Neisseria gonorrhoeae strains inhibited by vancomycin in selective media and correlation with auxotype

JOURNAL OF CLINICAL MICROBIOLOGY, July1981,p.94-99 0095-1137/81/070094-06$02.00/0

Neisseria gonorrhoeae

Strains

Inhibited

by

Vancomycin

in

Selective

Media and

Correlation

with Auxotype

STANLEYMIRRETT,1* L. BARTHRELLER,' AND JOAN S.KNAPP2

Departmentof Medicine, Clinical Microbiology Laboratory,DivisionofInfectiousDiseases, Universityof

Colorado HealthSciences Center, Denver,Colorado80262,1andDepartmentofMedicine, U.S.Public

HealthService Hospital,Seattle, Washington981142

Received17February1981/Accepted24April1981

StrainsofNeisseria gonorrhoeaethat failed to grow onThayer-Martin (T-M) andMartin-Lewis (M-L) media accounted for 2.0% of isolates at theUniversity ofColoradoHospital and itsVenerealDisease Clinic. Atotal of 31 inhibited and 31control strainswerecomparedby agar dilution testing for theirsusceptibilities

to13antimicrobialagentsused for treatmentorin selective media. All 62isolates

were resistant to lincomycin, colistin, nystatin, amphotericin B, trimethoprim

lactate,polymyxin B,and anisomycin. Vancomycin was the inhibitoryantibiotic

for N. gonorrhoeae in both T-M and M-L media. The vancomycin-inhibited strains were also significantly more sensitive to penicillin and ampicillin than were the control strains (P < 0.001). The presence of the other antibiotics in selective media didnotaffect the minimum inhibitory concentrations of

vanco-mycinforgonococci.All31inhibited strains weresensitive to 8.0

tig

ofvancomycin

perml,and26of theseweresensitiveto 2.0

ytg/ml.

Decreasing the size ofinoculum ofgonococci results in greater inhibition by any given concentration of

vanco-mycin.Thevancomycin-sensitive strainscontainedsignificantly more arginine-,

hypoxanthine-,anduracil-requiringauxotypes (28 out of 31) than did the control strains (9 out of 31). As with T-M medium, some strains of gonococci will be missed when M-L medium with4.0jigof vancomycin per ml is the only medium

usedfor thediagnosisofgonorrhea.This may be of particular importance in the

confirmation of disseminated infection with Arg- Hyx- Ura- auxotypes of N.

gonorrhoeae

when cultures ofblood, joint fluid,orskin lesions arenegative.

Theintroductionofaselective antibiotic-con-taining medium

capable

of

suppressing

theusual

flora present in theanalcanal, vagina, pharynx,

and other sites enabled improved isolation of

Neisseria gonorrhoeae.Theantibiotics used

ini-tially, ristocetinand

polymyxin

B,werereplaced

by vancomycin, colistimethate, and nystatin 2

yearslater (30). Subsequently,other

investiga-tors triedvarious antibioticcombinations in an

attempttofindamedium with minimal inhibi-tion of N.

gonorrhoeae

and maximum

suppres-sion ofcontaminatingflora (3, 6,22). Since 1970,

theaddition oftrimethoprim toThayer-Martin (T-M) medium to suppress swarming Proteus

spp. has gained widespread acceptance (19, 24, 26).

Reports of inhibition of N. gonorrhoeae by

selective media appear throughout the litera-ture, and inhibition caused

specifically

by van-comycin,trimethoprim,

lincomycin,

amphoteri-cin, andhigh concentrationsof

nystatin

hasbeen

documented (2, 5, 11, 23, 28). Recently, Martin and Lewis (16) suggested replacing nystatin,

which has beenreported to decrease the shelf life of selective media due to its instability (3), withanisomycin,anewand morestable

antifun-gal agent (15). Moreover, thevancomycin

con-tentin the currentformulation ofMartin-Lewis (M-L) medium has been increased from 3 to 4

ygto provide improved inhibition of gram-posi-tive cocci.

The routine use in ourlaboratory ofchocolate

agarand T-M medium or, more recently, M-L medium for isolation ofgonococci from nonster-ilesites enabled us to collect strains of N. gon-orrhoeae that grewonlyonchocolate agareven

after subculture. The objectives of this study were (i) to determine the proportion of strains of N.gonorrhoeaethat were sensitive to either

T-M or M-Lmedium, (ii) to determine, for

in-hibitedstrains andnoninhibited control strains, the minimalinhibitoryconcentrations(MICs)of anisomycin and other antibiotics used in selec-tive media or for therapy, (iii) to determine

I whether the inhibitory effect of T-M and M-L

media isinoculum dependent, and (iv) to com-94

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pare theauxotypes of the inhibited and control strains.

(This paperwaspresented in partatthe79th Annual Meeting of the American Society for Microbiology,LosAngeles,Calif.[S.Mirrettand L. B. Reller, Abstr. Annu. Meet. Am. Soc. Mi-crobiol. 1979,C43,p.41].)

MATERIALS AND METHODS

Bacterialstrains.Allspecimenssubmittedtothe

Clinical Microbiology Laboratory and the Venereal

Disease ClinicattheUniversityofColoradoHospital

duringa14-monthperiod in1976and1977for culture

of N.gonorrhoeaewerestudied. Specimenswere

in-oculated routinely by the house staffonto abiplate

containing chocolate agar on oneside and T-M

me-dium or,morerecently,M-L mediumontheotherside

(Pasco Laboratories, Denver, Colo.). T-M medium

contained thefollowing:vancomycin,3,ug/ml;colistin,

7.5

jtg/ml;

nystatin, 12.5 U/ml; and trimethoprim, 5

ug/ml. M-Lmedium contained thefollowing:

vanco-mycin,4,tg/ml;colistin,7.5,ug/ml;anisomycin,20

,g/

ml; andtrimethoprim,5ug/ml.Beforerelease for use,

all mediaweretestedasrecommended fortheirability

tosupportgrowthofgonococciandtoinhibitthe usual

flora (1). Primary isolates that grewonchocolate agar

butfailedtogrowontheselective mediawere

subcul-tured from chocolate agar to anadditional plate of

selective and nonselective media. Only isolates that

again failed to grow on the selective medium were

included inthisstudy. A heavy suspension froman

18-to24-hsubculture of thestudyisolateswasprepared

in amixturecontaining50%ofetal calfserumand 50%

Trypticase soy broth (BBL Microbiology Systems,

Cockeysville,Md.)with 1%yeastextractand frozenat

-70°C until needed. No more than oneisolate per

patientwasselected.

Eachtimeaninhibited strainwasisolated,another

gonococcal isolate from the clinical laboratory that

was notinhibitedby theselective medium wasalso

savedas acontrol strain.Controlswerehandled in the

same manner as the inhibited isolates. All isolates

wereconfirmedbycarbohydratedegradationtests(17)

and bythe fluorescentantibody technique (21) with

conjugate obtainedcommercially (Difco Laboratories,

Detroit, Mich.) as well as with conjugate obtained

from the Centers for Disease Control, Atlanta, Ga.

Control strains of Candidaalbicans,Escherichia coli

(ATCC25922),Staphylococcusaureus(ATCC25923),

and Proteus mirabiliswereusedthroughoutthestudy

asnecessarytodetermineappropriateactivityof the

antibiotics in themedia.

Agar dilution testing. (i) Medium A(GC

me-diumbase).MICs ofall antibioticsweredetermined

in GC medium base(DifcoLaboratories) agar

contain-ing 1%hemoglobin (Difco) and 1%IsoVitaleX (BBL

MicrobiologySystems)byamodificationofthe

tech-nique of Ronaldetal.(25). Serial twofold dilutionsof

thefreshlythawed antibioticwerepreparedindistilled

water and dispensed into individual plastic petri

dishes.Molten (55°C) chocolateagar(20ml/100-mm

plate) wasadded to each plate and mixed with the

antibiotic beforetheplateswere allowedtosolidify.

Plates were left at room temperature overnight and inoculated the next day.

The frozen organisms were thawed slightly, subcul-tured to chocolate agar plates, and incubated in CO2 at 35°C overnight. Suspensions of the fresh isolates

were prepared inMueller-Hinton broth containing 1%

IsoVitaleX to equal the density of a 0.5 McFarland standard. Since preliminary experiments

demon-strated no difference between an inoculum size of103

to 104organisms as recommended by some

investiga-tors (10) andan inoculum size of104to 105 organisms

as recommended by Ronald et al. (25), the higher

inoculum size was selected for simplicity. Actual viable colony-forming units (CFU) in the inoculum were

found to be about 104 when determined by plate

counts. A multipoint inoculator was used to transfer the organisms to the plates before incubation in 35°C

in 5%CO2 for 18 to24h. The MIC wasdetermined as

thelowest concentration of drug yielding no growth or

abarely visible haze.

(ii) Medium B (T-M agar base). Agar dilution MICs of vancomycin were determined in the same

manner asdescribed above (medium A) with the

ad-dition of 7.5,ug colistimethate per ml, 12.5 U of nystatin

perml, and5

jg

oftrimethoprim per mltodetermine

whether synergy or antagonism to vancomycin could be demonstrated in the presence of the antibiotics contained in the selective media.

(iii) Medium C (M-L agar base). Agar dilution

MICs of vancomycin weredeterminedasdescribed for

medium B, but anisomycin(20,ug/ml)wassubstituted

for nystatintodetermine whetherconditions for

syn-ergy orantagonism to vancomycin couldbe

demon-stratedinM-Lmedium.

Antibiotics. All 13 antibiotic powders were

sup-plied by the manufacturers and stored inadesiccator

at 4°C until used. The powders were weighed,

dis-solved in appropriate solvents, and diluted in sterile

water to a final concentration of 2,560,Lg/ml. These

solutions were frozen at -70°C until used.

Inoculum studies. The complete M-Lbase

me-dium containingcolistimethate (7.5

Ag/ml),

anisomy-cin (20

tig/ml),

trimethoprim lactate (5,ug/ml), and various concentrations of vancomycin was used to

determine the effect of increasing inoculum size on

isolates which failed to grow on selective media. A

multipoint inoculator was used to inoculate 102, 103,

104, and 105organisms to the agar surface. A 0.01-ml

sterile, calibrated loop was used to apply106 organisms

to the plate, andapipettewas used toinoculate 107

organisms. Plates were read after24hofincubation in

5%CO2at35°C. Plates thatwereinoculated with100

organisms were reexamined at 48 h when the sparse colonies were larger and the MIC endpoints were more distinct.

Auxotyping. Auxotyping of gonococcal isolates wasdone asdescribed previously (12).

Statisticalmethods.ThemeanMIC valueswere

compared by using a two-sample ttest, a chi-square

analysis, orboth, where appropriate.

RESULTS

Atotal of31 inhibited strains that grewonly

on chocolate agar and 31 control strains of N.

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gonorrhoeaewerecollected from theUniversity Hospital and its Venereal Disease Clinic. The

inhibited strains from both clinics were all

iso-lated from genital sites (cervical, vaginal, and urethral), except foragastricaspirate fromone

neonateandfivespecimens of which thesource

was unknown. None of the isolates was from patients with disseminated gonococcal infection. Of483culturesof N.gonorrhoeae collected dur-ing a 14-monthperiod (1976 to 1977) from the

UniversityHospital,19(3.9%)wereinhibitedby T-M or M-L medium. In contrast, 12 of 1,074

(1.1%) isolates ofgonococci from the Venereal Disease Clinic during the same period were

in-hibited by the selective media. The combined

percentage of inhibited strains was 2%. The sourcesofthe strainsareshown inTable 1.

The geometric .mean MICs for the inhibited strains and the controlstrainstothe13

antimi-crobialstestedareshowninTable2.

Vancomy-cin appeared to be the only inhibitory

antimi-crobial agent in theselectivemedium and hada

geometric meanMIC of 1.6

jIg/ml

for N.

gon-orrhoeae (0.5to8.0

jig/mil),

comparedwith >32

jig/ml

for control strains (P < 0.001). Strains inhibitedby vancomycinwerealsosignificantly

moresensitive topenicillinandampicillin (P <

0.001) and were somewhat more sensitive to

erythromycin (P<0.05).Noneoftheantifungal agents,

including

anisomycin,

had any effecton thestrains tested.

When MICstovancomycinweredetermined

for theinhibited strainsin T-Mand M-Lmedia,

thegeometricmeanMICof 1.7jig of vancomycin

per ml was similarto the MIC in nonselective

media.Thecontrol strainsshowedadecreasein

geometricmeanMIC from>32

,ug/ml

in choco-lateagar to

28jig/ml

in theselectivemedia.

Table 3showsthe effectofinoculumsize on

TABLE 1. SourcesofN.gonorrhoeaeisolates

No.ofisolatesoffollowing

Source -strains:

Inhibiteda Control'

Cervix 14 19

Urethra 10 8

Throat 0 1

Rectum 0 1

Gastricaspirate 1 0

Vagina 1 2

Unspecified 5 0

aInhibited strains of N.gonorrhoeae were those

thatwereisolatedonlyonchocolateagarandnot on

T-M orM-L medium. Strainswereisolated from 15

male patientsand 15female patients; 1 patient'ssex

wasnotspecified.

bStrainswereisolatedfrom 10maleand 21 female

patients.

TABLE 2. GeometricmeanMICsforinhibited and

control strainsofN.gonorrhoeaeto 13

antimicrobialagents

MIC(,g/ml)for

follow-Antimicrobial ing strains: agent Inhibiteda Control

(n=31) (n=31)

Vancomycin 1.6 >32 <0.001

Penicillin 0.04 0.12 <0.001

Ampicillin 0.08 0.21 <0.001

Tetracycline 0.4 0.4 NSb

Spectinomycin 30 30 NS

Erythromycin 0.4 0.7 <0.05

Lincomycin 30.4 >32 NS

Trimethoprim >64 >64 NS

Colistin >64 >64 NS

PolymyxinB >64 >64 NS

Nystatin >32 >32 NS

AmphotericinB >32 >32 NS

Anisomycin >64 >64 NS

aStrains of N. gonorrhoeae thatwereisolatedonly

onchocolateagarandnot onT-MorM-L medium.

bNS,Notsignificant (P>0.05).

TABLE 3. Vancomycin-inhibitedstrainsof N.

gonorrhoeae(n=31)growingatdifferent inoculum

sizesatvariousconcentrations of vancomycin

No.ofstrains atfollowingvancomycin Inoculum concn(Ag/ml):

(log,0CFU)

0.25 0.5 1.0 2.0 3.0 4.0

2 23 15 7 5 3 1

3 31 21 9 5 5 4

4 31 27 14 6 6 5

5 31 31 17 7 6 5

6 31 31 25 13 10 8

7 31 31 29 24 17 12

vancomycin susceptibility; as the inoculum

in-creased, the number of inhibited strains

de-creased, regardless of the initial vancomycin

concentration. For example, at 3 jig of vanco-mycinperml (theconcentrationofvancomycin

in T-M medium),only5 outof31 (16%) ofthe

strainsgrewwith aninoculumsize of 103

CFU,

whereas 17 outof31 (53%) of the strains grew

with an inoculum of

107

CFU/ml. With an

in-crease of vancomycin to 4 jig/ml, as recently

recommended by workers at the Centers for

Disease Control (16), only12 outof31 (38%) of the sensitive strainswere capable of growth at

the same high inoculum size. The decreased

number of strains growingat4jigofvancomycin

permlwasnotstatisticallysignificant (P<0.1)

when compared with the number of strains

growingat3jigof vancomycinperml,regardless

of the inoculum concentration. In contrast, the decreased growthat3 or 4 jigofvancomycinper

ml was statistically significant at all inoculum

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VANCOMYCIN-SENSITIVE N. GONORRHOEAE 97

concentrationswhencompared with the growth at 0.25 ,ugofvancomycin perml.

Table 4 shows the distribution of auxotypes among theinhibited and control strains. There weresignificantlymore arginine, hypoxanthine, and uracil (Arg- Hyx- Ura-) auxotypes in the inhibited group(28outof31) than in the control group (9 out of 31) (P < 0.001). Forty-eight

percent of the strains from male patients and sixty-seven percent of the strains from female patients were of the Arg- Hyx- Ura- auxotype. Table5shows that the distribution ofArg- Hyx-Ura-auxotypes of N.gonorrhoeae byraceand

sex was not significantly different between the

inhibited and control groups. Table 6compares the geometric mean MICs of antibiotics active against gonococci by Arg- Hyx- Ura- or

non-Arg- Hyx- Ura- auxotypes. The Arg-

Hyx-Ura- auxotypesweregenerallymoresensitiveto

vancomycin, penicillin, ampicillin, and

tetracy-cline. All of the Arg- Hyx- Ura- strains were

sensitiveto0.25jigofpenicillinpermlorless.

TABLE 4. AuxotypesofN.gonorrhoeaein the

vancomycin-inhibitedandvancomycin-resistant

groups

No. of isolates offollowing

Auxotypea strains:

Inhibited Control

Arg-Hyx- Ura- 28 9

Pro- 1 8

Pro- Arg- 1 0

Pro- Arg- Ura- 1 0

Prototrophic 0 11

Arg- 0 2

Undetermined 0 1

aArg-,Requiresarginineforgrowth;Hyx-, requires

hypoxanthine for growth; Ura-, requires uracil for

growth; Pro-,requiresproline forgrowth;and

proto-trophic,nogrowth requirement.

TABLE 5. DistributionofArg- Hyx- Ura-auxotypesofN.gonorrhoeaebyraceandsex

No. of Arg-Hyx-Ura- iso-Patient lates(%) of following strains: p

Inhibiteda Controlb

Whitemale 5(17.9) 0(0) NSc

Black male 5(17.9) 0(0) NS

Whitefemale 14(50.0) 5 (55.6) NS

Black female 2(7.1) 3 (33.3) NS

Unknown 2 (7.1) 1 (11.1) NS

a Total, 28.

bTotal,9.

'NS,Notsignificant (P- 0.1).

TABLE 6. GeometricmeanMICs of antibioticsfor

N. gonorrhoeae by auxotype

MIC(,ug/ml)

Antimicrobial Ag Hyx- Non-Ag P agent Ura- Hyx-

Ura-(n=37) (n=25)

Vancomycin 3.8 42.2 <0.001

Penicillin 0.04 0.16 <0.001

Ampicillin 0.08 0.3 <0.001

Tetracycline 0.3 0.5 <0.01

Erythromycin 0.4 0.7 NS

aNS, Not significant (P>0.05).

DISCUSSION

Aselective medium for isolation of gonococci is recommended for routine use because of its suppression of contaminating flora and its ability tosupport thegrowth of N. gonorrhoeae (11, 28, 29). However, thevalue of selective media over nonselective media must be weighed against the loss of recovery of N. gonorrhoeae due to the susceptibility of some strains to the antibiotics in the medium. Many laboratoriesutilize only a selective medium to isolate N.gonorrhoeae, and, thus, detection of these sensitive strains is im-possible. Several studies have reported this phe-nomenon,with a frequency as high as30% (33). Inoculum size accounts in part for this effect as shown by our data (Table 3) and byTaylorand

Phillips (28), who found that eight samples of urethral pus would yield growth on selective mediacontaining2

jg

of vancomycin per mlonly with an undiluted inoculum. Lowe and Kraus (14) reported that the quantity of N. gonor-rhoeaerecovered from thevaginalsecretions of womenrangesfrom 4.0x

102

to 1.8 x 107CFU, witha geometric mean of 1.45 x 105 CFU. Our inoculum studies showed that even with an in-oculum of 105 CFU, only 20% of the sensitive strainswerelikely to grow at the recommended vancomycin concentrations in selective media.

The increased susceptibility of vancomycin-sensitive strains to penicillin demonstrated in

this study agrees with the work of Reyn and Bentzon (23), who also showed that vancomycin-sensitive strains are very vancomycin-sensitive to other

an-tibiotics, includingpenicillin.Some authors have also reported isolates that are susceptible to trimethoprim (28), amphotericin B (20, 32), or lincomycin (6). None of our isolates was suscep-tible to any of these antibiotics.

Based on the decreased in vitro recovery of gonococciat 4

jig/ml

versus 3

jIg/ml,

onemight expectfewer primary isolates of the vancomycin-sensitive strains on M-L medium, but the advan-tagesofisolating fewer contaminating flora may outweigh this potential disadvantage. Although

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98 MIRRETT, RELLER, AND KNAPP

ourstudy did not look at contaminating flora,

the work of others suggests that M-L medium is more likely to reduce the contaminating flora (18). Only additional prospective studies with clinical specimens, such as that described by Smeltzer et al. (27), would determine which se-lective medium isoptimal.

The increased sensitivityofArg- Hyx- Ura-strains ofgonococci overnon-Arg- Hyx-

Ura-strains to penicillin and ampicillin has been shown by others (13, 31). The fact that these strains are also very sensitive to vancomycin

and, thus, are less likely to grow on selective media, isofconcernwhen one istryingtoisolate these strains. It is likely that, in surveys to

determine the frequency of these Arg-

Hyx-Ura- auxotypes, theuseof

vancomycin-contain-ingmedia may select for non-Arg- Hyx- Ura-auxotypes. Moreover, the presumptive

bacteri-ological diagnosis of disseminated gonococcal infectionoftendependsonisolationofgonococci

from the endocervix, anal canal, or pharynx,

sinceculturesof blood andjointfluid are infre-quently positive(4, 8,9). Inasmuch as most cases ofdisseminatedgonococcal infectionsarecaused

by Arg- Hyx- Ura- auxotypes ofgonococci (7, 12, 13), bacteriological diagnosis ofgonococcal infection may be impaired by selective media

thatfailtogrow these auxotypes.

Theidealsolution, althoughnotpracticaland cost effective in many laboratories, would in-clude the use ofbothinhibitoryand

noninhibi-tory media. Analternative solution is for clinical microbiologistsand clinicians to be aware of the

problemand consider a nonselective medium for patients suspected of having gonorrhea but whose initial culturesare negative.It would be prudent to use both chocolate agar and M-L

medium for endocervical,

urethral,

pharyngeal,

andanal canal cultures frompatients with the

syndromeof disseminatedgonococcalinfection.

ACKNOWLEDGMENTS

WethankZaigaT.JohnsonandSandraL.Broersmafor technicalhelp and FranklynN.JudsonandKingK.Holmes fortheir reviewof themanuscript.

ThisstudywassupportedinpartbyNational Institutes of Healthresearch grantAl12192.

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