that have been noted with various meningococcal test- ing systems are discussed below.

Several years ago, tentative break- points for disk diffusion testing of peni- cillin, chioramphenicoi, minocycline, rifampicin, and spiramycin were devel- oped by the World Health Organization in order to undertake global surveil- lance of resistant meningococci (10). However, several laboratories have questioned the effectiveness of the 10- IU (international unit) penicillin disk recommended for meningococcal test- ing.

In 1985, Campos et al. reported that a 2-IU penicillin G disk, as opposed to the 10-IU disk, more accurately distin-

Clinical Microbiology Newsletter 15:5,1993 © 1993 Elsevier Science Publishing Co., Inc. 0196-4399/93/$0.00 + 06.00 3 7

guished penicillin-susceptible strains of meningococci from those that were relatively penicillin-resistant (MICs between 0.1 and 1.0 I.tg/ml) when break- points of <26 mm for resistance and >28 mm for susceptibility were used (11). Campos et al. also evaluated a disk diffusion screening test by using a l-~g oxacillin disk in conjunction with zone diameters of<12 mm as an indica- tor of reduced susceptibility to penicil- lin, but found that it did not adequately discriminate between penicillin-suscep- tible and relatively penicillin-resistant isolates. In a more recent article, Cam- pos et al. reconsidered the oxacillin screen test and now support its use (12). The change was based on the discovery that the organisms that had previously produced false-positive results (i.e., those that were penicillin-susceptible but produced no zones of inhibition around oxacillin discs) had MICs to am- picillin of_>0.1 og/ml. Thus, if the ox- acillin screen test was used to indicate resistance to any beta-lactam drug, as opposed to penicillin resistance alone, the sensitivity and specificity of the test appeared to improve. However, the clinical utility of identifying penicillin- susceptible, relatively ampicillin-resis- tant strains of meningococci has not been established. Studies at the Centers for Disease Control and Prevention (CDC) that tested a large number of meningococci from several regions around the world showed far more false- positive results than the study reported by Campos et al.; thus, given our re- suits and the confusion surrounding the significance of the differential suscepti- bility of some strains of meningococci to beta-lactam agents, we would not recommend this screening test.

For laboratories that wish to pursue penicillin-susceptibility testing of men- ingococci, CDC recommends a broth microdilution technique as described by NCCLS (13), including use of cation- adjusted Mueller-Hinton broth and an inoculum of 5x 105 CFU/ml with incuba- tion in 5% CO2 for 18-20 h. While some microbiologists prefer to use a nu-

tritionally richer medium when testing meningococci, in most cases we have not found this to be necessary. For agar dilution testing, we recommend an in- oculum of 104 CFU and incubation of plates in 5% COz at 35°C. Plain Mueller- Hinton agar and chocolate Mueller- Hinton agar give comparable results.

In at least one hospital in Spain, approximately 48% of meningococcal isolates have penicillin MICs of 0.1-1.0 ~g/ml (12). Since the multiply anti- microbial-resistant pneumococci that are prevalent in Spain have been recog- nized throughout the United States (14), it is likely that more relatively penicillin-resistant isolates of meningo- cocci also will be detected in the United States during the next few years. How- ever, most isolates will still be highly susceptible to penicillin, and, in the absence of a standardized method, it is difficult to recommend widespread sus- ceptibility testing of meningococci except when a patient fails to respond to beta-lactam therapy or when an out- break requires prompt and effective therapy. In the latter instance, suscepti- bility testing should be performed by a dilution testing method and the results should be forwarded to state and local health departments and to the CDC.

Meningococci are the latest in a growing line of serious pathogens to develop resistance to commonly used antimicrobial agents. Microbiologists must be aware of the presence of such organisms and be on their guard against the development of resistance in menin- gococci in their area.

References

1. Pinner, R.W. et al. 1991. Meningococ- cal disease in the United States-1986. J. Infect. Dis. 164:368-374.

2. Counts, G. W. et al. 1984. Group A meningococcal disease in the U.S. Pacific Northwest: epidemiology, clini- cal features, and effect of a vaccination control program. Rev. Infect. Dis. 6:640- 648.

3. Bennett, J. V., H. M. Camp, and T. C. Eickhoff. 1968. Rapid sulfonamide disc

sensitivity test for meningococci. Appi. Microbiol. 16:1056-1060.

4. Morello, J. A., W. M. Janda, and G. V. Doem. 1991. Neisseria and Bran- hamella, p. 258-275. In A. Balows et al. (ed.) Manual of clinical microbiol- ogy, 5th ed. American Society for Mi- crobiology, Washington, D.C.

5. Dillon, J. R., M. Pauze, and K-H. Yeung. 1983. Spread of penicillinase- producing and transfer plasmids from the gonococcus to Neisseria meningiti- dis. Lancet i:779-781.

6. Botha, P. 1988. Penicillin-resistant Neis- seria meningitidis in Southem Africa. Lancet i:54.

7. S~ez-Nieto, J. A. et al. 1987. Isolation of Neisseria meningitidis strains with in- crease of penicillin minimal inhibitory concentrations. Epidemiol. Infect. 99:463-469.

8. Sutcliffe, E.M. et al. 1988. Penicillin-in- sensitive meningococci in the U. K. Lancet i: 657-658.

9. Woods, C., B. Wasilauskas, and L. Giv- ner. 1992. Meningitis caused by Neisse- ria meningitidis relatively resistant to penicillin (NmRRP) in North Carolina. ICAAC Abstract (late breaker).

10. World Health Organization, Expert Committee on Antibiotic Susceptibility Tests. 1991. Antibiotic sensitivity stand- ard technique. WHO/BS 76:1129.

11. Campos, J. et al. 1987. Detection of relatively penicillin G-resistant Neisse- ria meningitidis by disk susceptibility testing. Antimicrob. Agents Chemother. 31:1478-1482.

12. Campos, J. et al. 1992. Discriminative criteria for Neisseria meningitidis isolates that are moderately susceptible to penicillin and ampicillin. Antimi- crob. Agents Chemother. 36:1028- 1031.

13. National Committee for Clinical Labo- ratory Standards. 1990. Methods for di- lution antimicrobial susceptibility tests for bacteria that grow aerobically. 2nd ed. Approved standard M7-A2. Na- tional Committee for Clinical Stand- ards, Villanova, PA.

14. McDougal, L. K. et al. 1992. Analysis of multiply antimicrobial-resistant iso- lates of Streptococcus pneumoniae from the United States. Antimicrob. Agents Chernother. 36:2176-2184.

3 8 0196-4399/93/$0.00 + 06.00 © 1993 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 15:5,1993