0095-1137/84/010063-05$02.00/0
Copyright ©1984, American Societyfor 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,2AND BARBARAH. MINSHEW3 Neisseria ReferenceLaboratory and Department of Medicine,Univ'ersity
of Washington, Seattle, Washington 981951;DepartmentofMedicine,
Tuifts-New
England Medical Center Hospital, Boston, Massachiusetts 021112; and ClinicalMicrobiology Laboratory, Department of Pathology, SeattlePlublicHealth 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 thecervix of apatient attending an arthritisclinic at Seattle Public Health Hospital, Seattle, Wash.
Thisstrain,NRL32165, did not producedetectableacid from glucose, maltose, sucrose, fructose,mannitol,
orlactose in eithercystineTrypticase agar (BBL Microbiology Systems, Cockeysville, Md.) or modified
oxidation-fermentation medium and was identified presumptively as aglucose-negative Neisseria
gonor-rhoeaestrain,but wasidentifiedlater asNeisseriacinerea on the basis of itsbiochemical reactions. Nitrate
was notreduced,nitrite(0.001%,wt/vol) wasreduced,andpolysaccharidewas notproduced fromsucrose.
Proline, arginine,andcystine-cysteine wererequired for growth on defined medium. Strain NRL 32165 did
not react with antigonococcal protein I monoclonal antibodies and did not produce immunoglobulin A
protease.InDNA:DNA homology studieswith N.gonorrhoeae NRL8038(F62) andN.cinerea type strain
NRL30003, strain NRL 32165 showed95%homologyrelative to N. cinereaand 44% homology relative to
N. gonorrhoeae. Thus, the identity of strain NRL 32165 was confirmed as N. cinerea (von Lingelsheim
1906)Murray 1939. Of allNeisseriaspp., N. cinerea is most likely to bemisidentifiedas aglucose-negative
N. gonorrhoeae strain.
Commensal
colistin-susceptible
Neisseria spp. andBran-hamellacatarrhalis areonlyrarelyisolatedon media
selec-tive for the pathogenic Neisseria spp., Neisseria
gonor-rhoeae andNeisseria meningitidis, andaredifferentiatedon
the basis of their cultural characteristics, distinctive sugar
utilization reactions, ability toreduce nitrate,and ability to
produce
polysaccharide
fromsucrose. Accordingto currentliterature(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
namedMicrococcus
cinereius.
Itwasdescribed 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 theepithet
cinereahad
priority
taxonomically. Since then,manystrains appear to have been isolated butincorrectly identifiedasB.catarrhalis(8, 11). Thesespecies differ biochemically onlyin
their ability to reduce nitrate, a test which was not
intro-ducedintotheclassification ofNeisseria spp. until1961(1).
B. catarrhalis strains reduce nitrate, whereas N. cinerea
strains do not. Bergerand Paepcke (3) "rediscovered" and
describedN. cinerea in 1962 andshowed thattherewas no
antigenic relatedness between N. cinerea and Neisseria
catarrhalis;the latter
species
wassubsequently reassigned
tothe genus Branhamella (5) in 1970. N. cinerea was not
described inBergey's Manual
of
DeterminativeBacteriolo-gy, 8thed. (33)ortheManual
of
ClinicalMicrobiology,
3rded.(23),butit will bedescribedin the9th edition of
Bergey's
Manual(N.A. Vedros,
personal
communication).
Early studies indicated thatN. cinerea strains colonized
the nasopharynx (2, 8, 11). Commensal Neisseria spp. and
*Correspondingauthor.
B. catarrhalis were isolated only rarely fromthe
genitouri-nary tract (12, 14, 36, 38). The prevalence ofN. cinereais
uncertain because early investigators identified N. cinerea
strains as a subtype ofB. catarrhalis (8, 11). Berger and
Paepcke (3) foundthat N. cinereastrains accounted forca.
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 wereidentifiedas N.
flavescens,
N.gonorrhoeae,
orB.catarrha-lis, butreevaluation ofthese
publications
(2) indicates thatthey wereprobably N. cinerea strains.
Inthisstudy,wedescribetheisolationon
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 whoseculturalcharacteristics
resembled those ofN.gonor-rhoeae. This strain was
subsequently
identified as N.cin-erea(von
Lingelsheim
1906) Murray1939(27).
Weevaluatedbiochemicaltestsandreference
techniques
whichwillaidinidentifying N. cinerea strains and
differentiating
them fromN.
gonorrhoeae.
MATERIALSAND METHODS
Isolation andmaintenance. An
asaccharolytic,
gram-nega-tive, oxidase-positive diplococcus was isolated on
Martin-Lewis medium (Prepared Media Laboratory,
Tualatin,
Oreg.) fromthecervix ofa
19-year-old
womansubsequently
diagnosedashaving
psoriatic
arthritis. The strain,designat-ed NRL 32165, wassubcultured onchocolateagarmedium.
Sincereceiptin theNeisseriaReference
Laboratory (NRL),
63
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TABLE 1. Summary ofdifferential characteristics for N. gonorrhoeae, asaccharolyticNeisseria spp.,and B.catarrhalisa
Acid 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 fromsu- cystine- IgApro- 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. +,Positiveforallstrains
tested; -, negativefor all strains tested. DataforN.gonorrhoeaearebasedonliterature citations(2,6, 23, 33).
the strain has been cultured on GC base medium (Difco
Laboratories, Detroit,
Mich.) plus
defined supplement(GCK) (37)at36°CinaCO2-enriched
atmosphere.
The strainwas stored in50%gammaglobulin-freehorse 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 asreferencestrains for DNA:DNA homology studies. Species used in
comparative studies to determine differential biochemical
characteristics arelisted in Table 1.Two N. cinerea strains
(NRL 32824andNRL32828)isolatedonsheep bloodagar in Seattle in 1981 werealsostudied. Inaddition, 10strains ofB. catarrhalis and the taxonomic type strain ofN.flavescens
(NRL 30009) were studied.
Colonymorphology. Observations of the colonial
morphol-ogy of strains were made on GCK medium, human and sheep blood agar, and chocolateagar. Strains were streaked
onto 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 agarmedium(BBLMicrobiology Systems,
Cock-eysville, Md.) for theirabilitytoproduce acidfromglucose,
maltose, lactose, and sucrose. Subsequent
confirmatory
tests were made inmodifiedoxidation-fermentationmedium
containingglucose, maltose, sucrose,fructose, mannitol, or
lactose (19). Modified oxidation-fermentation base medium
has thefollowing composition (per liter): 0.2% Difco
prote-asepeptone no. 3-0.5% NaCI-0.03%dipotassium hydrogen
phosphate-0.3%agar-0.5 ml of a0.17%phenol red solution.
Additional biochemical tests. Tests to detect nitrate and
nitrite reduction and polysaccharide production from
su-crose wereperformed as described previously (3, 19). Auxotyping. Strains were tested on chemically defined
medium (NEDA) as described previously (18).
Require-ments for proline, arginine, hypoxanthine, uracil,
methio-nine,andcystine-cysteine were determined. Type strains of
asaccharolytic species N. flavescens and B.
catarrhalis
were
compared with N. cinerea strains.
Antibioticsusceptibility tests. Susceptibility ofN. cinerea strains topenicillin,tetracycline, spectinomycin,
erythromy-cin, and vancomycin were determined as described previ-ously (18). Colistin susceptibility was determined by disk diffusion tests on GCK agarwith BBL disks (10
jig).
Serology. Serogrouping ofgonococciwasperformed using
thecoagglutination 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 boiled
organism
with one drop of a suspension of the reagentstaphylococci,rotating for 2min,andobserving the mixture
under 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 be used to identify gonococci since cross-reactions with other Neisseria spp. have not been observed (29).
Detectionof IgA protease.Immunoglobulin A(IgA) prote-ase was detected using sodium dodecyl
sulfate-polyacryl-amidegel electrophoresisas describedpreviously (20,25).
Isolationof DNA. DNA was isolated bythe procedure of Brenneretal. (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 ex-periments, whole-cell DNA from strain NRL 32165 was labeled to a specific activity of 2 x 106cpm/Lgof DNA by
the nick translation technique of Maniatis et al. (21) with
[3H]deoxyribosylthymine triphosphate (77 Ci/mM; New
En-gland 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 to500 base pairs.
Whole-cell DNA:DNA homology studies. Whole-cell DNA was hybridized overnight, and homology was analyzed by thesingle-strand endonuclease procedure of Crosa et al. (7) asmodifiedby Piotet al. (30).Homologieswere determined afterhybridization at 65°C overnight in 0.42 MNaCl, which were optimal conditions for hybridization, i.e., 25 to 30°C
belowthemelting point ofthe probe DNA.
RESULTS
An asaccharolytic, gram-negative, oxidase-positive
diplo-coccus was isolated on Martin-Lewis medium from the cervix ofa patient attendingan arthritis clinic. This strain, NRL32165, was tested in the clinical laboratory for its sugar
utilization pattern in cystine Trypticase agar and failed to
produce acid fromglucose,maltose, sucrose, or lactose. The strain was also resistant to erythromycin by disk diffusion
testingand was studied further.
Colonialmorphology. N. cinerea colonies on GCK
medi-umincubatedat36°C for 24 h weregold-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
coloniesofN.gonorrhoeae) and weretranslucent and easily
emulsified in broth. Cellsshowed the diplococcal morpholo-gy, in whichcells hadadjacentsides flattened and varied in
size. Pairs or clumps predominated over individual
diplo-cocci. Giant cells were sometimesseenin 48-hcultures(2).
Coloniesonsheepand humanbloodagar and chocolateagar
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were grey-white. Cell and colonial morphology conformed with the description of N. cinerea (3).
Biochemical characteristics. The biochemical characteris-tics 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 con-taining 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 require-ment 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 re-quired 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 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), 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. Resist-ance to erythromycin was higher than normally found forN. gonorrhoeae strains (13, 28). Colistin disk susceptibility
tests with N. cinerea strains showed that zonesofinhibition were :10 mm in diameter with anunstandardized 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 find-ings, the strains of N. cinerea examined in this study failed to produce IgA protease, thus providing anadditional refer-ence test which can aid in their identification.
DNA:DNA homologystudies. Homology studieswere per-formed 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 (calfthymus) DNA averaged
75 and 7%, respectively. Although thetypestrain N. cinerea
NRL 30003 was tested, we did not use it as the reference
strain 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 were concerned that the presence ofthisplasmidmight affect the homology results measurably. Results ofhomology studies are summarized below; each value istheaverageof four or five determinations. The type strain NRL30003 and strain NRL 32824 showed95and 94%relative homology, respec-tively, with strain NRL 32165; strain NRL 32828 showed
81% homology relative to strain NRL32165. The
compara-tively low value of 81% relative homology between strains
NRL 32828and NRL 32165compared withhigher values for
theother strains might be accounted for inpart
by
the3.2-Mdal plasmidcarried by strain NRL 32828.If thisplasmidis presentin 20 to 30copies percell, it wouldaccountforca.
6% ofthe DNA in this strain (22). In contrast, N.
gonor-rhoeae 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(vonLingelsheim 1906) Murray 1939.
DISCUSSION
A strain of N. cinerea (von
Lingelsheim 1906)
Murray
1939 wasisolatedfromaclinical
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 UnitedStates,
although it appears that strains of this
species
have beenisolated from clinical
specimens
in the United States andEurope on severaloccasions but
incorrectly
identifiedasB. catarrhalis (14),N.flavescens (31),N.gonorrhoeae
(32),
orN. pseudocatarrhalis (Huntoon, Abstr. Annu. Meet. Am.
Soc. Bacteriol. 1934).
Previoushomology studieshave shownthat strains
classi-fiedin thesame
species
show70%or morerelative homolo-gyunderoptimal conditions(4). DNA:DNAhomology
stud-ies by different methods have
reported
different relativehomology values for
interspecific
reactionsbetween strainsrepresentative ofNeisseria spp. Hoke andVedros
(15)
usedthe thermal renaturation
technique
andreported
60%rela-tive 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 relativehomology
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 nottested in that
study.
Although
no DNA:DNAhomology
studiesby theS1 endonuclease
procedure
have beenreport-edpreviously betweenN.
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
relatedtoN.gonorrhoeae
infatty acid
composition
rather than to the other commensalNeisseria spp. and B. catarrhalis has been
reported
previ-ously (16, 17).
Theisolation ofN. cinerea
emphasized
weaknesses inonereference test used to confirm the identification of
gono-cocci. Catlin(6) noted that
although
all strains ofN.gonor-rhoeaerequired
cystine-cysteine
forgrowth
onNEDAmedi-um, more than90% ofN.
meningitidis
strains did not, andsome N. lactamica strains
required
cystine-cysteine,
where-asothers didnot. Ourobservationsinauxotyping
N.gonor-rhoeae,N.
meningitidis,
andN. lactamica strains since 1973haveconfirmed thoseofCatlin. In
addition,
in thisstudy
wefound thatN. cinerea andN.
flavescens
strainsrequired
not onlycystine-cysteine
forgrowth
on NEDAmedium,
but N. cinerea strains alsorequired
proline
andarginine
(Pro-Arg-)forgrowth,thus
contributing
furthertoconfusion withgonococci.
Consequently,
therequirement
forcystine-cyste-ine cannot be used to differentiate between N. cinerea and
N. gonorrhoeae.
Although in
coagglutination
tests allgonococci
reactedwith at least 1 of 10 research
antigonococcal
protein
I monoclonal antibodies(29, 34),
no cross-reactions wereon April 10, 2020 by guest
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observed with other Neisser-ia spp., including N. (inerea strains and B. (at(lrrhalis. However, because commercial serological diagnostictestsforN.
gonorrhoeae
donot detect allstrains (10;J. P. Libonati,R. L. Leilich, and L. Loomis. Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C19, p.314), commercial diagnostic reagents are oflimited 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. cinerea
strains to grow on simple media such as tryptic soy agar (Prepared Media Laboratory)orMueller-Hintonagar(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 tocolistin (10-pLg disk; BBL) but resistant
toerythromycin and of intermediate resistance topenicillin. Although N. cinerea appears to be an extremely rare isolate fromgenital 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, andE. W. Hook
III, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C26, p. 316). The presence of N. cinerca in these sites poses two
areasofpotential confusion for clinical microbiologists iden-tifyingNeisseriaspp.First,since N. cinerea coloniesclosely
resemble N. gonorrhoeae colonies, these species may be confusedwhenacid productionfromglucose is weak. Thus, N. cinerea strains may be misidentified as N. gonorrhoeae when isolated on selective media oron nonselective media often used nowto detect vancomycin-susceptible
gonococ-cal strains. Second,N. cinerea strainsmaybeconfusedwith
B. catarrhalis strains when isolated from pharyngeal speci-mens onnonselective media employedtoisolate Haemophi-lus influenzae. Since B. catarrhalis is being increasingly implicatedas arespiratory pathogen, it is important that the
etiological agents in theseinfections be identified correctly. AlthoughN. cinereacolony morphology is distinctfrom that
ofB.catarrhalis strains,N. ciner-eastrainswereconsistently misidentified as aB. 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 withN.
flai'escens
(31) resulted because the strains exhibit similar colony morphologies and also because pro-duction ofpolysaccharide from sucrose was not introduced into Neisseria spp. classification until 1961.SinceN. (cinerea isnotlistedamonghumanNeisseriaspp.
incurrentreference texts, asummary of differential
charac-teristics useful for differentiating between asaccharolytic species of N. (inerea, N. flaves(cens, B. caitarrhalis, and weakacid-producing N. gonorrhoeae strains is provided in Table 1. Unfortunately, DNA:DNA homology studies
pro-videthe onlyconfirmatorytestavailablecurrentlytoidentify N. cinerea. Tests used in clinical laboratories permit the
identification ofN. cinerea onlyby the elimination ofother
currently recognized Neisseria spp. or B. catarrhalis. In
viewof thelegal and scientific implications of misidentifying N. (cinerea strainsas N. gonorrhoeae orB. catallhalis, we
wish toalert clinical microbiologists to the existence ofN.
(inerea and to advise caution when identifying apparently
glucose-negative, gram-negative, oxidase-positive
diplo-cocci.
ACKNOWLEDGMENT
This researchwas supported by Public Health Servicegrant
Al-12191fromthe National Institutes of Health.
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