Copyright © 1986, American Society for Microbiology
Susceptibility Testing
of Slowly Growing Mycobacteria
by
a
Microdilution
MIC Method with
7H9
Broth
RICHARD J. WALLACE, JR.,* DONALDR. NASH, LORRAINE C. STEELE, AND VINCENTSTEINGRUBE
DepartmentofMicrobiology, The University ofTexasHealth CenteratTyler, Tyler, Texas 75710 Received 28 May 1986/Accepted 2 September1986
Basedonprevioussuccesswith rapidly growing mycobacteria,amicrodilution MICsystemwasdevisedfor
slowly growing mycobacterial species using 7H9 broth. Test drugs included isoniazid, rifampin, ethambutol,
streptomycin, clofazamine, and sulfamethoxazole. Sixty isolates of four mycobacterial species, including
Mycobacterium tuberculosis, frompatients who hadneverreceived drug therapywereevaluated in thesystem,
aswell as25drug-resistant isolatesand 11 control strains. MICswereread when good macroscopic control
growth wasevident,aperiodwhich varied with each species. Most species exhibiteda narrow rangeofMICs witheasilydiscernible growth endpoints.Theaminoglycosides,ethambutol, clofazamine, and sulfamethoxazole weretheonly drugswith activity against all speciesatclinically achievable levels inserum.Correlationbetween
susceptibilities by theproportion method inagarwith single drug concentrations and the broth methodwere
excellentforM. tuberculosis, M. kansasii, and M. marinum for isoniazid, rifampin, and ethambutol. Isolates of theM. avium complex were much more susceptible in broth than in agarfor rifampin, ethambutol, and streptomycin. Given the successful transition of most microbiology laboratories to MIC plates for other
bacterial species, thismethodwouldallowfortestingofmultiple drugsatmultiple concentrations and has good potential for evaluation of drug combinations anddrug-resistant isolates.
Currentmycobacterial susceptibilitytesting in theUnited
Statesuses theproportion method, whereby the numberof
mycobacterial colonies growing on 7H10 agarcontaining a
fixed concentrationof drug is compared with the number of
colonies growing on control agar without drug. If the
pro-portion ofresistantcolonies isless than or equal to1%,the
organism is considered susceptible (2, 8). The system was devised for Mycobacterium tuberculosisand was based on thepremisethatall strainsof tubercle bacilli, including wild
strains, contain some mutants resistant to the test drug. A
proportion of resistant colonies above 1% defined drugs
which were unlikely to be effective in therapy, whereas
ratios of
.1S%
identified drugs which would be successful against that specific organism. Although clinical data to supportthisbreakpoint(asopposedto10% resistance, e.g.) have notbeen presented, the system has provenuseful.The major advantages ofthe system arethat susceptibili-ties canbe tested directly, the method avoids any problem withtheinoculumsize effect(sinceonly tests with 100 to 300
coloniesoncontrolplatesareused),andan
organism
control isprovidedwith eachtest.Problems with the systemarethat agardilution is cumbersomefortestingofmultiple drugsatmultiple concentrations, and evaluation ofmultiple drugsin combination at various concentrations involves a
mind-boggling number ofplates. Current single-drug concentra-tions used in
proportion-method
work were chosen forM. tuberculosis and accurately separate wild strains from posttreatmentisolates butoften failtoprovide
thisinforma-tion forothermycobacterial species.
Recently, some laborato
ies
have begun to use theBACTEC broth susceptibility method, a method that in-volves measurement oftheproduction ofradiolabeled CO2 bymycobacteria growingin brothcontainingaradiolabeled fatty acid as a substrate (6, 7, 11). The advantages ofthis
*Correspondingauthor.
system are that many mycobacteria, including M.
tubercu-losis,grow morerapidly in broth, and the system allowsfor
early detection of growth; thus, susceptibility results are
obtained much earlier. However, the BACTEC machine is
expensive, and testing of more than one drug at multiple concentrations is time consuming and costly. The system has proven effective for testing M. tuberculosis but not
nontuberculous mycobacteria.
AbrothmicrodilutionMIC method has been describedby
Swenson et al. (10) forrapidly growingmycobacteria. This system has proved very reliable, reproducible, and easily quality controlled. A recent treatment study of more than 100patients,inwhich thesesusceptibilityresultswereused,
showed the methodtoprovide good
predictability
of clinicalresponseto specific drug therapy(12).
Thus, we electedto evaluate a similar systemfor slowly growing mycobacteria by using 7H9 broth instead of the
cation-supplemented
Mueller-Hintonbroth (MHB)used fortesting rapidlygrowing mycobacteria (10).
MATERIALS AND METHODS
MIC plates were prepared with 96-well microdilution
plates and the Mini Quick Spense II (Bellco Glass, Inc., Vineland, N.J.) system. Antimicrobial agents were
dis-solved, twofold dilutions of thedrugsin 7H9 broth
(pH
6.8)
weremade, and 0.1-ml volumesweredispensedinto wells of
microdilutionplates (5).Theantimicrobial agents tested
(and
their concentrations) included isoniazid
(INH;
0.25 to 32pjg/ml),
rifampin (RIF;0.125to16,ug/ml),
ethambutol(EMB;
0.5 to 64
,ug/ml),
streptomycin (STR;
0.5 to 64,ug/ml),
gentamicin(GEN;0.5to64
p.g/ml),
amikacin(AMI;
0.5to64,ug/ml), kanamycin (KAN;0.5 to 64
,ug/ml),
cefoxitin(2
to128
jig/ml),
sulfamethoxazole(SMX;
1 to 128jig/ml),
ansamycin (LM427;
ANS;
0.125 to 16jig/ml),
andclofazamine
(CLF;
0.25to32pg/ml).
MICplates
weremade upandstoredat-70°untiluse.Severalinvestigational drugs
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2
. 125
125 125
*125
.5.25
FIG. 1. Microdilution MIC plate fora susceptible strain ofM. tuberculosis. The wells contained twofold dilutions (rows Ato H) of antimicrobialagents,withthelowestdrug concentration (row H) listedinmicrogramspermilliliter. The well in theupperleftcorner isthe positive control, whereas the well inthelowerrightcorner is the broth control. Notethesharpendpointsfor alldrugsexceptSMX. FOX =
Cefoxitin; ANS, ansamycin; CLF,clofazamine;DOX,doxycycline.
were studiedin one ortwoofthewells butare notreported
here because of either inadequate numbers or inability to compare results with susceptibility by other methods. For
thisreason, data on fewerthan 12 drugs are reported. For
comparison
of controlstrains,
theaminoglycosides
andSMX were alsoprepared in cation-supplemented MHB.
Sixty wild strains ofmycobacteria referred to one ofus
(R.J.W.) forsusceptibility testingorfromtheclinical
myco-bacteriology laboratory ofThe University ofTexas Health
Center at Tyler were used. These included 17 cutaneous
isolates ofM. marinum, 13 sputum isolates of M. kansasii,
10 sputumisolates of M.tuberculosis, and20isolates ofthe M. avium complex(10from patients with acquired immune
deficiency syndrome, 6 skin or soft tissue isolates, and 4
pulmonary isolates). The latterwereallrecentisolates,and most were ofthe smooth, transparent colony type. Clinical
historieswereobtainedfor each patient from whom isolates wereobtained to be certain they had not received prior drug
therapy. Inaddition,relapse or known drug-resistant strains
ofM.kansasii(14isolates) and M. tuberculosis (11 isolates) were also tested. Organisms were identified to species by standardmethods at TheUniversity of Texas Health Center
clinicallaboratory, The Texas Department of Health Labo-ratories, Austin, and The Mycobacterial Reference Section of the Centers forDisease Control, Atlanta, Ga.
Organisms for testing were
initially
grown toturbidity,
without shaking, in 7- or 15-ml tubes containing 5-mm
(diameter) glass beads and 7H9 broth. The cultures were
vortexed for20 to30sbeforeuseandthenleftfor3to5s to
allow for settling of heavy
particles.
Thesuspension
of organisms was then matchedtotheoptical density
ofa0.5McFarland standard. Serial 10-fold dilutions were then
made, and appropriate dilutionswere addedtothe wellsof
the microtiter plates containing the different drug dilutions by using a disposable inoculator
which
delivers approxi-mately 0.01 ml (MIC-2000 inoculator; Dynatech Laborato-ries, Inc., Alexandria, Va.). Colony counts wereperformed for each inoculum. Theplatesweresealedinplastic bags and incubatedat30°C(M.marinum) or35°C (all other species) inroomair in a moisturized incubator. The plastic
bags
were essential to prevent evaporationin the wells and were alsouseful for biologic containment. In the latter part of the
study, plates were also taped at both ends to prevent accidental opening and to minimize the risk of accidental spillage with handling. Plates of M. tuberculosis, M.
kansasii, and the M. avium complexwereremoved from the bags only in the biologiccabinet wheretheywereread. All
microdilutionplates were readafter 7, 10, 14, and 21 days by
lookingformacroscopic growthwithanindirectlightsource. MICswere the lowestdilutions exhibitingno growth for all
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TABLE 1. MIC50s and MIC9osfor 60 wild strains of four mycobacterial species inhibited by antituberculosis and antibacterial agents in7H9 broth
MICs(ptg/ml)for(no.of strains):
Drug M. marinum (17) M.kansasii(13) M.intracellulare(20) M. tuberculosis (10)
50% 90% 50% 90%o 50% 90% 50% 90%
INH 16 16 2.0 4.0 8.0 >16 s0.25 s0.25
RIF 0.5 1.0 s0.25 0.5 0.5 2.0 sO.125 0.125
Ansamycin s0.25 s0.25 s0.25 s0.25 0.25 1.0 .O.125 sO.125
Clofazamine 1.0 2.0"a 0.25 0.5 1.0 2.0b 1 2
GEN 8.0 16.0" 8.0 16.0 8.0 160b 4 8
AMI s0.5 0.5 2.0 4.0 4.0 8.0 s0.5 s0.5
KAN 1.0 2.0 8.0 16.0 8.0 16.0 1 1
STR 1.0 2.0 2.0 4.0 4.0 16.0 0.5 s0.5
EMB sO.5 1.0 1.0 2.0 4.0 4.0 2 2
SMX .<1.0 2.0 .<1.0 2.0 4.0 8.0 4 8
Cefoxitin >128 >128 64 128b >128 >128
"For M. marinum, only 13 strains were tested with CLF and 14 strains weretestedwith GEN.
bFor M.intracellulare,only 10 strains were tested with CLF,GEN,and FOX.
drugs except SMX, for which 80% inhibition of growth was bility if a heterogeneous population were present. For this
used (1). reason, a 3-week reading time was used for M. kansasii and
Each isolate was also tested for susceptibility to M. tuberculosis. For the former, most strains could be read antituberculosis drugs by the standard method of proportion easily at 10 days, and all could be read by 14 days. More than in7H10agar. Theconcentrations tested were: INH, 1 p.g/ml; half of the strains had no change in MIC between 10 days and RIF, 1 ,ug/ml; EMB, 5 ,ug/ml;STR, 2,ug/ml. Resistance was 3 weeks, with almost 90% of the changes involving only 1 defined asgrowth on drug-containing plates of greater than dilution. For M. tuberculosis strains, most could also be read 1% of the growth on drug-free control medium which con- after 14 days. However, we chose to use the3-weekreading tained between 100 and 500 colonies (8). time for these two species until more resistant strains are
The controlstrains used were M. tuberculosis H37Rv; M. studied.
fortuitum ATCC 6841 (TMC 1529); M. marinum ATCC 927 Several different dilutions were tried for each organism. and 35780(TMC 1218 and 1219); M. avium-M. intracellulare For thenontuberculousmycobacterial species, large inocula ATCC 35718, 35761, and 35847(TMC 721, 1403, and 1476); (1:10 and 1:100 dilutions) of the starting suspensions(.5 X M. smegmatis ATCC 14468 (TMC 1546); M. chelonae
105
CFU/ml in each well) resulted in a muchfaster reading ATCC 35751 (TMC 1542); Escherichia coli ATCC 25922: and time but yielded high MICs for those drugs, such as EMBPseudomonas aerugihosa ATCC 27853. and SMX,which are known to be affected by inoculum size.
For instance, with M. kansasii, 4 of 14 tests involving the
RESULTS large inoculum showed resistance to SMX, and 6 of 11
showed resistance to EMB. With counts of slO4 CFU/ml, All speciesgrew well in the broth medium and produced however, noresistance was observed to either drug.
Subse-sharp, easily discernible growth endpoints (Fig. 1). MICs quently, 10-3 and 10-4 dilutions of the original dilution were generally read when organisms had reached good matching the optical density of a 0.5 McFarland standard macroscopic growth in control wells. This turned out to be7 were used toinoculate plates, with the lowestdilutionwhich days forM. marinumand 14daysfor theM. avium complex. produced goodcontrol growthin the plates being used. This Because of the possible presence of drug-resistant strains resulted in an inoculumof103 to 104 CFU/mlasdetermined amongisolates ofM. tuberculosis and M. kansasii, a con- by colonycounts.
cern was that early readings might result in false suscepti- Thedrug concentrationsthat inhibited 50(MIC50)and 90%
TABLE 2. MICs formycobacterialand bacterial control strainsin themicrodilutionbroth system
MIC(pg/ml)of:
Organism
INH RIF Ansamycin Clofazamine GEN AMI KAN STR EMB SMX
M.tuberculosis H37Rv .0.25 .0.25 .0.25 .0.25 8 .0.5 2 1 2 8
M. marinumATCC 927 32 0.5 .0.25 4 8 .0.5 2 4 4 .1
M. marinumATCC 35780 16 .0.25 .0.25 2 2 .0.05 2 4 0.5 1
M.avium complexATCC 35718 >32 1 0.5 0.5 2 2 2 1 2 4
M. aviumcomplexATCC 35761 16 0.25 1 4 2 1 2 1 1 4
M. aviumcomplex ATCC35847 16 1 1 1 2 1 1 .0.5 2 2
M. smegmatis ATCC 14468 >32 >16 4 8 2 .0.25 1 1 1 l1
M.fortuitum ATCC 6841 4 8 4 32 16 1 8 16 4 <1
M. chelonae ATCC 35751 >32 >32 >32 >32 64 16 16 64 32 128
E. coli ATCC 25922 >32 0.5 1 32 1 1 2 2 >64 2
P.aeruginosa ATCC 27853 >32 8 8 >32 2 1 >64 8 >64 >128
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TABLE 3. Modal MICs ofaminoglycosides and sulfamethoxazole in MHB and 7H9 for six control strains
ModalMIC (range)of thefollowing in indicated medium:
Organism Amikacin Kanamycin Gentamicin Tobramycin Sulfamethoxazole
MHB 7H9 MHB 7H9 MHB 7H9 MHB 7H9 MHB 7H9
E. coliATCC 2(1-2) 1(1) 2(2) 2(1-2) 1 (0.5-2) 1(1) 1(0.5-2) 2(2) 4(2-8) 2('1-2) 25922
P.aeruginosa 16 (4-16) 1(1) >32(>32- >64(>64) 16(4-32) 1(1-2) 4(1-4) >128(>128) >128(>128)
ATCC27853 >1,024)
M.fortuitum 1(0.5-1) 1 ( 0.5-2) 16(8-16) 16(8-32) 16(8-16) 16(16-64) 16(16-32) <32(>32) 2(2-8) '1 (-1) ATCC 6841
(type strain)
M. chelonae 4 (4-8) 16(16-32) 4 (4) 16(16-32)32(8-32) 32(>32-64) 8(8-16) >32(>32) >128 (64- >128(>128)
ATCC 35751 >128)
M.marinum 1(1) 1(1) 2(2) 2(2) 8(8) 16(16) 16(16) 8(8) 4(4)
MM-42
M. marinum 1 (1) 0. 5(<0.5) 2(2) 1(1) 32(32) 4(4) 16(16) 16(16) 4(4) MM-44
(MIC90)
of the wildstrains of the four mycobacterial species their publishedMICs (3). Theremaining
six control strainsstudied are shown in Table 1. Each species exhibited a either would not grow adequately in MHB or were not narrow rangeof MICsforeachdrug,with nodrugexhibiting tested.
more than a 2-dilution difference between the MIC50 and Acomparisonof the resultsobtained in broth and in agar
MIC%0
andonlyfour(8%) exhibitingasmuchas a2-dilution bythestandardproportionmethodforINH,RIF, EMB,anddifference. MICs for the 27 isolates of M. kansasii by this STRisshown in Table4.ThecorrelationforM.tuberculosis
method have been reported previously (C. H. Ahn, R. J. for all of the drugs was 100%. For M. kansasii, major Wallace, Jr., L. C. Steele, and D. T. Murphy, Am. Rev. discrepancieswerenotedforINH,and for bothM. kansasii
Respir. Dis., in press). andM.marinum,discrepancieswerenoted for STR. For the
MICs for the 11 bacterial and mycobacterial control M.
av'ium
complex, good correlation betweenthetwo meth-strains are listed in Table 2. As expected, M. tuberculosis ods was observed only for INH. In each instance, the H37Rv was highly drug susceptible except for GEN. The discrepancy was a result of lowerMICsin broth than in agar. three strains of the M. avium complex were mixtures ofopaque and transparent colony types and were relatively
drug susceptibleexceptforINH.Fivecontrol strains grew in
MHB,and theirMICs were determined three to five times in This study was not an attempt to compare MICsobtained
this medium.The results were comparedwith MICs in 7H9 by the microdilution broth system with MICs obtained by
broth fortheaminoglycosides and SMX(Table 3). Withthe theproportion method in agar because testing eightdilutions exception ofP. aeruginosa, which was 8- to 16-fold more of 12 different drugs for 96 mycobacterial strains would have active in7H9broth, the MICs for the other organisms were required a prohibitive number of plates. Instead, the feasi-comparable in the two mediaandwere within 2 dilutions of bility of the microdilution method for determining MICs in
TABLE 4. Comparison ofsusceptible andresistantstrains tested in broth and in agar by the standard proportion method
Organism Drug(concn in No. of strains classified
as:%
Agreement(no. ofstrains) agar[,Lg/mll)- A B C D
M.tuberculosis (21) INH(1) 10 11 0 0 100
RIF(1) 13 8 0 0 100
EMB(5) 17 4 0 0 100
STR(2) 17 4 0 0 100
M. kansasii(27) INH (1) 1 20 5 1 78
RIF(1) 16 10 1 0 96
EMB(5) 20 6 0 1 96
STR (2) 9 2 16 0 59
M.marinum (17) INH (1) 0 17 0 0 100
RIF(1) 17 0 0 0 100
EMB (5) 17 0 0 0 100
STR(2) 6 1 9 1 41
M.avium complex (20) INH (1) 0 20 0 0 100
RIF(1) 0 5 15 0 30
EMB(5) 5 3 12 0 40
STR(2) 0 13 7 0 65
aComparative concentrationsin brothwerethesameas in agar except for EMB, forwhich8,Lg/mlwasusedas theresistance breakpoint.
bA,Susceptible bybothmethods;B, resistantbyboth methods;C.susceptibleinbrothbutresistantinagar;D,resistantin broth butsusceptibleinagar.
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7H-9 broth for slowly growing mycobacteria was evaluated. The results of these studies were then compared with the results of single-drug susceptibility assays in agar using dilutions of antituberculosis drugs as performed in most state tuberculosis laboratories (8). With minor exceptions, the results of microdilution MICs and susceptibility results for antituberculosis drugs correlated well and justified this type of comparison.
Major discrepancies between the two methods were seen with STR for both M. marinum and M. kansasii. In each case, the isolates tended to be resistant in agar but suscep-tible in broth. Many of the cultures showed borderline resistance in agar, however, with only 1 to 10% of the population resistant to the drug. Many of the isolates sub-jected to multiple susceptibility tests for the same drug produced variable results (i.e., some tests indicated an isolate to be susceptible, whereas a duplicate test with the same isolate indicated resistance). This probably resulted from the fact that the test concentration in agar was very close to the real MIC for these organisms, and minor variations in method could have resulted in similar isolates being interpreted as susceptible or resistant. Hawkins and Gross noted a similar poor correlation for STR when com-paring susceptibility measured in broth (BACTEC) with standard agar dilution for the same two species. As was observed in our MIC system, their method, using different isolates, showed the latter to be susceptible to STR in broth but resistant in agar (J. E. Hawkins and W. M. Gross, Program Abstr. 24th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1200, 1984).
Similar discrepancies were noted for the M. avium com-plex when it was tested against RIF, EMB, and STR. Isolates were usually resistant to the single, fixed drug concentration in agar but were inhibited at MICs wellbelow this concentration in broth. Almost identical levels of dis-crepancy for these drugs was reported bySteadham et al. (9) for the M. avium complex when they compared the BACTEC broth method with standard agardilutions. Other authors have made similar but less detailedobservations (6). The reason for these discrepancies is not readily apparent, but they appear to be a very consistent finding.
Of interest was the excellent activity of SMX against all strains of mycobacteria, including M. kansasii and M. tuber-culosis, and the activity of AMI, which was comparable to STR in activity against most species and was fourfold more active against isolates of M. marinum.Activity of both SMX and AMI against nontuberculous mycobacteria has been previously demonstrated (4, 10, 13).
We studied only a small number of drug-resistant strains of M. tuberculosis, although this appears to be the most useful area for this susceptibility testing method. Ability to test second-lineantituberculosis drugs such ascapreomycin, pyrazinamide, cycloserine, andethionamide will need eval-uation. Although this method is not likely to allow direct susceptibility testing because of the problem of contamina-tion, it would allow for easy evaluation of all first- and second-line drugs, as well as levels of resistance.
Another useful area for this method would be evaluation of levels of resistance in posttreatment strains of M. kansasii. As previously noted, the current single-drug con-centrations of INH and STR which are used in the propor-tion method usually do notallow one todistinguish primary drug resistance from acquired resistance after unsuccessful drug therapy
(i.e.,
most wild strains are resistant to the test concentrations in agar when first tested). Using this MIC method, we were able to demonstrate acquired drugresist-ance, in treatment failure isolates ofM. kansasii, to RIF,
INH,
and EMB and to suggest optimal retreatment drug regimens.This resulted in successful sputum conversions in sevenofeight
patientswith RIF-resistantM. kansasii(Ahnet
al.,
inpress).Itisalso
likely
that thecurrentmethod wouldproveusefulfor the M. avium complex, although acquired (secondary) drug resistance has notbeendemonstrated for thisspecies. This method would allow for evaluation of multiple drug combinations, as well asfacilitate attempts at correlation of
results of drug therapy with levels of susceptibility of the
infecting strain.
TheMICsforthevarious controlstrains were comparable in 7H9 broth and MHB despite the marked difference in pH
(6.8 and 7.4, respectively). The valuesforE. coli were also
comparable to published MICs (3). The major difference
between the twomediawas seenfor P. aeruginosa with the
aminoglycosides. This probably relates to the differencesin cation content between the two broths. MHB is
Mg2'
andCa2+
supplemented to a concentration of 25 and 50 mgIl,respectively, whereas 7H9 broth contains 50mg ofMg2'but
only 0.5 mg of
Ca2+
per ml. A similar difference in MICresults between the two broths was noted for the M. chelonae control strain, although the reason for this
differ-ence is less well worked out than with P. aeruginosa. The low concentrations of
Ca2+
in 7H9 broth did not affect the other control ogranisms, although a comparison of MICs with and without the cations may be necessary for organ-isms, suchasM. tuberculosis, which wouldnot growinboth media and hence forwhich no comparison could be made.LITERATURE CITED
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