The Identification of Pathogenic Neisseria Spp. and Branhamella Catarrhalis

Evaluation of the RIM-N, Gonochek II, and Phadebact Systems for the Identification of Pathogenic Neisseria spp. and Branhamella catarrhalis

WILLIAM M. JANDA,1,2* MYRNA G. ULANDAY,' MARJORIE BOHNHOFF,3 AND LEON J. LEBEAU' The University of Illinois Hospital,' The University of Chicago Medical Center,3 and The Howard Brown Memorial Clinic,2 Chicago, Illinois 60612 Received 6 November 1984/Accepted 22 January 1985

Methods for identifying Neisseria spp. include conventional and modified carbohydrate degradation procedures, chromogenic enzyme substrate tests, and immunologic coagglutination tests for Neisseria gonorrhoeae. In this study, we evaluated the abilities of the RIM-N carbohydrate degradation system (American MicroScan, Campbell, Calif.), the Gonochek II enzymatic identification system (Du Pont Co., Wilmington, Del.), and the Phadebact Gonococcus coagglutination test (Pharmacia Diagnostics, Piscataway, N.J.) to identify pathogenic Neisseria spp. and Branhamella catarrhalis. Both stock strains and clinical isolates, including 176 N. gonorrhoeae, 173 Neisseria meningitidis, 48 Neisseria lactamica, and 12 B. catarrhalis strains, were tested. The RIM-N identified 98% of the gonococci, 99% of the meningococci, 94% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 1 h. The Gonochek II system identified 99% of the gonococci, 97% of the meningococci, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 30 min. Phadebact coagglutination provided clearly positive results for only 77% of the N. gonorrhoeae strains, producing negative or equivocal results with 23% of the strains. The RIM-N and Gonocheck II tests generally produced clear-cut reactions. An additional advantage of the Gonocheck II system was the small inoculum required for the performance of the test compared with the other systems, thus allowing the identification of N. gonorrhoeae directly from the primary isolation medium.

Current methods for identifying Neisseria spp. isolated (Pharmacia Diagnostics, Piscataway, N.J.), a COA proce- from clinical specimens include conventional and modified dure for the identification of N. gonorrhoeae. carbohydrate degradation procedures, enzymatic tests with (This work was presented at the 24th Interscience Confer- chromogenic substrates, and immunologic coagglutination ence on Antimicrobial Agents and Chemotherapy, 8 to 10 (COA) tests for confirmation of Neisseria gonorrhoeae. October 1984, Washington, D.C.) Whereas conventional sugar utilization tests may require prolonged incubation for organism growth and test results, MATERIALS AND METHODS the modified procedures rely on a heavy bacterial inoculum for rapid identification (2, 6, 8). Chromogenic enzyme sub- Table 1 lists the sources of the stock strains and clinical strate tests, in combination with certain modified conven- isolates examined in this evaluation. tional tests, have also proven useful for identification of Clinical isolates. Clinical isolates of N. gonorrhoeae (102 Neisseria spp. and other bacteria, and commercial systems strains), Neisseria meningitidis (101 strains), Neisseria lac- in which this method is used generally provide results in 4 h tamica (2 strains), and B. catarrhalis (3 strains) were ob- (3, 5, 10). In COA test methods, antibody directed against tained from patients at the University of Illinois Hospital and gonococcal antigens is utilized to produce specific agglutina- the University of Chicago Medical Center and from homo- tion reactions, allowing rapid confirmation of N. gonor- sexual men at the Howard Brown Memorial Clinic in Chi- rhoeae (1, 2, 7). Although not currently commercially avail- cago. Specimens from the University of Illinois Hospital and able, specific agglutination of N. gonorrhoeae by lectins has the University of Chicago Medical Center were received on also been proposed as a useful test for confirmatory identi- JEMBEC plates (Ames Co., Division of Miles Laboratories, fication of gonococci (4). The use of inoculum directly from Elkhart, Ind.) containing modified Thayer-Martin (MTM) the primary selective medium is a distinct advantage with medium (Remel, Lenexa, Kans.) or in swab transport sys- many of these test procedures. tems. Swab specimens were inoculated onto MTM medium In the present study, the rapid identification method for and chocolate (CH) agar (BBL Microbiology Systems, Co- Neisseria (RIM-N; American MicroScan, Campbell, Calif.), ckeysville, Md.) in the laboratory. Specimens from the a rapid carbohydrate test system, and the Gonochek II Howard Brown Memorial Clinic were inoculated directly system (GCII; Du Pont Co., Wilmington, Del.), a rapid onto MTM medium. After incubation in a CO2 environment chromogenic substrate system, were evaluated for the iden- at 35°C, colonies of oxidase-positive, gram-negative diplo- tification of pathogenic Neisseria spp. and Branhamella cocci were subcultured onto CH agar. Growth from the first catarrhalis. Both stock strains and fresh clinical isolates or second subculture was used to inoculate all the test were included in this study. In addition, all strains were systems. In one part of the study, 38 urethral and 6 rectal tested for reactivity with the Phadebact Gonococcus test gonococcal isolates were tested on all of the identification systems with growth from the primary MTM medium. On the following day, conventional identification tests and re- peat tests with all of the systems were performed from CH * Corresponding author. agar subcultures. The identities of all of the isolates used in 734 VOL. 21, 1985 IDENTIFICATION OF NEISSERIA SPP. 735

TABLE 1. Sources of clinical isolates and stock strains used in nitroanilide, and a P-naphthyl amino acid derivative) in a the evaluation of the RIM-N, GCII, and COA tests single tube are utilized to detect enzymatic activities asso- Organism Total no. Source (no.) ciated with N. gonorrhoeae, N. meningitidis, and N. lactamica. After rehydration of the dried substrates in the tube N. gonorrhoeae with 4 drops of phosphate-buffered saline (pH 7.4), 5 to 10 Clinical isolates 102 Anogenital (100) Oropharyngeal (1) medium-to-large colonies of oxidase-positive, gram-negative Systemic (1) diplococci from a pure culture growing on selective medium Stock strains 74 Anogenital (33) or a CH agar subculture are emulsified in the tube with a Systemic (38) wooden applicator stick. The tube is capped with the paired Oropharyngeal (2) stopper and stored at 35°C for 30 min. Specific color reac- Umbilicus (1) tions in the bacterial suspension confirm the isolate as N. meningitidis (hydrolysis of y-glutamyl-p-nitroanilide, yel- N, meningitidis low) or N. lactamica (hydrolysis of 5-bromo-4-chloro-3- Clinical isolates 101 Oropharyngeal (97) indoyl-,3-D-galactopyranoside, blue). If the suspension is Anogenital (3) Systemic (1) colorless at the end of the incubation period, the paired Stock strains 72 Systemic (37) stopper is split apart and the topmost stopper is inserted into Anogenital (31) the tube. The tube is then inverted so that the bacterial Oropharyngeal (2) suspension comes in contact with the diazo dye coupler Pericardial fluid (1) (o-aminoazotoluene diazonium salt [fast garnet]) present on Lymph node biopsy (1) the stopper. The detection of P-naphthylamine released by bacterial prolyliminopeptidase activity (pink) identifies the N. lactamica isolate as N. gonorrhoeae. The absence of a colored product Clinical isolates 2 Oropharyngeal (2) at the completion of the testing and reading steps is a Stock strains 46 Oropharyngeal (45) presumptive identification of B. catarrhalis. Cervix (1) The Phadebact Gonococcus test is a COA test for the B. catarrhalis identification of N. gonorrhoeae (1, 2, 8). This test was Clinical isolates 3 Conjunctival (3) performed from primary MTM or CH agar subcultures by Stock strains 9 Conjunctival (4) the boiled bacterial suspension technique recommended by Tracheal aspirate (3) the manufacturer. The bacterial suspension is allowed to Oropharyngeal (1) react with the antigonococcal antibody COA reagent (test Systemic (1) reagent) and with the control COA reagent (no antibody) on a cardboard slide. Agglutination within 2 min in the test COA reagent and not in the control COA reagent identifies this evaluation were confirmed by colony and Gram stain the organism as N. gonorrhoeae. All isolates in this study morphology, ability to grow on MTM medium, oxidase were tested with both control and test COA reagents, and reactivity, and acid production from appropriate carbohy- agglutination was scored as 2+ or greater (definite aggluti- drates by the rapid technique described previously (6). nation), ± (slight or equivocal agglutination), or - (no Stock strains. All stock strains had been isolated from a agglutination). variety of clinical specimens and were maintained frozen at -86°C. These included 74 N. gonorrhoeae, 72 Neisseria RESULTS meningitidis, 46 Neisseria lactamica, and 9 B. catarrhalis Table 2 summarizes the data for the identification of strains. All N. gonorrhoeae stock strains had been previ- gonococci, meningococci, N. lactamica, and B. catarrhalis ously auxotyped for their nutritional requirements on a modified Catlin medium (9). For testing on the various identification systems, stock strains were subcultured twice on CH agar before inoculation onto the systems. TABLE 2. Identification of Neisseria spp. and B. catarrhalis by Description of test systems. The RIM-N is a rapid carbo- the RIM-N and GCII systems hydrate degradation system that relies on a heavy bacterial Identification by: inoculum to produce detectable acid from sugars. Substrate RIM-N GCII solutions containing 2% each of glucose, maltose, sucrose, Organism No. tested and lactose with a phenol red indicator, plus a substrate No. (%) Total No. (%) Total blank lacking carbohydrate, are dispensed into five nonster- M ~ ~~~~~~~~~~~~~ ile, specially buffered microtubes in 3-drop volumes. Inocula N. gonorrhoeae 98.3 98.9 from a pure 18- to 24-h culture of oxidase-positive, gram- Clinical isolates 102 101 (99.0) 100 (98.0) negative diplococci growing on selective medium or a CH Stock strains 74 72 (97.3) 74 (100) agar subculture are delivered into each of the tubes with N. meningitidis 99.4 97.1 small, disposable plastic loops. After inoculation, the tubes Clinical isolates 101 101 (100) 99 (98.0) are agitated on a Vortex mixer (The Vortex Manufacturing Stock strains 72 71 (98.6) 69 (95.8) Co., Cleveland, Ohio) for 10 s and incubated in air at 35°C for 1 h. Acid production is indicated by a change in the N. lactamica 93.8 100 phenol red indicator from red-pink to yellow or yellow-or- Clinical isolates 2 1 (50) 2 (100) ange. Negative carbohydrate reactions remain red-pink or Stock strains 46 44 (95.7) 46 (100) turn slightly pink-orange. In this study, the RIM-N reactions were read and interpreted after 30 min and 1 h of incubation. B. catarrhalis 100 100 In the GCII system, three chromogenic substrates (5-bro- Clinical isolates 3 3 (100) 3 (100) mo-4-chloro-3-indoyl-3-D-galactopyranoside, y-glutamyl-p- Stock strains 9 9 (100) 9 (100) 736 JANDA ET AL. J. CLIN. MICROBIOL. by the RIM-N and GCII systems. The RIM-N identified TABLE 4. Identification of AHU- and non-AHU-requiring strains 98.3% of 176 N. gonorrhoeae, 99.4% of 173 N. meningitidis, of N. gonorrhoeae by the RIM-N. GCII, and COA tests 93.8% of 48 N. lactamica, and 100% of 12 B. catarrhalis strains within No. No. (%) identified by: 60 min. Of those organisms correctly identified tested RIM-N GCII COA by the RIM-N after 60 min, 93.6% of the gonococci, 98.8% of the meningococci, and 84.4% of the N. lactamica strains AHU and AHU plus 40 38 (95.0) 40 (100) 39 (97.5) were correctly identified after only 30 min of incubation. The other requirements" gonococci not identified by RIM-N (three strains) were all Non-AHU' 34 34 (100) 34 (100) 23 (67.6) glucose negative after 60 min. The single N. meningitidis Total % 97.3 100 83.8 strain not identified was a stock strain originally recovered " Isolates were tested for their requirements for arginine, proline, methio- from blood and cerebrospinal fluid as a small colony variant nine, histidine, leucine, lysine, valine, hypoxanthine, uracil, and thiamine PP,. " Includes 25 strains requiring AHU-O+. 8 requiring AHU-O+-leucine, 2 of a group B meningococci. This strain was also asaccharo- requiring AHU-O-, 2 requiring AHU-O+-proline, 2 requiring arginine-uracil- lytic in the conventional sugar utilization test. The three N. O+, and 1 requiring arginine-uracil-O-proline, where 0 denotes replacement lactamica strains not identified by the RIM-N were lactose of arginine with ornithine. negative after 60 min of incubation. ` Includes 11 strains with no requirement, 9 requiring arginine, 8 requiring proline, 3 requiring arginine and proline, and 3 requiring proline and hypoxan- The GCII system identified 98.9% of the N. gonorrhoeae thine. strains, 97.1% of the N. meningitidis strains, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains (Table 2). Two clinical isolates of N. gonorrhoeae failed to produce the pink compound after inversion of the tube and tures. The RIM-N identified 86.8 and 100% of the 44 isolates contact with the developing reagent on the stopper. The when inoculated from primary MTM medium and CH agar meningococcal strains not identified by the GCII system did subcultures, respectively. The GCII system identified 95.5% not produce a discernible yellow color within the 30-min of the isolates when the inoculum was derived from either incubation period. These included two fresh oropharyngeal medium. The COA test identified 68.2% of the N. gonor- isolates, one stock urethral isolate, and both the large and rhoeae isolates when the test suspension was prepared from small cblony variants of the meningococcal strain discussed either the primary MTM medium or a subculture. COA above. reactivity was dependent upon the individual strain and not Table 3 shows the results of the COA test for all of the on the source of the suspension used for testing. isolates tested. Only 83.8% of the 74 N. gonorrhoeae stock Since multiply auxotrophic gonococci may present diffi- strains and 72.5% of the 102 recent clinical isolates produced culties in identification by conventional test methods (2, 8, easily interpretable positive results with COA. Negative or 9), a collection of prototrophic and auxotrophic strains equivocal results or both were observed for 27.5 and 16.2% having single and multiple nutritional requirements for of the clinical isolates and stock strains, respectively. Of the growth were tested on each of the identification systems 48 N. lactamica strains, 23 (47.8%) produced positive COA (Table 4). The RIM-N identified 95% of the strains requiring reactions with either the test COA reagent or both the test arginine-hypoxanthine-uracil (AHU) for growth and 100% of and the control COA reagents. None of the N. meningitidis the non-AHU-requiring strains; 100% of the strains in both or B. catarrhalis strains agglutinated with either the test or groups were correctly identified by the GCII system. The the control COA reagents. COA test identified 83.8% of these organisms, with more of A total of 44 strains of N. gonorrhoeae (38 urethral and 6 the AHU-requiring strains (97.5%) than the non-AHU-re- rectal isolates) were tested on all of the identification sys- quiring strains (67.6%) producing clear-cut reactions. tems from both primary MTM medium and CH agar subcul- Over a 2-week period during this evaluation, seven isolates belonging to the Neisseria sicca-Neisseria subflava group were recovered from oropharyngeal subcultures of Howard Brown Memorial Clinic patients. All grew luxuri- TAALE 3. Reactions of Neisseriia spp. and B. C'atarr-halis with antly on MTM medium and produced acid from glucose, COA maltose, and sucrose, but not from lactose, in the conven- No. () of strains producing tional identification system. All of the isolates synthesized Organism No. tested following reaction (')" iodine-positive polysaccharide on 5% sucrose agar and did -/- or -1+ -/2 or 3+ Other" not reduce nitrate. Of the seven strains, five produced acid from glucose, maltose, and sucrose within 1 h with the N. gonorrhoeae were Clinical isolates 102 28 (27.5) 74 (72.5) 0 RIM-N, and two of these also positive with the COA Stock strains 74 12 (16.2) 62 (83.8) 0 test reagent. All of these strains produced reaction results in the GCII tube that were identical with those of N. gonor- N. meningitidis rhoeae, i.e., an initial colorless reaction with the develop- Clinical isolates 101 101 (100) 0 0 ment of a pink color upon inversion of the tube and exposure Stock strains 72 72 (100) 0 0 of the bacterial suspension to the developing reagent. Since N. sic-ca-N. s1ibflav,a strains usually do not grow on media N. lactamnica selective for the pathogenic Neisseria spp., these strains are Clinical isolates 2 0 0 2 (100) being characterized further in our laboratories. Stock strains 46 25 (54.3) 12 (26.1) 9 (19.6) B. catarrhalis DISCUSSION Clinical isolates 3 3 (100) 0 0 Stock strains 9 9 (100) 0 0 In this study, both the RIM-N carbohydrate degradation system and the GCII chromogenic substrate system per- " Reactions with the control COA reagent/reactions with the test COA regeant. formed well for the identification of both stock strains and " Strains agglutinated with both the test and control COA reagents. clinical isolates of pathogenic Neisseria spp. and B. catar- VOL. 21, 1985 IDENTIFICATION OF NEISSERIA SPP. 737 rhalis. Of 409 strains tested, 98.3% were correctly identified substrate methods, respectively, are used for the identifica- by both systems (Table 2). Test results were generally clear tion of Neisseria spp. and B. catarrhalis. Both of these cut and easy to interpret. In addition, both of these systems systems would be of value for clinical laboratories perform- provided reliable results when the inoculum was derived ing confirmatory tests on Neisseria isolates. Because of from either the primary isolation medium or a subculture. equivocal and false-negative results, particularly with fresh On the other hand, the COA test did not perform as reliably clinical isolates, the COA test was not reliable for the rapid as did the other two systems for the confirmation of N. confirmation of N. gonorrhoeae. gonorrhoeae. Overall, only 77.3% of 176 strains produced unequivocal COA reactions (Table 3). Strains that were ACKNOWLEDGMENTS COA positive or negative with primary isolation medium We thank Neil Forster, Les Gravesen, and the laboratory staff of showed similar reactivities when tested from subcultures. the Howard Brown Clinic for assistance in the processing of clinic The relatively large number of gonococci not reactive with specimens. the COA test in this study may reflect an antigenic noniden- This work was kindly supported by DuPont Co. Diagnostic tity of the current strains with the antibodies now constitut- Systems, Wilmington, Del. ing the reagents. The higher percentage of AHU-requiring LITERATURE CITED strains identified with the COA test as compared with the 1. Anand, C. M., and E. M. Kadis. 1980. Evaluation of the non-AHU-requiring strains (Table 4) may reflect the use of Phadebact Gonococcus Test for confirmation of Neisseria gon- this clinically significant subpopulation of gonococci in the orrhoeae. J. Clin. Microbiol. 12:15-17. development of antibodies for COA reagents. COA reactiv- 2. Arko, R. J., K. G. Finley-Price, K.-H. Wong, S. R. Johnson, and ity and its relation to gonococcal auxotypes and serotypes G. Reising. 1982. Identification of problem Neisseria gonor- are currently being investigated in our laboratories. As rhoeae cultures by standard and experimental tests. J. Clin. reported by others, about one-half of the N. lactamica Microbiol. 15:435-438. strains tested were also positive with one or both COA 3. D'Amato, R. F., L. A. Eriquez, K. M Tomfohrde, and E. Singerman. 1978. Rapid identification of Neisseria gonorrhoeae reagents, again emphasizing the particular need to perform and Neisseria meningitidis by using enzymatic profiles. J. Clin. o-nitrophenyl-,-D-galactopyranoside or lactose utilization Microbiol. 7:77-81. tests on oropharyngeal neisserial isolates (1, 8). 4. Doyle, R. J., F. Nedjat-Haiem, K. F. Keller, and C. E. Frasch. The GCII system presented certain advantages over the 1984. Diagnostic value of interactions between members of the other systems examined in this evaluation. The single-tube family Neisseriaceae and lectins. J. Clin. Microbiol. 19:383-387. format was easy to handle and required less than 30 s for 5. Janda, W. M., J. A. Morello, and M. Bohnhoff. 1984. Use of the rehydration and inoculation, whereas the RIM-N required API Neldent system for identification of pathogenic Neisseria significant time for dispensing the individual substrates and spp. and Branhamella catarrhalis. J. Clin. Microbiol. 19:338- for inoculating the five microtubes. Another advantage of 341. 6. Kellogg, D. S., Jr., K. K. Holmes, and G. A. Hill. 1976. the GCII system is the small inoculum required. Whereas Cumitech 4, Laboratory diagnosis of gonorrhea. Coordinating only 5 to 10 colonies were needed for the GCII system, both ed., S. Marcus and J. C. Sherris. American Society for Mi- the RIM-N and the COA test required sufficient inoculum to crobiology, Washington, D.C. provide a dense suspension in each of the five microtubes or 7. Lawton, W. D., and G. J. Battaglioli. 1983. Gono Gen coagglu- for reaction with both test and control COA reagents. Such tination test for confirmation of Neisseria gonorrhoeae. J. Clin. abundant growth of gonococcal colonies is frequently not Microbiol. 18:1264-1265. available on primary isolation medium, thus necessitating 8. Morello, J. A., and M. Bohnhoff. 1980. Neisseria and Branha- the subculture of the few colonies that are present before the mella, p. 111-130. In E. H. Lennette, A. Balows, W. J. Hausler, performance of either the RIM-N or COA test. Although the Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd GCII system is more ed. American Society for Microbiology, Washington, D.C. slightly expensive ($1.98 per test) than 9. Morello. J. A., S. A. Lerner, and M. Bohnhoff. 1976. Charac- the RIM-N ($1.34 per test) or the COA test ($1.26 per test), teristics of atypical Neissera gonorrhoeae from disseminated the time required for preparation, inoculation, and interpre- and localized infections. Infect. Immun. 13:1510-1516. tation is considerably less. In short, the RIM-N and the GCII 10. Robinson, M. J., and T. R. Oberhofer. 1983. Identification of system represent two reliable and novel methods in which pathogenic Neisseria species with the RapID NH system. J. rapid carbohydrate utilization and chromogenic enzyme Clin. Microbiol. 17:400-404.