Isolated on Martin-Lewis Medium Selective for Pathogenic Neisseria spp. JOAN S. KNAPP,'* ..... Mayer, L. D., K. K. Holmes, and S. Falkow. 1974. Characteriza-.
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 gonorrhoeae 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. B. catarrhalis were isolated only rarely from the genitourinary 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. cinerea 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. catarrhalis, but reevaluation of these publications (2) indicates that they were probably N. cinerea strains. In this study, we describe the isolation on colistin-containing Martin-Lewis medium of a colistin-susceptible, asac-
Commensal colistin-susceptible Neisseria spp. and Branhamella catarrhalis are only rarely isolated on media selective for the pathogenic Neisseria spp., Neisseria gonorrhoeae 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 introduced 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 Bacteriology, 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.
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. gonorrhoeae. This strain was subsequently identified as N. cinerea (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-negative, oxidase-positive diplococcus was isolated on MartinLewis medium (Prepared Media Laboratory, Tualatin, Oreg.) from the cervix of a 19-year-old woman subsequently diagnosed as having psoriatic arthritis. The strain, designated NRL 32165, was subcultured on chocolate agar medium. Since receipt in the Neisseria Reference Laboratory (NRL),
63
64
J. CLIN. MICROBIOL.
KNAPP ET AL.
TABLE 1. Summary of differential characteristics for N. gonorrhoeae, asaccharolytic Neisseria spp., and B. catarrhalisa Acid from: Species Species
G G
M M
S S
N. gonorrhoeae N. cinerea N. flavescens B. catarrhalis
+
-
-
F F
Ma Ma
L
Nitrate reduction
Polysac-
Require-
charide from su-
ment for
crose
-
-
-
-
-
-
cystinecysteine + + +
Production of
Crlistin
tease
ance
+
+
IgA pro-
reit
gron
onM
h
agar
+ + + + + ' Abbreviations: G, glucose; M, maltose; S, sucrose; F, fructose; Ma, mannitol; L, lactose; MH, Mueller-Hinton. +, Positive for all strains tested; -, 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 (Difco Laboratories, Detroit, Mich.) plus defined supplement (GCK) (37) at 36°C in a CO2-enriched atmosphere. The strain was stored in 50% gamma globulin-free horse serum (GIBCO Laboratories, 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 reference strains for DNA:DNA homology studies. Species used in comparative studies to determine differential biochemical characteristics are listed in Table 1. Two N. cinerea strains (NRL 32824 and NRL 32828) isolated on sheep blood agar in Seattle 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 morphology of strains were made on GCK medium, human and sheep blood agar, and chocolate agar. Strains were streaked onto each medium and colony morphology was described after incubation for 24 h at 36°C in a CO--enriched atmosphere. Sugar utilization tests. Strains were tested in cystine Trypticase agar medium (BBL Microbiology Systems, Cockeysville, Md.) for their ability to produce acid from glucose, maltose, lactose, and sucrose. Subsequent confirmatory tests were made in modified oxidation-fermentation medium containing glucose, maltose, sucrose, fructose, mannitol, or lactose (19). Modified oxidation-fermentation base medium has the following composition (per liter): 0.2% Difco protease peptone no. 3-0.5% NaCI-0.03% dipotassium hydrogen phosphate-0.3% agar-0.5 ml of a 0.17% phenol red solution. Additional biochemical tests. Tests to detect nitrate and nitrite reduction and polysaccharide production from sucrose were performed as described previously (3, 19). Auxotyping. Strains were tested on chemically defined medium (NEDA) as described previously (18). Requirements for proline, arginine, hypoxanthine, uracil, methionine, and cystine-cysteine were determined. Type strains of
asaccharolytic species N. flavescens and B. catarrhalis were compared with N. cinerea strains.
Antibiotic susceptibility tests. Susceptibility of N. cinerea strains to penicillin, tetracycline, spectinomycin, erythromycin, and vancomycin were determined as described previously (18). Colistin susceptibility was determined by disk diffusion tests on GCK agar with BBL disks (10 jig). Serology. Serogrouping of gonococci was performed using the coagglutination technique. Reagent staphylococci (provided 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 performed by mixing one drop of a suspension of boiled organism with one drop of a suspension of the reagent
staphylococci, rotating for 2 min, and observing the mixture under oblique transmitted light for agglutination. The reagents used were chosen to subgroup gonococci into serogroups WI, WII, and Wlll, but cumulatively they may be used to identify gonococci since cross-reactions with other Neisseria spp. have not been observed (29). Detection of IgA protease. Immunoglobulin A (IgA) protease was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (20, 25). Isolation of DNA. DNA was isolated by the procedure of Brenner et al. (4), and the final concentration was calculated from 260 to 280 nm absorption in a Gilford model 2400 spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio). Preparation of radiolabeled DNA. For hybridization experiments, whole-cell DNA from strain NRL 32165 was labeled to a specific activity of 2 x 106 cpm/Lg of DNA by the nick translation technique of Maniatis et al. (21) with [3H]deoxyribosylthymine triphosphate (77 Ci/mM; New England Nuclear Corp., Boston, Mass.). Nick translation was performed with the DNase concentration adjusted to yield DNA with a final fragment size of 3,000 base pairs, after which the DNA was sonicated to 500 base pairs. Whole-cell DNA:DNA homology studies. Whole-cell DNA was hybridized overnight, and homology was analyzed by the single-strand endonuclease procedure of Crosa et al. (7) as modified by Piot et al. (30). Homologies were determined after hybridization at 65°C overnight in 0.42 M NaCl, which were optimal conditions for hybridization, i.e., 25 to 30°C below the melting point of the probe DNA. RESULTS An asaccharolytic, gram-negative, oxidase-positive diplococcus was isolated on Martin-Lewis medium from the cervix of a patient attending an arthritis clinic. This strain, NRL 32165, was tested in the clinical laboratory for its sugar utilization pattern in cystine Trypticase agar and failed to produce acid from glucose, maltose, sucrose, or lactose. The strain was also resistant to erythromycin by disk diffusion testing and was studied further. Colonial morphology. N. cinerea colonies on GCK medium incubated at 36°C for 24 h were gold-brown; the cell mass
was often more distinctly gold-brown than N. gonorrhoeae. Colonies were ca. 1 mm in diameter with a glistening surface, entire margin, and convex elevation (similar to T3 colonies of N. gonorrhoeae) and were translucent and easily emulsified in broth. Cells showed the diplococcal morphology, in which cells had adjacent sides flattened and varied in size. Pairs or clumps predominated over individual diplococci. Giant cells were sometimes seen in 48-h cultures (2). Colonies on sheep and human blood agar and chocolate agar
VOL. 19, 1984
were grey-white. Cell and colonial morphology conformed with the description of N. cinerea (3). Biochemical characteristics. The biochemical characteristics of strains NRL 32165, NRL 32824, and NRL 32828 agree with those reported previously for N. cinerea (3). Sugar utilization tests performed in both cystine Trypticase agar medium and modified oxidation-fermentation medium containing glucose, maltose, sucrose, lactose, mannitol, or fructose confirmed that the strains did not produce acid from any of these carbohydrates even after incubation for 14 days. Strains did not reduce nitrate or produce polysaccharide from sucrose, but they did reduce nitrite (0.001%, wt/vol), as did N. gonorrhoeae strains (J. S. Knapp, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, D27, p. 63). On the basis of biochemical tests, the strains were identified as N. cinerea. Nutritional requirements. Determination of the requirement of the strains for cysteine-cystine was made after incubation for 48 h at 36°C in a CO2-enriched atmosphere. All N. cinerea strains required cysteine-cystine for growth on NEDA medium. In addition, all strains also required proline and arginine (Pro- Arg-). N. flavescens also required cysteine-cystine for growth, but B. catarrhalis did not. Serology. N. cinerea, B. catarrhalis, and N. flavescens strains did not react with any of 10 antigonococcal protein I monoclonal antibodies in coagglutination tests. Antibiotic susceptibility patterns. N. cinerea strains were susceptible to various antibiotics measured in minimal inhibitory concentrations (micrograms per milliliter) as follows. Penicillin (0.125 to 1.0), tetracycline (0.125 to 0.5), vancomycin (32 to .64), erythromycin (2.0 to 4.0), and spectinomycin (16 to .32). The results demonstrated that antibiotic susceptibility patterns for N. cinerea strains were not notably different from those for N. gonorrhoeae. Resistance to erythromycin was higher than normally found for N. gonorrhoeae strains (13, 28). Colistin disk susceptibility tests with N. cinerea strains showed that zones of inhibition were :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 commensal Neisseria spp. do not (26). In accordance with these findings, the strains of N. cinerea examined in this study failed to produce IgA protease, thus providing an additional reference test which can aid in their identification. DNA:DNA homology studies. Homology studies were performed using 3H-labeled whole-cell DNA from N. cinerea NRL 32165 and unlabeled DNA from the test organisms. The actual extent of reassociation of homologous (strain NRL 32165) and heterologous (calf thymus) DNA averaged 75 and 7%, respectively. Although the type strain N. cinerea NRL 30003 was tested, we did not use it as the reference strain for these studies because it contained a small, 2.0megadalton (Mdal) plasmid. Since most small plasmids occur in multiple copies (often 20 to 30) per cell, we were concerned that the presence of this plasmid might affect the homology results measurably. Results of homology studies are summarized below; each value is the average of four or five determinations. The type strain NRL 30003 and strain NRL 32824 showed 95 and 94% relative homology, respectively, with strain NRL 32165; strain NRL 32828 showed 81% homology relative to strain NRL 32165. The comparatively low value of 81% relative homology between strains
ISOLATION OF N. CINEREA
65
NRL 32828 and NRL 32165 compared with higher values for the other strains might be accounted for in part by the 3.2Mdal plasmid carried by strain NRL 32828. If this plasmid is present in 20 to 30 copies per cell, it would account for ca. 6% of the DNA in this strain (22). In contrast, N. gonorrhoeae NRL 8038 showed only 44% homology relative to strain NRL 32165. These studies confirm that strain NRL 32165 and other recently isolated strains were N. cinerea (von Lingelsheim 1906) Murray 1939. DISCUSSION A strain of N. cinerea (von Lingelsheim 1906) Murray 1939 was isolated from a clinical specimen on Martin-Lewis medium selective for pathogenic Neisseria spp. This isolation is noteworthy since N. cinerea is not currently listed among human Neisseria spp. (23, 33). N. cinerea has not been correctly identified previously in the United States, although it appears that strains of this species have been isolated from clinical specimens in the United States and Europe on several occasions but incorrectly identified as B. catarrhalis (14), N. flavescens (31), N. gonorrhoeae (32), or N. pseudocatarrhalis (Huntoon, Abstr. Annu. Meet. Am. Soc. Bacteriol. 1934). Previous homology studies have shown that strains classified in the same species show 70% or more relative homology under optimal conditions (4). DNA:DNA homology studies by different methods have reported different relative homology values for interspecific reactions between strains representative of Neisseria spp. Hoke and Vedros (15) used the thermal renaturation technique and reported 60% relative homology between N. gonorrhoeae and N. cinerea, Neisseria sicca, Neisseria subflava, and N. flavescens. In contrast, Elwell and Falkow (9), using the more restrictive S1 endonuclease assay, demonstrated relative homology values for interspecific reactions between the DNA of N. gonorrhoeae, N. sicca, N. subflava, and N. flavescens ranging between 24 and 29%; N. cinerea strains were not tested in that study. Although no DNA:DNA homology studies by the S1 endonuclease procedure have been reported previously between N. gonorrhoeae and N. cinerea, the relative homology of 44% between these species suggests that N. cinerea may be more closely related to N. gonorrhoeae than are other commensal Neisseria spp. Evidence that N. cinerea is more closely related to N. gonorrhoeae in fatty acid composition rather than to the other commensal Neisseria spp. and B. catarrhalis has been reported previously (16, 17). The isolation of N. cinerea emphasized weaknesses in one reference test used to confirm the identification of gonococci. Catlin (6) noted that although all strains of N. gonorrhoeae required cystine-cysteine for growth on NEDA medium, more than 90% of N. meningitidis strains did not, and some N. lactamica strains required cystine-cysteine, whereas others did not. Our observations in auxotyping N. gonorrhoeae, N. meningitidis, and N. lactamica strains since 1973 have confirmed those of Catlin. In addition, in this study we found that N. cinerea and N. flavescens strains required not only cystine-cysteine for growth on NEDA medium, but N. cinerea strains also required proline and arginine (ProArg-) for growth, thus contributing further to confusion with gonococci. Consequently, the requirement for cystine-cysteine cannot be used to differentiate between N. cinerea and N. gonorrhoeae. Although in coagglutination tests all gonococci reacted with at least 1 of 10 research antigonococcal protein I monoclonal antibodies (29, 34), no cross-reactions were
66
J. CLIN. MICROBIOL.
KNAPP ET AL.
observed with other Neisser-ia spp., including N. (inerea strains and B. (at(lrrhalis. However, because commercial serological diagnostic tests for N. gonorrhoeae do not detect all 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 for distinguishing N. gon0orrh-loeae strains from N. ( inerei strains. Additional tests which proved useful in differentiating N. (cinerei(l from N. gono-rrhoeie were the ability of N. c inerea strains 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 and Mueller-Hinton agar supported good growth of N. cinerea strains but not N. gonor-hoeae strains. N. (inerea strains were susceptible to colistin (10-pLg disk; BBL) but resistant to 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 isolated more frequently from the nasopharynx (3) and oropharynx (J. S. Knapp, P. A. Totten, B. H. Minshew, and E. W. Hook III, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C26, p. 316). The presence of N. cinerca in these sites poses two areas of potential confusion for clinical microbiologists identifying Neisseria spp. First, since N. c inerea colonies closely resemble N. gonorrhoeae colonies, these species may be confused when acid production from glucose is weak. Thus, N. cinerea strains may be misidentified as N. gonorrhoeae when isolated on selective media or on nonselective media often used now to detect vancomycin-susceptible gonococcal strains. Second, N. cinerea strains may be confused with B. catarrhalis strains when isolated from pharyngeal specimens on nonselective media employed to isolate Haemophilus influenzae. Since B. catarrhalis is being increasingly implicated as a respiratory pathogen, it is important that the etiological agents in these infections be identified correctly. Although N. c inerea colony morphology is distinct from that of B. catarrhalis strains, N. ciner-ea strains were consistently misidentified as a B. catarrhalis subtype (8, 11) before the introduction of nitrate reduction as a differential test in Neisseria spp. classification in 1961 (1). Confusion of N. (ine)(ea with N. flai'escens (31) resulted because the strains exhibit similar colony morphologies and also because production of polysaccharide from sucrose was not introduced into Neisseria spp. classification until 1961. Since N. (cinerea is not listed among human Neisseria spp. in current reference texts, a summary of differential characteristics useful for differentiating between asaccharolytic species of N. (inerea, N. flaves(cens, B. caitarrhalis, and weak acid-producing N. gonorrhoeae strains is provided in Table 1. Unfortunately, DNA:DNA homology studies provide the only confirmatory test available currently to identify N. cinerea. Tests used in clinical laboratories permit the identification of N. c inerea only by the elimination of other currently recognized Neisseria spp. or B. catarrhalis. In view of the legal and scientific implications of misidentifying N. (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 apparently glucose-negative, gram-negative, oxidase-positive diplococci.
ACKNOWLEDGMENT This research was supported by Public Health Service 12191 from the National Institutes of Health.
grant
Al-
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. Mikrobiol. Immunitaetsforsch. 36:97-167. 3. Berger, U., and E. Paepcke. 1962. Untersuchungen uber die asaccharolytischen Neisselrien des menschlichen Nasopharynx. Z. Hyg. 148:269-281. 4. Brenner, D. J., A. C. McWhorter, J. K. L. Knutson, and A. G. Steigerwalt. 1982. Escherichia iulnaris: a new species of Eliterobacteriaceae associated with human wounds. J. Clin. Microbiol. 15:1133-1140. 5. Catlin, B. W. 1970. Transfer of the organism named Neisseria c-atatrrhhalis to Brcanhainella gen. nov. Int. J. Syst. Bacteriol. 20:155-159. 6. Catlin, B. W. 1973. Nutritional profiles of Neisseriia gonorrhoeae, Neisserii ineningitidis, Neisseria Iactainica in chemically defined media and the use of growth requirements for gonococcal typing. J. Infect. Dis. 128:178-194. 7. Crosa, J. H., D. J. Brenner, and S. Falkow. 1973. Use of singlestrand specific nuclease for analysis of bacterial plasmid deoxyribonucleic acid homo- and heteroduplexes. J. Bacteriol. 115:904-911. 8. Elser, W. J., and F. M. Huntoon. 1909. Studies on meningitis. J. Med. Res. 20:371-541. 9. Elwell, L. P., and S. Falkow. 1977. Plasmids of the genus Neisseria, p. 127-154. In R. B. Roberts (ed.). The gonococcus. John Wiley & Sons, Inc.. New York. 10. Futrovsky, S. L., C. A. Gaydos, and J. Keiser. 1981. Comparison of the Phadebact gonococcus test with the rapid fermentation method. J. Clin. Microbiol. 14:89-93. 11. Gordon, J. E. 1921. The gram-negative cocci in "colds" and influenza. VII. Influenza studies. J. Infect. Dis. 29:462-494. 12. Gurd, F. B. 1908. A contribution to the bacteriology of the female genital tract with special reference to the detection of the gonococcus. J. Med. Res. 18:291-303. 13. Hall, W. H., E. A. Schierl, and J. E. Maccani. 1979. Comparative susceptibility of penicillinase-positive and -negative Neisseria gonorrhoeie to 30 antibiotics. Antimicrob. Agents Chemother. 15:562-567. 14. Henriksen, S. D. 1946. Isolation of atypical strains of Neisselia catarrhalis from the genito-urinary tract. Acta Derm. Venereol. (Stockholm) 26:506-514. 15. Hoke, C., and N. A. Vedros. 1982. Taxonomy of the Neisseriae: deoxyribonucleic acid base composition, interspecific transformation. and deoxyribonucleic acid hybridization. lnt. J. Syst. Bacteriol. 32:57-66. 16. Jantzen, E., K. Bryn, T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. IV. A procedure for fractionation and identification of fatty acids monosaccharides of cellular structures. Acta Pathol. Microbiol. Scand. Sect. B 82:753-766. 17. Jantzen, E., K. Bryn, T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. V. Fatty acid composition of neisseria and moraxellae. Acta Pathol. Microbiol. Scand. Sect. B 82:767-779. 18. Knapp, J. S., and K. K. Holmes. 1975. Disseminated gonococcal infections caused by Neisseria gonorrhloeae with unique nutritional requirements. J. Infect. Dis. 132:204-208. 19. Knapp, J. S., and K. K. Holmes. 1983. A modified oxidationfermentation medium for detection of acid production from carbohydrates by Neisserial spp. and Brain/wilcnella catarrhalis. J. Clin. Microbiol. 18:56-62. 20. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of the bacteriophage T4. Nature (London) 227:680-685. 21. Maniatis, T., A. Jeffery, and A. G. Kleid. 1975. Nucleotide sequence of the rightward operator of phage lambda. Proc. NatI. Acad. Sci. U.S.A. 72:1184-1188. 22. Mayer, L. D., K. K. Holmes, and S. Falkow. 1974. Characterization of plasmid deoxyribonucleic acid from Neisseriia gonorrholecae. Infect. Immun. 10:712-717.
VOL. 19, 1984
ISOLATION OF N. CINEREA
23. Morello, J. A., and M. Bohnnoff. 1980. Neisseria and Branh/amella, p. 111-130. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 24. Morello, J. A., S. A. Lerner, and M. Bohnhoff. 1976. Characteristics of atypical Neisseria gonorrhoeae from disseminated and localized infections. Infect. Immun. 13:1510-1516. 25. Mulks, M. H., S. J. Kornfeld, and A. G. Plaut. 1980. Specific proteolysis of human IgA by Streptococclus pneiurnoniiae and Haemnopli/us infliuenzae. J. Infect. Dis. 141:450-456. 26. Mulks, M. H., and A. G. Plaut. 1978. IgA protease production as a characteristic distinguishing pathogenic from harmless Neisseriaceae. N. Engl. J. Med. 299:973-976. 27. Murray, E. G. D. 1939. Genus Neisseria Trevisan, p. 278288. In D. H. Bergey, R. S. Breed, E. G. D. Murray, and A. P. Hitchins (ed.). Bergey's manual of determinative bacteriology. 5th ed. The Williams & Wilkins Co.. Baltimore. 28. Ng, W. S., P. Anton, and K. Arnold. 1981. Neisserial gonorr-lioeae strains isolated in Hong Kong: in vitro susceptibility to 13 antibiotics. Antimicrob. Agents Chemother. 19:12-17. 29. Nowinski, R. C., M. R. Tam, L. C. Goldstein, L. Stong, C.-C. Kuo, L. Corey, W. E. Stamm, H. H. Handsfield, J. S. Knapp, and K. K. Holmes. 1983. Monoclonal antibodies for diagnosis of infectious diseases in humans. Science 219:637-644. 30. Piot, P., E. Van Dyck, M. Goodfellow, and S. Falkow. 1980. A
31. 32. 33.
34.
I.
35.
36.
37. 38.
67
taxonomic study of Gardnerella ivaginalis (Haetnophillus iagina/lis) Gardner & Dukes 1955. J. Gen. Microbiol. 119:373-396. Prentice, A. W. 1957. Neisseria flai'escens as a cause of meningitis. Lancet i:613-614. Reyn, A. 1948. Atypical gonococcal strains. Acta Derm. Venereol. (Stockholm) 28:381-386. Reyn, A. 1974. Genus 1. Neisseria Trevisan, p. 428-432. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co.. Baltimore. Tam, M. R., T. M. Buchanan, E. G. Sandstrom, K. K. Holmes, J. S. Knapp, A. W. Siadak, and R. C. Nowinski. 1982. Serological classification of Neisseriia gonorrhoeae with monoclonal antibodies. Infect. Immun. 36:1042-1053. Von Lingelsheim, W. 1906. Die bakteriologischen Arbeiten der Kgl Hygienischen Station zu Beuthen 0. Schl. wahrend der Genickstarreepidemie in Oberschlesien im Winter 1904/05. Klin. Jahrb. 15:373-489. Wax, L. 1950. The identity of Neisseriia other than the gonococcus from the genito-urinary tract. J. Vener. Dis. Inf. 31:208. White, L. A., and D. S. Kellogg, Jr. 1965. Neisseria gonorrhloeae identification in direct smears by a fluorescent antibodycounterstain method. Appl. Microbiol. 13:171-174. Wilkinson, A. E. 1952. Occurrence of Neisseria other than the gonococcus in the genital tract. Br. J. Vener. Dis. 28:24-27.
