0095-1137/88/122544-05$02.00/0
Copyright © 1988,American Society for Microbiology
Selective Medium
for Branhamella catarrhalis with Acetazolamide
as a
Specific Inhibitor of
Neisseria
spp.
MARIO VANEECHOUTTE,* GERDA VERSCHRAEGEN, GEERT CLAEYS,AND
ANNE-MARIE VAN DENABEELE
DepartmentofMedicalMicrobiology, UniversityHospital, DePintelaan 185, B-9000Ghent, Belgium Received 21 June 1988/Accepted 1 September 1988
Several semiselective media for Branhamella catarrhalishave been proposed. Thesemedia allow growth of
all members of the family Neisseriaceae, and further differentiation is necessary. By addition of 10 ,g of
acetazolamide, a carbonicanhydrase inhibitor, per mland incubation in air, a medium wascreated which
reducedgrowthof Neisseriaspp.Whensaliva samples from 178healthy schoolchildrenwerescreenedfor the
presence ofB. catarrhalis, the carrier ratefor this organism was estimated tobe 48.9% with the selective
mediumcomparedwith12.4% whenasemiselective medium, which containsonly 10,ugofvancomycin, 5,ug
of trimethoprim, and2,ug of amphotericinBperml,wasused and6.2% whenanonselectivebloodagarplate
was used. The number of Neisseriaspp. isolated dropped from 297 on thesemiselective agar to55 on the
selectiveagar.
Branhamella catarrhalis is now generally considered an
importantpathogen indifferentdiseases. It isresponsiblefor an overall rate of 2 to 26% of the cases of respiratory diseases(8, 11, 23, 28, 29, 35) and wasfoundto bepresent in 22of40 casesof acutelaryngitis inotherwisehealthy adults in Sweden (27) andinacute maxillary sinusitis (6, 21). It is
alsoreported as thepathogenicagent in 6 to17%ofcultures
ofacute and chronic otitis media (19, 21, 22, 23). Several
reports of ophthalmia neonatorum (15), conjunctivitis (25), and keratitis (16) have been published. Finally, sporadic cases of urethritis (12), peritonitis (10), and systemic dis-eases such as meningitis, endocarditis, and septicemia (13) areknown.
Several authorshaveproposed media which allow growth
of members of the family Neisseriaceae and which can
differentiate between Neisseria spp. and B. catarrhalis. Jennison et al. (18) and Berger (3) used, respectively,
glu-cose and maltose in the presence of a pH indicator for differentiation. CorkillandMakin(9)proposedthe combina-tion of the antibiotics fromThayer-Martin medium(31) and DNase test agar, andSoto-Hernandezetal.(30)developeda
medium based on this idea. Van Hare et al. (33) used a
semiselective broth to study nasopharyngeal samples in acute otitis mediapatients. This paperdescribes the
devel-opment of afully selective mediumforB. catarrhalisbased on the addition ofacetazolamide
(2-acetylamino-1,3,4-thio-diazol-5-sulfonamide), a synthetic sulfonamide (4, 14, 26),
which inhibits thegrowth ofNeisseria spp. Theuse ofthis
medium in a screening study of healthy schoolchildren is evaluated.
(Thiswork waspresentedin part at the 28th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
LosAngeles, Calif., 1988.)
MATERIALSANDMETHODS
Organisms. B. catarrhalis strains were obtained from E. Falsen, Culture Collection of the University ofGoteborg,
Goteborg, Sweden; E. Van Dyck, Institute for Tropical
Medicine, Antwerp, Belgium;B. Davies,De Wever
Zieken-huis, Heerlen, TheNetherlands;M. A. Calder, City
Hospi-*Corresponding author.
tal, Edinburgh, Scotland; U. Berger, University of
Heidel-berg, Heidelberg, Federal Republic of Germany; T.
Nagatake, Institute for Tropical Medicine, Nagasaki,Japan;
I.Eliasson, Universityof Lund, Lund, Sweden; C. Gilquin, Institute for HygieneandEpidemiology, Brussels, Belgium;
M. Piessens-Denef, O. L. V. Ziekenhuis, Mechelen, Bel-gium; and fromourownlaboratory.
B.catarrhalis (ATCC 25238),B. caviae(ATCC 14659), B.
cuniculi (ATCC 14688), and B. ovis (ATCC 19575) type
strains and Neisseria spp. were kindly supplied by U. Berger,C. Gilquîn,andE. VanDyck. Othermembersofthe
Neisseriaceae were isolated from clinical samples sent to ourlaboratory. These strainswere usedtodetermine MICs oftheantibiotics uspd in the mediaand tofind the optimal combinations ofbasic media, antibiotics, andconditions of incubation.
Antibioticsusceptibility testing. MICs ofvancomycin, tri-methoprim,amphotericin B,andacetazolamidewere
deter-mined by the agar dilution technique with Trypticase soy agar(BBLMicrobiology Systems,Cockeysville, Md.),
sup-plemented with 5% human or ovine blood. For acetazola-mide, plates wereincubated in both airandaC02-enriched atmosphere.
P-Lactamase
productionwastestedwithnitro-cefin (Oxoid Ltd., Basingstoke, England).
Compositionofselectiveand semiselective media and incu-bation conditions. (i) Selective and semiselective agars. The
basic media tested were Trypticase soy agar and brucella
agar(BBL), whichweresupplementedwith5%
human
bloodor5% ovineblood. Antibioticsaddedwere 10 ,ug of
vanco-mycin (Vancocin HCI; Eli Lilly Benelux, Brussels,
Bel-gium), 5 ,ugoftrimethoprim(Wellcome Reagents,
Erembo-degem, Belgium), 2 ,ug ofamphotericin B (SigmaChemical Co., St. Louis, Mo.), and 10 ,ug of sodium acetazolamide (Diamox; Lederle, Etten-Leur, The Netherlands) per ml. When these mediaare incubated in air, Neisseria spp. are
inhibited, while incubation in 5% C02 neutralizesthe inhi-bition of carbonicanhydrase by acetazolamide, resultingin normal growth of Neisseria spp. Thus, according to the
incubation conditions, a selective or semiselective medium iscreated.
(ii)Selectiveandsemiselectivebroths. Brain heartinfusion broth(DifcoLaboratories,Detroit,Mich.)andtryptone soya 2544
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TABLE 1. Resultsofsusceptibilitytesting of the antibiotics used in selective medium
MIC(,ug/ml)"
Antibiotic Organism(s) tn Incubation in air Incubation
in 5% C0
Range 50% 905% (range)
Vancomycin B. catarrhalis 60 32-128 32 64
Trimethoprim B. (atarrlalis 60 32->64 64 >64
Amphotericin B B. catairhlalis 60 >32 >32 >32
Acetazolamide B. catbarrhalis 60 50->200 100 200 25-100
OtherNeisseriac eae
Acinetobacter 4 50->200 50->200
c(alco(aceticits
Branhamellacaviae 1 >200 >200
B. (Ufittic-iili 1 100 100
B. ovis 2 200 100
Moraxella osloensis 2 6 6->200
Neisseria canis 1 0.8 >200
N. cinerea 5 3-6 100->200
N.flavesc ens 4 1.5-3 50->200
N.gonorrhoeae 2 Ob 50-100
N. laatalnica 4 0-0.8 0.2-100
N. mneningitidis 13 0-0.8 0.2-100
N. ilrnosa 2 0.4-1.5 50->200
N.polysacc/harea 1 0 0.2
N. siecca 3 0.8-1.5 100->200
N.subflav'a subsp.petflava 3 0.4-0.8 100->200
N. subflava subsp. flava 1 0.4 >200
"50%and90%, MIC for 50 and90%of strainstested,respectively.
h Nogrowth when incubated in air.
broth (Oxoid Ltd.) to which 5 jtg ofvancomycin, 3 p.g of
trimethoprim, 2 Fg ofamphotericin B, and 10 or 15 p.g of
acetazolamide per ml were added were evaluatedas
selec-tive broths. Omission ofacetazolamide creates a
semiselec-tive broth, comparable to the one used by Van Hareet al. (33). Broths were incubated on a shaking platform in air.
After 18 h, the brothswere inoculatedontoblood agarwith
a 10-,tl calibrated loopand incubated in 5%CO,.
Study group. Saliva samplesfrom a groupof 178
school-children(653 yearolds, 84 7yearolds,and 29 12 yearolds)
were obtained inApril 1988 tocompare the selectivebroth
and agar with the semiselective broth and agar and with
nonselective blood agar plates. Paired saliva and throat
samples were taken from 75 children. The children were
asked to spit saliva into a sterile receptacle. One- to 3-ml portions were obtained. Throat swabs were takenfrom the
tonsils with sterile cotton swabs. A transport medium was
not used, since specimens wereinoculated within 4 h.
Isolation andidentification of membersof theNeisseriaceae.
All oxidase-positive colonies which were isolated on the
various media used to screen the study groups were Gram
stained. Gram-negative diplococci positive for oxidase,
4-methylumbelliferyl butyrate (32), and DNase and glucose
negative were identified as B. catarrhalis. Other gram-negative,oxidase-positive diplococciwereconsidered
Neis-seria spp., without further identification to specieslevel.
Statisticalanalysis.Statisticalanalysiswasdonebyx2with
Yates correction.
RESULTS
Susceptibility testing. Table 1 presents the results of the susceptibility testingofthevariousantibiotics. AllB.
catarr-halisstrainswerehighlyresistantto thefourantibiotics used
in the selective medium. All strains belonging tothe genus
Neisseria were susceptible to <10 p.gofacetazolamide per
mlwhen incubated in air. When the incubationatmosphere
wasenriched with carbon dioxide, most strains could grow at concentrations of100 ,ugof acetazolamideper ml.
Laboratory testingofselectiveagar.Different combinations ofagar base, bloodsupplement, and antibiotics were inocu-lated with 59 B. catarrhalis strains and 33 Neisseria sp.
strains. The addition ofacetazolamide reduced the number of Neisseria spp. able to grow from 30 (medium 2) to 1
(media 3, 4, and5)(Table 2). A combination of brucella agar
with 5% sheepbloodsupported the growth of allB. cattarrh-alis strains. Sheepblood was animportantfactor in enhanc-ing thegrowth of B. catarrhalis in air, as canbe concluded from the significant riseof viable cultures when Trypticase soy agarwith humanblood iscompared with thatcontaining sheep blood
(X2
= 13.55; P < 0.005).TABLE 2. Number of strains of B. catarrhalisand Neisseria spp. abletogrowondifferentcombinations of agar, blood
supplement,andantibiotics"
Mediumcomposition' No.of strains abletogrow Medium
no. Agar Blood Antibiotics Neisseriaspp. B. (atarrll(llis
base (n = 33)` (n= 59)
1 TSA H 31 59
2 TSA H VTA 30 59
3 TSA H VTA,AA 1 42
4 TSA S VTA,AA 1 56
5 BA H VTA,AA 1 51
6 BA S VTA,AA 2 59
`Incubation in air.
^TSA,Trypticasesoy agar;BA, brucellaagar;H.human blood;S.sheep
blood; VTA, 10 ,ug ofvancomycin, 5 ,ug oftrimethoprim, and 2 ,ug of
amphotericinBperml;AA,10 ,ug ofacetazolamide per ml.
`N. canis(1 strain),N. cinzerea(5 strains),N.flav'escens (4strains), N.
gonorrhoeae(2strains),N.lauctanica(4strains),N.ineningitidis(8strains),
N. inucosa (2strains), N.polysaccharea(1strain), N. sicca (3strains),N.
subfloia subsp.perflava (3strains).
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TABLE 3. Comparison of five media for selective recovery of B. catarrhalis from 178 saliva samples and 75 throat swabs obtainedfrom children (3 to 12 yearsold)
Samples andorganism(s) No. of isolates on: Total no. of
recovered Nonselective agar Selective agar Semiselectiveagar Selective broth Semiselective broth isolates Saliva (total)
B. catarrhalis il 87 22 43 1 90
Neisseria spp. NDa 55 297 167 253 297
Other ND 54 18 38 18 54
Saliva (from boys)
B. catarrhalis i1 35 12 15 0 38
Neisseria spp. ND 33 113 72 103 113
Other ND 23 7 18 7 25
Throat swabs (from boys)
B. catarrhalis 7 23 17 18 2 28
Neisseria spp. ND 5 109 27 89 109
Other ND 0 12 10 7 15
a ND, Not determined.
Laboratory testing ofselectivebroth. Testing ofthe
selec-tive broth combinations revealed that brain heart infusion
broth supported the growth of B. catarrhalis better than tryptone soyabroth. Thefinal combination contained brain
heart infusion broth and 5 ,ug of vancomycin, 3 ,ug of
trimethoprim, 2 ,ug of amphotericin B, and 15 ,ug of
aceta-zolamide perml.
Testing of selective media on saliva samples and throat swabs fromschoolchildren.Selective andsemiselective solid and liquid mediawereused forthe systematic screening of
healthy schoolchildren.For recovery of B. catarrhalisfrom
salivasamples and throat swabs,selectiveagar wasfoundto
be farsuperiortononselective and semiselective mediaand toselective broth(Table3).
Saliva samples. Of allB. catarrhalis strains isolated from saliva, 96.7%weredetectedon selectiveagar. Theisolation
efficiencywas raised bya factor of7.9 in comparison with nonselective agar and by afactor of4.0incomparisonwith
semiselective agar. Inonly25% of theselective plates with
growth ofB.catarrhaliswasthenumberof coloniescounted
<5, and such samplesweremostlyfrom children from whom
no B. catarrhalis could be detected on the other media.
Recovery from semiselective broth was minimal (1 of 90
isolations), while the selective broth was only half as
effi-cient asthe selective agar(43 of90isolations) butwas still
twiceas efficientas thesemiselective agar(22 isolations). Also, reduction of the number of Neisseriaspp. was more
importantontheselectiveagarthanontheother media.Not
onlywasthe numberof isolated strainsreduced,butalsothe
number of colonies per plate was low. Dense growth of
Neisseria spp., as seen on the other media, was never
observed.
Comparisonof throat swabs andsaliva samples. When all media weretaken intoaccount, B. catarrhalis was isolated
from 42 boys (56%) from whom both throat and saliva sampleswere tested. For 24 of the 42boys with apositive
culture, B. catarrhalis was isolated fromboth samples. In
four children, only the throat harbored this organism,
whereas 14additional isolations couldbe made from saliva
samples. This difference in number of isolations between throat and saliva issignificant
(X2
= 8.6;P < 0.005).Comparison ofcarriership, asestablished by different
au-thors. Table 4 presents the estimates of carrier rates for
adults and children as reported by different authors, using
selective and nonselective media. For adults, rates ranging from 0.6to6.8% (1, 2, 5, 20, 27) have been reported. The
estimatesfor childrenrange from18% (33) to56%(present
study). Althoughtheseestimates were mostlyobtainedwith nonselectivemedia,the percentages reported are rather high (18 to39%) comparedwith those (6.2 to9.3%)found in this study when only the nonselective agar was taken into account.
PI-Lactamase
production.
In94ofthe 178children(52.8%),
B. catarrhalis was isolatedfrom atleast one site. Foreachchild,oneisolatewastestedfor
P-lactamase
production,and 73 (77.7%) were found to be ,B-lactamase positive. This percentageof,-lactamase production is comparable(X2 = 1; P>0.1) to that(69.8%)found for a totalof83 strainsisolated inourlaboratory during 1987frompatients with respiratorytractdiseases.
DISCUSSION
Although carbonic anhydrase is rare in bacteria, it is
commonlyproduced byNeisseriaspp.(34),andalthoughits role is barely understood, it is vital for the growth and multiplication of these organisms (26). Sanders and Maren
(26) showed that this enzyme was absent in B. catarrhalis.
Acetazolamide, a syntheticsulfonamide, was foundto be
the mostpotent inhibitor of theenzyme(26). Theinhibition
of the enzyme, however, can be overcome by a carbon
dioxide-rich atmosphere (14).
ThoughBergerand Issi(4)havepointedtothepresenceof
carbonic anhydraseas an importantcharacteristicto
distin-guish the genera Moraxella (Branhamella) and Neisseria, thepossibility ofinhibitingNeisseria spp. on a medium for
the selective recovery of B. catarrhalis by the use of a
carbonic anhydrase inhibitor thus far has not been tried.
Onlytheantibioticsvancomycin,trimethoprim,and
ampho-tericin B, which are known not to inhibit Neisseria and Branhamella spp., have been used (20, 30, 33).
Thecombinationof thesefourantibioticswastested under
laboratory conditionsand resulted inanimportantreduction ofthe number of Neisseria spp. However, itwas observed thatTrypticase soy agar, supplemented with humanblood,
to which the antibioticswere added, could not supportthe
growth of 29% of the B. catarrhalis strains tested. This
probablywas notduetoantibioticinhibition,but ratherwas
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TABLE 4. Carrier rates forB. catarrhalis inhealthyadults and childrenasestimatedby differentauthors Reference Yr Geographicalorigin Isolation site Studygroup individuo.s cultures Remarks
studied
5 1962 FederalRepublic Throat Adults 172 0.6 Selective medium (3)
ofGermany
2 1973 Sweden Nose Adultpatients with noninfectious 100 4.0
diseases
27 1980 Sweden Nasopharynx Adults 103 3.9
1 1985 Scotland Pernasal swabs Staff members 59 6.8
20 1988 Washington Oropharynx Aduits 202 4.5 Semiselectivemedium
7 1981 Sweden Nasopharynx Children, 1-9yr 67 28.3
17 1982 Sweden Nasopharynx Children,0-7 yr 180 36.0
24 1984 Sweden Nasopharynx Children, 1-5yr 36 39.0 9.3cultures perchild
33 1987 Ohio Nasopharynx Children 18.0 Semiselective broth
Thisstudy 1988 Belgium Saliva Children,3-12yr 178 6.2 Nonselective agar
12.2 Semiselective agar 48.9 Selective agar
0.6 Semiselective broth 24.2 Selective broth
Throat Boys, 7-12 yr 75 9.3 Nonselective agar
22.7 Semiselective agar 30.7 Selective agar
2.7 Semiselective broth 24.0 Selective broth
Throatand Boys,7-12 yr 75 56.0 Nonselective agar,
saliva semiselective agar,
selectiveagar
relatedto theincubationinair, whichisnecessaryto obtain
growth inhibition ofNeisseriaspp. Itisgenerally knownthat
organisms from the upper respiratory tract are cultured easier in a carbon dioxide-rich atmosphere. Replacing hu-manblood withovine blood neutralized greatly thenegative effect of the reduced atmospheric C02 content, as the
numberof strainsgrowingonthisversion ofthemediumwas
comparable to that on a control blood agar plate. Still, coloniesgrewbetterwhenthe mediumwasincubated in5%
C02.
To evaluate the suitability of the selective medium, we
compared it with a semiselective medium, which supports
growth of bothNeisseria spp. and Branhamella spp., in a
screening of saliva samples and throat swabs from healthy schoolchildren. The solid selective medium provedtobefar superior indetectingB.catarrhalisstrains thantheselective
broth andsemiselective andnonselective media.
The increasedisolation ratio ofB.catarrhalisfrom saliva by use ofamedium containing acetazolamide seems to be correlated with the reduction ofgrowth ofNeisseria spp.
This can bededuced from the decline ofNeisseria spp. on
theselectiveagarby81%incomparisonwith the
semiselec-tiveagar,which corresponds to a72%rise in B. catarrhalis isolations. Also,theassessmentthat asolid mediumis more
suitable than a liquid medium points to overgrowth as the
responsiblefactor for the reduced number ofB.catarrhalis
strains on semiselective and nonselective media. Different
media have beenproposedfor the selective recovery of B. catarrhalis (3, 18, 30, 33). Differentiation directlyon
isola-tion medium, asestablished bydifferent authors(3, 18, 30),
is notnecessarywiththe mediumdescribed here, sincethe
colony morphology of B. catarrhalis is very typical,
espe-ciallywhensheep blood isaddedtothe medium: the colonies are sliding, coherent, and butter colored. Moreover, the results ofthis study indicate that allowing growth of Neis-seria spp. caninhibit growth of B. catarrhalis; thus, direct
differentiation alone is not sufficient to ensure a good esti-mate of the carrierrate.
When throat swabs and saliva sampleswerecomparedfor 75children, the samplingofsaliva, which revealed 38 of 42
detected isolates, gave a better estimate of the general carrierrate ofB. catarrhalis. Thisfinding isprobably dueto the apparent differences in the sampling of both sites, resulting indifferent inocula.
Thedifferent carrierratesfor adults and childrenmay be
explained by differences in immunity. Leinonen et al. (22)
showed how immunity against B. catarrhalis is builtup in
young children.
The current overall rate of
P-lactamase
production by strains of B. catarrhalis may be estimated at 75% (35). Previous reports dealt with hospitalized patients, patients withahistory ofrecurrent respiratory diseases, orchildrenwith otitis media, whichare all groups for which antibiotic
therapy iscustomary.Thus, itwasbelievedthatthehighrate of
P-lactamase-producing
strains was related to the use of1-lactam
antibiotics in the treatment. Thehigh
percentagefor healthy children, assessedinthis study, is unexpected,
however. Previousantibiotic usebythese childrenwas not
investigated.
It can be concluded that the increased isolation of B.
catarrhalis onthismedium,when saliva andthroatsamples
areunderstudy, indicatesthatsuppressionofNeisseria spp. is necessary whenabetter estimate of the carrierrateofB.
catarrhalisis desired. Thefindingthatsampling saliva gives
abetterestimate ofthecarriership than swabbing throats is interesting because of the quicker and easier way these
samples canbecollected.
Severalpossibilities exist forthe useofaselective medium forB. catarrhalis. Studiesofseasonalvariations, asnoticed byothers (22, 23, 33), and agedistribution andexamination
andcomparison ofdifferent sitessuch as thenasopharynx, throat, oropharynx (saliva), conjunctivae, and urogenital
tract are possible, especially because large series can be screened in arapid,nonlaboriousway. Its use in the study of sputa is underevaluation.
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ACKNOWLEDGMENTS
This work was supported by grant 820286 of the Instituut ter Bevordering van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.
Wethank Jeannette Calle, Noella Paepe, and Remi Van der Sypt for technical assistance and the directorates of the St. Lievens Instituut and the St. JozefInstituut in Ghent, Belgium, for their willingcooperation.
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