Characterization of Neisseria cinerea, a Nonpathogenic ... · N. gonorrhoeae. Thus, the identity of...

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Vol. 19, No. 1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1984, p. 63-67 0095-1137/84/010063-05$02.00/0 Copyright © 1984, American Society for Microbiology Characterization of Neisseria cinerea, a Nonpathogenic Species Isolated on Martin-Lewis Medium Selective for Pathogenic Neisseria spp. JOAN S. KNAPP,'* PATRICIA A. TOTTEN,1 MARTHA H. MULKS,2 AND BARBARA H. MINSHEW3 Neisseria Reference Laboratory and Department of Medicine, Univ'ersity of Washington, Seattle, Washington 981951; Department of Medicine, Tuifts-New England Medical Center Hospital, Boston, Massachiusetts 021112; and Clinical Microbiology Laboratory, Department of Pathology, Seattle Plublic Health Hospital, and Department of Laboratory Medicine, University of Washington, Seattle, Washington 981143 Received 16 June 1983/Accepted 3 October 1983 An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash. This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol, or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified oxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonor- rhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitrate was not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose. Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 did not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A protease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strain NRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative to N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim 1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negative N. gonorrhoeae strain. Commensal colistin-susceptible Neisseria spp. and Bran- hamella catarrhalis are only rarely isolated on media selec- tive for the pathogenic Neisseria spp., Neisseria gonor- rhoeae and Neisseria meningitidis, and are differentiated on the basis of their cultural characteristics, distinctive sugar utilization reactions, ability to reduce nitrate, and ability to produce polysaccharide from sucrose. According to current literature (23, 33), among commensal species isolated from humans, only Neisseria flavescens and B. catarrhalis are asaccharolytic. Another asaccharolytic Neisseria sp., Neisseria cinerea, was first described by von Lingelsheim in 1906 (35) and named Micrococcus cinereius. It was described subsequently as Neisseria pseudocatarrhalis (F. M. Huntoon, Abstr. Annu. Meet. Am. Soc. Bacteriol. 1934, M50, p. 108) but was assigned to the species N. cinerea (27) since the epithet cinerea had priority taxonomically. Since then, many strains appear to have been isolated but incorrectly identified as B. catarrhalis (8, 11). These species differ biochemically only in their ability to reduce nitrate, a test which was not intro- duced into the classification of Neisseria spp. until 1961 (1). B. catarrhalis strains reduce nitrate, whereas N. cinerea strains do not. Berger and Paepcke (3) "rediscovered" and described N. cinerea in 1962 and showed that there was no antigenic relatedness between N. cinerea and Neisseria catarrhalis; the latter species was subsequently reassigned to the genus Branhamella (5) in 1970. N. cinerea was not described in Bergey's Manual of Determinative Bacteriolo- gy, 8th ed. (33) or the Manual of Clinical Microbiology, 3rd ed. (23), but it will be described in the 9th edition of Bergey's Manual (N. A. Vedros, personal communication). Early studies indicated that N. cinerea strains colonized the nasopharynx (2, 8, 11). Commensal Neisseria spp. and * Corresponding author. B. catarrhalis were isolated only rarely from the genitouri- nary tract (12, 14, 36, 38). The prevalence of N. cinerea is uncertain because early investigators identified N. cinerea strains as a subtype of B. catarrhalis (8, 11). Berger and Paepcke (3) found that N. cinerea strains accounted for ca. 93% of 27 asaccharolytic Neisseria (Branhamella) spp. isolated from the nasopharynx of healthy subjects. N. cin- erea is regarded as a nonpathogenic species, although it appears to have been isolated from the genitourinary tract (14, 36) and from the cerebrospinal fluid of a patient with acute meningitis (31). In some cases, the isolates were identified as N. flavescens, N. gonorrhoeae, or B. catarrha- lis, but reevaluation of these publications (2) indicates that they were probably N. cinerea strains. In this study, we describe the isolation on colistin-contain- ing Martin-Lewis medium of a colistin-susceptible, asac- charolytic, gram-negative, oxidase-positive diplococcus whose biochemical characteristics did not conform to the description of either N. flavescens or B. catarrhalis, but whose cultural characteristics resembled those of N. gonor- rhoeae. This strain was subsequently identified as N. cin- erea (von Lingelsheim 1906) Murray 1939 (27). We evaluated biochemical tests and reference techniques which will aid in identifying N. cinerea strains and differentiating them from N. gonorrhoeae. MATERIALS AND METHODS Isolation and maintenance. An asaccharolytic, gram-nega- tive, oxidase-positive diplococcus was isolated on Martin- Lewis medium (Prepared Media Laboratory, Tualatin, Oreg.) from the cervix of a 19-year-old woman subsequently diagnosed as having psoriatic arthritis. The strain, designat- ed NRL 32165, was subcultured on chocolate agar medium. Since receipt in the Neisseria Reference Laboratory (NRL), 63 on March 27, 2021 by guest http://jcm.asm.org/ Downloaded from

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Vol. 19, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1984, p. 63-670095-1137/84/010063-05$02.00/0Copyright © 1984, American Society for Microbiology

Characterization of Neisseria cinerea, a Nonpathogenic SpeciesIsolated on Martin-Lewis Medium Selective for Pathogenic Neisseria

spp.JOAN S. KNAPP,'* PATRICIA A. TOTTEN,1 MARTHA H. MULKS,2 AND BARBARA H. MINSHEW3

Neisseria Reference Laboratory and Department of Medicine, Univ'ersity of Washington, Seattle, Washington 981951;Department of Medicine, Tuifts-New England Medical Center Hospital, Boston, Massachiusetts 021112; and ClinicalMicrobiology Laboratory, Department of Pathology, Seattle Plublic Health Hospital, and Department of Laboratory

Medicine, University of Washington, Seattle, Washington 981143

Received 16 June 1983/Accepted 3 October 1983

An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis mediumfrom the cervix of a patient attending an arthritis clinic at Seattle Public Health Hospital, Seattle, Wash.This strain, NRL 32165, did not produce detectable acid from glucose, maltose, sucrose, fructose, mannitol,or lactose in either cystine Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modifiedoxidation-fermentation medium and was identified presumptively as a glucose-negative Neisseria gonor-rhoeae strain, but was identified later as Neisseria cinerea on the basis of its biochemical reactions. Nitratewas not reduced, nitrite (0.001%, wt/vol) was reduced, and polysaccharide was not produced from sucrose.Proline, arginine, and cystine-cysteine were required for growth on defined medium. Strain NRL 32165 didnot react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin Aprotease. In DNA:DNA homology studies with N. gonorrhoeae NRL 8038 (F62) and N. cinerea type strainNRL 30003, strain NRL 32165 showed 95% homology relative to N. cinerea and 44% homology relative toN. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim1906) Murray 1939. Of all Neisseria spp., N. cinerea is most likely to be misidentified as a glucose-negativeN. gonorrhoeae strain.

Commensal colistin-susceptible Neisseria spp. and Bran-hamella catarrhalis are only rarely isolated on media selec-tive for the pathogenic Neisseria spp., Neisseria gonor-rhoeae and Neisseria meningitidis, and are differentiated onthe basis of their cultural characteristics, distinctive sugarutilization reactions, ability to reduce nitrate, and ability toproduce polysaccharide from sucrose. According to currentliterature (23, 33), among commensal species isolated fromhumans, only Neisseria flavescens and B. catarrhalis areasaccharolytic.Another asaccharolytic Neisseria sp., Neisseria cinerea,

was first described by von Lingelsheim in 1906 (35) andnamed Micrococcus cinereius. It was described subsequentlyas Neisseria pseudocatarrhalis (F. M. Huntoon, Abstr.Annu. Meet. Am. Soc. Bacteriol. 1934, M50, p. 108) but wasassigned to the species N. cinerea (27) since the epithetcinerea had priority taxonomically. Since then, many strainsappear to have been isolated but incorrectly identified as B.catarrhalis (8, 11). These species differ biochemically only intheir ability to reduce nitrate, a test which was not intro-duced into the classification of Neisseria spp. until 1961 (1).B. catarrhalis strains reduce nitrate, whereas N. cinereastrains do not. Berger and Paepcke (3) "rediscovered" anddescribed N. cinerea in 1962 and showed that there was noantigenic relatedness between N. cinerea and Neisseriacatarrhalis; the latter species was subsequently reassigned tothe genus Branhamella (5) in 1970. N. cinerea was notdescribed in Bergey's Manual of Determinative Bacteriolo-gy, 8th ed. (33) or the Manual of Clinical Microbiology, 3rded. (23), but it will be described in the 9th edition of Bergey'sManual (N. A. Vedros, personal communication).

Early studies indicated that N. cinerea strains colonizedthe nasopharynx (2, 8, 11). Commensal Neisseria spp. and

* Corresponding author.

B. catarrhalis were isolated only rarely from the genitouri-nary tract (12, 14, 36, 38). The prevalence of N. cinerea isuncertain because early investigators identified N. cinereastrains as a subtype of B. catarrhalis (8, 11). Berger andPaepcke (3) found that N. cinerea strains accounted for ca.93% of 27 asaccharolytic Neisseria (Branhamella) spp.isolated from the nasopharynx of healthy subjects. N. cin-erea is regarded as a nonpathogenic species, although itappears to have been isolated from the genitourinary tract(14, 36) and from the cerebrospinal fluid of a patient withacute meningitis (31). In some cases, the isolates wereidentified as N. flavescens, N. gonorrhoeae, or B. catarrha-lis, but reevaluation of these publications (2) indicates thatthey were probably N. cinerea strains.

In this study, we describe the isolation on colistin-contain-ing Martin-Lewis medium of a colistin-susceptible, asac-charolytic, gram-negative, oxidase-positive diplococcuswhose biochemical characteristics did not conform to thedescription of either N. flavescens or B. catarrhalis, butwhose cultural characteristics resembled those of N. gonor-rhoeae. This strain was subsequently identified as N. cin-erea (von Lingelsheim 1906) Murray 1939 (27). We evaluatedbiochemical tests and reference techniques which will aid inidentifying N. cinerea strains and differentiating them fromN. gonorrhoeae.

MATERIALS AND METHODS

Isolation and maintenance. An asaccharolytic, gram-nega-tive, oxidase-positive diplococcus was isolated on Martin-Lewis medium (Prepared Media Laboratory, Tualatin,Oreg.) from the cervix of a 19-year-old woman subsequentlydiagnosed as having psoriatic arthritis. The strain, designat-ed NRL 32165, was subcultured on chocolate agar medium.Since receipt in the Neisseria Reference Laboratory (NRL),

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TABLE 1. Summary of differential characteristics for N. gonorrhoeae, asaccharolytic Neisseria spp., and B. catarrhalisaAcid from: Polysac- Require- Produc-

Species G M S F Ma Nitrate charide ment for tion of Crlistin gron hSpecies

G M S F Ma L reduction from su- cystine- IgA pro-reit onM

crose cysteine tease ance agar

N. gonorrhoeae + - - - - - - - + + +N. cinerea - - - - - - - - + - - +N. flavescens - - - - - - - + + - - +B. catarrhalis - - - - - - + - - - - +

' Abbreviations: G, glucose; M, maltose; S, sucrose; F, fructose; Ma, mannitol; L, lactose; MH, Mueller-Hinton. +, Positive for all strainstested; -, negative for all strains tested. Data for N. gonorrhoeae are based on literature citations (2, 6, 23, 33).

the strain has been cultured on GC base medium (DifcoLaboratories, Detroit, Mich.) plus defined supplement(GCK) (37) at 36°C in a CO2-enriched atmosphere. The strainwas stored in 50% gamma globulin-free horse serum (GIBCOLaboratories, Grand Island, N.Y.) in tryptic soy broth(Difco) at -70°C.

Strains. N. cinerea type strain NRL 30003 (ATCC 14685)and N. gonorrhoeae NRL 8038 (F62) were used as referencestrains for DNA:DNA homology studies. Species used incomparative studies to determine differential biochemicalcharacteristics are listed in Table 1. Two N. cinerea strains(NRL 32824 and NRL 32828) isolated on sheep blood agar inSeattle in 1981 were also studied. In addition, 10 strains of B.catarrhalis and the taxonomic type strain of N. flavescens(NRL 30009) were studied.

Colony morphology. Observations of the colonial morphol-ogy of strains were made on GCK medium, human andsheep blood agar, and chocolate agar. Strains were streakedonto each medium and colony morphology was describedafter incubation for 24 h at 36°C in a CO--enriched atmo-sphere.Sugar utilization tests. Strains were tested in cystine

Trypticase agar medium (BBL Microbiology Systems, Cock-eysville, Md.) for their ability to produce acid from glucose,maltose, lactose, and sucrose. Subsequent confirmatorytests were made in modified oxidation-fermentation mediumcontaining glucose, maltose, sucrose, fructose, mannitol, orlactose (19). Modified oxidation-fermentation base mediumhas the following composition (per liter): 0.2% Difco prote-ase peptone no. 3-0.5% NaCI-0.03% dipotassium hydrogenphosphate-0.3% agar-0.5 ml of a 0.17% phenol red solution.

Additional biochemical tests. Tests to detect nitrate andnitrite reduction and polysaccharide production from su-crose were performed as described previously (3, 19).

Auxotyping. Strains were tested on chemically definedmedium (NEDA) as described previously (18). Require-ments for proline, arginine, hypoxanthine, uracil, methio-nine, and cystine-cysteine were determined. Type strains ofasaccharolytic species N. flavescens and B. catarrhalis werecompared with N. cinerea strains.

Antibiotic susceptibility tests. Susceptibility of N. cinereastrains to penicillin, tetracycline, spectinomycin, erythromy-cin, and vancomycin were determined as described previ-ously (18). Colistin susceptibility was determined by diskdiffusion tests on GCK agar with BBL disks (10 jig).

Serology. Serogrouping of gonococci was performed usingthe coagglutination technique. Reagent staphylococci (pro-vided by Lars Rudin, Pharmacia AB, Uppsala, Sweden)were sensitized with each of 10 monoclonal antibodies(provided by Milton Tam, Genetic Systems Corp., Seattle,Wash.) against N. gonorrhoeae (34). Each test was per-formed by mixing one drop of a suspension of boiledorganism with one drop of a suspension of the reagent

staphylococci, rotating for 2 min, and observing the mixtureunder oblique transmitted light for agglutination. The re-agents used were chosen to subgroup gonococci into sero-groups WI, WII, and Wlll, but cumulatively they may beused to identify gonococci since cross-reactions with otherNeisseria spp. have not been observed (29).

Detection of IgA protease. Immunoglobulin A (IgA) prote-ase was detected using sodium dodecyl sulfate-polyacryl-amide gel electrophoresis as described previously (20, 25).

Isolation of DNA. DNA was isolated by the procedure ofBrenner et al. (4), and the final concentration was calculatedfrom 260 to 280 nm absorption in a Gilford model 2400spectrophotometer (Gilford Instrument Laboratories, Inc.,Oberlin, Ohio).

Preparation of radiolabeled DNA. For hybridization ex-periments, whole-cell DNA from strain NRL 32165 waslabeled to a specific activity of 2 x 106 cpm/Lg of DNA bythe nick translation technique of Maniatis et al. (21) with[3H]deoxyribosylthymine triphosphate (77 Ci/mM; New En-gland Nuclear Corp., Boston, Mass.). Nick translation wasperformed with the DNase concentration adjusted to yieldDNA with a final fragment size of 3,000 base pairs, afterwhich the DNA was sonicated to 500 base pairs.

Whole-cell DNA:DNA homology studies. Whole-cell DNAwas hybridized overnight, and homology was analyzed bythe single-strand endonuclease procedure of Crosa et al. (7)as modified by Piot et al. (30). Homologies were determinedafter hybridization at 65°C overnight in 0.42 M NaCl, whichwere optimal conditions for hybridization, i.e., 25 to 30°Cbelow the melting point of the probe DNA.

RESULTSAn asaccharolytic, gram-negative, oxidase-positive diplo-

coccus was isolated on Martin-Lewis medium from thecervix of a patient attending an arthritis clinic. This strain,NRL 32165, was tested in the clinical laboratory for its sugarutilization pattern in cystine Trypticase agar and failed toproduce acid from glucose, maltose, sucrose, or lactose. Thestrain was also resistant to erythromycin by disk diffusiontesting and was studied further.

Colonial morphology. N. cinerea colonies on GCK medi-um incubated at 36°C for 24 h were gold-brown; the cell masswas often more distinctly gold-brown than N. gonorrhoeae.Colonies were ca. 1 mm in diameter with a glisteningsurface, entire margin, and convex elevation (similar to T3colonies of N. gonorrhoeae) and were translucent and easilyemulsified in broth. Cells showed the diplococcal morpholo-gy, in which cells had adjacent sides flattened and varied insize. Pairs or clumps predominated over individual diplo-cocci. Giant cells were sometimes seen in 48-h cultures (2).Colonies on sheep and human blood agar and chocolate agar

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ISOLATION OF N. CINEREA 65

were grey-white. Cell and colonial morphology conformedwith the description of N. cinerea (3).

Biochemical characteristics. The biochemical characteris-tics of strains NRL 32165, NRL 32824, and NRL 32828 agreewith those reported previously for N. cinerea (3). Sugarutilization tests performed in both cystine Trypticase agarmedium and modified oxidation-fermentation medium con-taining glucose, maltose, sucrose, lactose, mannitol, orfructose confirmed that the strains did not produce acid fromany of these carbohydrates even after incubation for 14 days.Strains did not reduce nitrate or produce polysaccharidefrom sucrose, but they did reduce nitrite (0.001%, wt/vol), asdid N. gonorrhoeae strains (J. S. Knapp, Abstr. Annu.Meet. Am. Soc. Microbiol. 1983, D27, p. 63). On the basis ofbiochemical tests, the strains were identified as N. cinerea.

Nutritional requirements. Determination of the require-ment of the strains for cysteine-cystine was made afterincubation for 48 h at 36°C in a CO2-enriched atmosphere.All N. cinerea strains required cysteine-cystine for growthon NEDA medium. In addition, all strains also requiredproline and arginine (Pro- Arg-). N. flavescens also re-quired cysteine-cystine for growth, but B. catarrhalis didnot.

Serology. N. cinerea, B. catarrhalis, and N. flavescensstrains did not react with any of 10 antigonococcal protein Imonoclonal antibodies in coagglutination tests.

Antibiotic susceptibility patterns. N. cinerea strains weresusceptible to various antibiotics measured in minimal in-hibitory concentrations (micrograms per milliliter) as fol-lows. Penicillin (0.125 to 1.0), tetracycline (0.125 to 0.5),vancomycin (32 to .64), erythromycin (2.0 to 4.0), andspectinomycin (16 to .32). The results demonstrated thatantibiotic susceptibility patterns for N. cinerea strains werenot notably different from those for N. gonorrhoeae. Resist-ance to erythromycin was higher than normally found for N.gonorrhoeae strains (13, 28). Colistin disk susceptibilitytests with N. cinerea strains showed that zones of inhibitionwere :10 mm in diameter with an unstandardized inoculum.In contrast, N. gonorrhoeae strains showed no inhibition.These results are consistent with those reported by Berger(2).IgA protease activity. None of the N. cinerea strains

produced IgA protease. Strains of N. gonorrhoeae and N.meningitidis produce IgA proteases, whereas commensalNeisseria spp. do not (26). In accordance with these find-ings, the strains of N. cinerea examined in this study failedto produce IgA protease, thus providing an additional refer-ence test which can aid in their identification.DNA:DNA homology studies. Homology studies were per-

formed using 3H-labeled whole-cell DNA from N. cinereaNRL 32165 and unlabeled DNA from the test organisms.The actual extent of reassociation of homologous (strainNRL 32165) and heterologous (calf thymus) DNA averaged75 and 7%, respectively. Although the type strain N. cinereaNRL 30003 was tested, we did not use it as the referencestrain for these studies because it contained a small, 2.0-megadalton (Mdal) plasmid. Since most small plasmids oc-cur in multiple copies (often 20 to 30) per cell, we wereconcerned that the presence of this plasmid might affect thehomology results measurably. Results of homology studiesare summarized below; each value is the average of four orfive determinations. The type strain NRL 30003 and strainNRL 32824 showed 95 and 94% relative homology, respec-tively, with strain NRL 32165; strain NRL 32828 showed81% homology relative to strain NRL 32165. The compara-tively low value of 81% relative homology between strains

NRL 32828 and NRL 32165 compared with higher values forthe other strains might be accounted for in part by the 3.2-Mdal plasmid carried by strain NRL 32828. If this plasmid ispresent in 20 to 30 copies per cell, it would account for ca.6% of the DNA in this strain (22). In contrast, N. gonor-rhoeae NRL 8038 showed only 44% homology relative tostrain NRL 32165. These studies confirm that strain NRL32165 and other recently isolated strains were N. cinerea(von Lingelsheim 1906) Murray 1939.

DISCUSSIONA strain of N. cinerea (von Lingelsheim 1906) Murray

1939 was isolated from a clinical specimen on Martin-Lewismedium selective for pathogenic Neisseria spp. This isola-tion is noteworthy since N. cinerea is not currently listedamong human Neisseria spp. (23, 33). N. cinerea has notbeen correctly identified previously in the United States,although it appears that strains of this species have beenisolated from clinical specimens in the United States andEurope on several occasions but incorrectly identified as B.catarrhalis (14), N. flavescens (31), N. gonorrhoeae (32), orN. pseudocatarrhalis (Huntoon, Abstr. Annu. Meet. Am.Soc. Bacteriol. 1934).

Previous homology studies have shown that strains classi-fied in the same species show 70% or more relative homolo-gy under optimal conditions (4). DNA:DNA homology stud-ies by different methods have reported different relativehomology values for interspecific reactions between strainsrepresentative of Neisseria spp. Hoke and Vedros (15) usedthe thermal renaturation technique and reported 60% rela-tive homology between N. gonorrhoeae and N. cinerea,Neisseria sicca, Neisseria subflava, and N. flavescens. Incontrast, Elwell and Falkow (9), using the more restrictiveS1 endonuclease assay, demonstrated relative homologyvalues for interspecific reactions between the DNA of N.gonorrhoeae, N. sicca, N. subflava, and N. flavescensranging between 24 and 29%; N. cinerea strains were nottested in that study. Although no DNA:DNA homologystudies by the S1 endonuclease procedure have been report-ed previously between N. gonorrhoeae and N. cinerea, therelative homology of 44% between these species suggeststhat N. cinerea may be more closely related to N. gonor-rhoeae than are other commensal Neisseria spp. Evidencethat N. cinerea is more closely related to N. gonorrhoeae infatty acid composition rather than to the other commensalNeisseria spp. and B. catarrhalis has been reported previ-ously (16, 17).The isolation of N. cinerea emphasized weaknesses in one

reference test used to confirm the identification of gono-cocci. Catlin (6) noted that although all strains of N. gonor-rhoeae required cystine-cysteine for growth on NEDA medi-um, more than 90% of N. meningitidis strains did not, andsome N. lactamica strains required cystine-cysteine, where-as others did not. Our observations in auxotyping N. gonor-rhoeae, N. meningitidis, and N. lactamica strains since 1973have confirmed those of Catlin. In addition, in this study we

found that N. cinerea and N. flavescens strains required notonly cystine-cysteine for growth on NEDA medium, but N.cinerea strains also required proline and arginine (Pro-Arg-) for growth, thus contributing further to confusion withgonococci. Consequently, the requirement for cystine-cyste-ine cannot be used to differentiate between N. cinerea andN. gonorrhoeae.

Although in coagglutination tests all gonococci reactedwith at least 1 of 10 research antigonococcal protein Imonoclonal antibodies (29, 34), no cross-reactions were

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observed with other Neisser-ia spp., including N. (inereastrains and B. (at(lrrhalis. However, because commercialserological diagnostic tests for N. gonorrhoeae do not detectall strains (10; J. P. Libonati, R. L. Leilich, and L. Loomis.Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C19, p. 314),commercial diagnostic reagents are of limited usefulness fordistinguishing N. gon0orrh-loeae strains from N. ( inereistrains.

Additional tests which proved useful in differentiating N.(cinerei(l from N. gono-rrhoeie were the ability of N. c inereastrains to grow on simple media such as tryptic soy agar(Prepared Media Laboratory) or Mueller-Hinton agar (Difco)and their susceptibility to colistin. Tryptic soy agar andMueller-Hinton agar supported good growth of N. cinereastrains but not N. gonor-hoeae strains. N. (inerea strainswere susceptible to colistin (10-pLg disk; BBL) but resistantto erythromycin and of intermediate resistance to penicillin.Although N. c inerea appears to be an extremely rare

isolate from genital sites (12, 14, 36, 38), it has been isolatedmore frequently from the nasopharynx (3) and oropharynx(J. S. Knapp, P. A. Totten, B. H. Minshew, and E. W. HookIII, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C26, p.

316). The presence of N. cinerca in these sites poses twoareas of potential confusion for clinical microbiologists iden-tifying Neisseria spp. First, since N. cinerea colonies closelyresemble N. gonorrhoeae colonies, these species may beconfused when acid production from glucose is weak. Thus,N. cinerea strains may be misidentified as N. gonorrhoeaewhen isolated on selective media or on nonselective mediaoften used now to detect vancomycin-susceptible gonococ-

cal strains. Second, N. cinerea strains may be confused withB. catarrhalis strains when isolated from pharyngeal speci-mens on nonselective media employed to isolate Haemophi-lus influenzae. Since B. catarrhalis is being increasinglyimplicated as a respiratory pathogen, it is important that theetiological agents in these infections be identified correctly.Although N. cinerea colony morphology is distinct from thatof B. catarrhalis strains, N. ciner-ea strains were consistentlymisidentified as a B. catarrhalis subtype (8, 11) before theintroduction of nitrate reduction as a differential test inNeisseria spp. classification in 1961 (1). Confusion of N.(ine)(ea with N. flai'escens (31) resulted because the strainsexhibit similar colony morphologies and also because pro-

duction of polysaccharide from sucrose was not introducedinto Neisseria spp. classification until 1961.

Since N. (cinerea is not listed among human Neisseria spp.

in current reference texts, a summary of differential charac-teristics useful for differentiating between asaccharolyticspecies of N. (inerea, N. flaves(cens, B. caitarrhalis, andweak acid-producing N. gonorrhoeae strains is provided inTable 1. Unfortunately, DNA:DNA homology studies pro-

vide the only confirmatory test available currently to identifyN. cinerea. Tests used in clinical laboratories permit theidentification of N. cinerea only by the elimination of othercurrently recognized Neisseria spp. or B. catarrhalis. Inview of the legal and scientific implications of misidentifyingN. (cinerea strains as N. gono rrhoeae or B. catallhalis, we

wish to alert clinical microbiologists to the existence of N.(inerea and to advise caution when identifying apparentlyglucose-negative, gram-negative, oxidase-positive diplo-cocci.

ACKNOWLEDGMENTThis research was supported by Public Health Service grant Al-

12191 from the National Institutes of Health.

LITERATURE CITED

1. Berger, U. 1961. Reduktion von nitrat and nitrit durch Neisseria.Z. Hyg. 148:45-50.

2. Berger, U. 1963. Die anspruchslosen Neisserien. Ergeb. Mikro-biol. Immunitaetsforsch. 36:97-167.

3. Berger, U., and E. Paepcke. 1962. Untersuchungen uber dieasaccharolytischen Neisselrien des menschlichen Nasopharynx.Z. Hyg. 148:269-281.

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