effect of media on growth rate and susceptibility testing of cryptococcus neoformans : der einfluß...

Effect of media on growth rate and susceptibility testing of Cryptococcus neoformans

Der Einflun des Nahrmediums auf die Wachstumsrate und die Empfindlichkeitstestung von Cy$~tococcus neoformans

G. St-Germain and Christiaiie Dion

Key words. C t y p t o c o c ~ u ~ ntuformmi\. antifiinqil aqeiit5, wsccptihilitv testinq

Schliisselworter. Cn,j,tot o(c /LJ w o / o t m m \ , AntimyLotikn, Enipfindlic hhritspru funq

Summary. The effect of four media or media cotnhinatioIis on the growth and the z n riilro susceptibility to amphoteririn B, 5-fluorocytosine, flucoiiazole and itracoiiazole of 5 1 isolatea of Cr. neo_formanJ was studied. 'The drug concentration producing a 50Oh decrease in OD,,, (ICc,n) was determined in microtiter format. The following MOPS buffered media or media combinations (antifungal drug diluent/inoculum dilueiit) were used: RPMI 1640 (RPMI)/RPMI, Yeast nitrogen base (YNB)/YNB, RPMI/YNB and RPMI/High resolution broth. Amphotericin B was firtlier tested in M3 broth in the microdilution format and on RPMI agar with the E-test. Growth rates (OD I,5) were higher in YNB, RPMI/YNB, RPMI/HR aiid M3 as compared to RPMI alone. After 48 h of incubation, ICso values obtained with RPMI alone and with RPMI/YNB correlated within two dilutions for 98, 95, 95 and 100% of test strains with amphotericin B, 5-fluorocytosiiie, fluconazole and itraconazole respectively. The E-test provided the widest range of IC: values with amphotericin B. Growth of Cr. iie$oorrnanJ can be enhanced in susceptibility testa using microtiter plates with drugs diluted in RPhlI by preparing the irioculum in YNB. However, when using spectrophotometric endpoint determinations, sufficient growth is obtain-

Laboratoire dc Santi. Publique du Qui.bec, Sainte-Anne-dc- Belle\w, Quebec, Canada.

Corrqxmdence: hlr Guy St-Gerrnain, Laboratoire de Sariti. Publique du Qukbec, 20045 Saintc-Marie, Saintc-Ann(.-De- Bellevue, Qiikhec, Canada H9X 3R5.

able with most isolates for test interpretation at 48 h in RPMI alone.

Zusammenfassung. An 5 1 Isolalen von (,?@to- rocrus neofbrnzonr wurde der Eiiifld3 von vier Niihr- medieri oder NBlirniedienkombiiiationcn auf das Wachstum uiicl die Empfiiidlichkcit fur Amphoteri- cin B, 5-Fluorcytosin, Fluconazol und Itraconazol untersucht. In Microtiterplatten wurde die Wirk- stoffkonzentration als IC,,, hestimmt, die eiiie 50% ige Waclistumshemmung, gemcssen als OD4,),, verursacht. Es wurden die folgeriden RIOPS- gepufferten Medien oder Medienkombiiiatiotieii eiiigesetzt (M.'irkstoffVerdiinliungsmittel/Inoku- lurn-Suspensionsniittel): RPMI 1(,4O/RPhII, Yeast nitrogen base/YNB, RPMUYNB und RPh4I/ High Resolution Bouillon. Amphotcriciii B wurde claruber hiiiaus in M3-Bouillon irn Microtitersy- stein urid auf RPMI-Agar iin E-Test gepriift. Die Wachstumsratcn (OD,,,) wareii in YNB, RPMI/ YNB, RPMI/HR und M3 heher als in RPMI allein. Nach einer Inkubationszeit ~011 48 h korre- lierten die IC,,-Werte niit KPhll allein und RPMI/YNR iiiiierhalb zwcier Verdunnungsstufen bei 98, 95, 95 und 100O/0 der lestst3mme fur Amphotericin B, 5-Fluorcytosir1, Fluconazol bzw. Itraconazol. Der E-Test zeigte die groI3ten Schwankungen der IC-Werte bci Ainphotericin B. Das Wachstum voii CI-. nerlforinans kaiiii bei Empfindlichkeitstestung irii Microtitersystem ver- bessert werden, weiiii die Wirkstoffe in RPhlI verduiint uiid das Inokulum in YNB suspendiert wircl. Bei spektrophotometrischeii Endpunktbe- stiminurigeii kann jedoch bei den meisteii Isolaten hinreichendes Wachstum fur die Ergebnisinterpre- tation nach 48 h in RPMI allein erreicht werden.

202 G . ST-GEKMAIN C:. DION

Introduction Antiji~ngal drug5 and mirrodzlution tray5

The antifungal agents used were ampliotcricin B and 5-fluorocytosine (Sigma Chemical Co., St Louis, MO, USA), itraconazole (Janswn Pharniaceutica, Mississauga, Ontario, Canada) and Auconazole (PfiLer Pharmaceuticals, Kirkland, Quebec, Canada). E-test strips with amplio- tericin B werc supplied by AB Biodisk ( S o h , Sweden).

In the effort to develop a standardimd susceptihil- i ty test for yeasts, concerns have been expressed to tlie effect that the use of RPMI 1640 broth, as 4uggested in the NCCLS proposed method M27P [ I ] , may not be appropriatc for tlie growth and susceptibility testing of Cg$tococ(u) neqformanr [2 -41. A study by Ghannoum et al. [2] with 21 strains of Cr, neoformans, indicated that buffered yeast nitrogen base (pH 7.0) was a more suitable medium for the growth of this organism. More recently, Odds el al. [5] investigated the role of oxygen as a limiting nutricnt for the growth of C I . rieojormans and observed an increase of up to two-fold in growth turbidity due to incubation under an atmosphere or oxygen or with agitation. With regard to the compression of the MIC raiige obtained with amphotericin €3, for yeasts in general, it is now apparent that method M27P will have to bc modified to allow better detection of rcsistant isolates [ 3 , 41. When using a tnicroclilutiori method with antifungal dilutions prepared in advance, the use of a different intdiurn for a particular species of yeast complicates testing pro- ccdures in that separate sets of microtiter plates must be prcpared. To avoid this difficulty we investigated the effect of preparing thc yeast inoculum in various broths while retaining the use of RPMI 1640 for the antifungal dilutions, a pro- cedure which resultcd in a 1 : 1 combination of these media with RPMI 1640. We also studied the effect of M3 broth and the use of the E-test on the range of MICs obtained with amphotericin R .

Materials and methods

Irnsf iaolates

Fifty-one strains of Cr. neqformani were tested: 2 quality control strains (ATCC '30 1 12 and '30 1 13) recommended by the NCCLS [ 1 , 61, 2 other A'lCC strains (A'I'CC 28957 and 66033) and 47 clinical isolates. Two of these isolates were kindly provided b y W. Powderly; they were isolated from an AIDS patient before (CNI) and after (CN3) fluconazole and amphotericin B treatment [7, 01. Overall, isolates were identified to species level with the API 20C: yeast identification system (BioMkrieux Vitek Inc., Haselwood, MO, USA) supplemented with a urease test and a morphology evaluation on cornmeal Tween 80 agar. All isolates were stored at - 70 "C in 10% glycerol and were passaged twice 011 Sabouraud glucose agar prior to testing.

Culture media

The following three media wcre buffered with morpliolinrpropanesLilplionic acid (1LZOPS) (0.165 M, pH 7.0): RPMI 1640 with glutaniine (RPMI) (GIBCO, Grand Island, NY, USA), yeast nitrogen base (YNB) (Difco, Dctroit, MI, USA) with 1 '%, glucow and 0.15'% asparagine and High resolution broth (HK) (Oxoid I,td., Basingstokc, UK). h13 broth (Difco) containcd 2 % glucose with a pH of 6.9. RPMI agar (RPMI 1640 powder 10.4 g, glucose 20 g, agar 15 g, glutaniine 0.3 g, watcr 1 1 ) was used in the performance of the E-test.

ilniijmgal diluiiona

Two-fold dilutiotis of' anipliotcricin B (0.016- 16 mg I - ' ) , 5-fluorocytosine (0.06-64 tng l - ' ) , fluconazole (0.03-32 tng I - ' ) ancl itraconazole (0.03-8 nig I - ' ) were prepared in RPhlI and YNB. Amphotericin B was also dilutcd in M3 broth. Dilutions were dispensed in 50 111 aliquot, in round-bottom 96-well assay plates which were kept fi-ozen at -70 "C in sealed plastic bags until use.

Inoculm prrparatzon

Cr. neofoiman~ strain\ werc grown 48-72 11 on Sabouraud glucose agar at 35 'C;. Spectro- photometrically standardized 1 x 10' to 3 x 10' cells ml - ' suspensions were prepared in saline, using five or inore colonies. 'I'he5e were further diluted 1 : 100 and 1 : 5 in the appropriate media (RPMI, YNB, HR or M3 broth) to obtain 2 x 10' to 6 x 10' cells ml - I.

Inoculation mid incubation

Antifungal assay plates prepared with RPMI were inoculated in duplicate with 50 pl of inocula prcpared in RPMI, YNB and HK, respectively, whereas plates already containing YNB or M3 were inoculated with cells suspended in YNB or M3 only, respectively. Plates were incubated at 35 'C, for a maximum period of 72 11.

mycoves 39, 20 1 -206 ( 1996)

Determination endpoint3

After 48 and 72 h, plates were agitated for 3 min at 900 rpm with a shaker (SLT Lab Instruments, model EAS 2/4, Grodig, Austria) and inhibitory concentrations corresponding to a 50?h decrease in turbidity (IC5,,) as compared to a growth control were determined with the i i ~ e of an automatic plate reader set at 405 mrii (Pasteur Diagnostic LP400, ildil Instruments, Strasliourg, France).

E-ie<t

The E-test was performed according to thc manufacturer's instructions (AR Biodisk). Well-isolated 48-72 h colonies of Cr. neoforman, were suspended in 0.85% NaCI to achieve a 110. 1 McFarland turhidity. A cotton swab was used to apply a uniform lawn of organisms onto 90 mm plates of KPMI agar. A single strip of amphotericin €3 was

Table 1. Elfect of different media and media cornl)inationh on thr growth cif 5 1 isolatrs of C r . 11~0fimiaiis a f~a r 48 h or inculiation

Gro\\tli rnccliuin ,1lraii OI),,jjS Numl)rr of strains with growth < 0.0 I OD,,,,

Kl'hll 0 093 h YNR 0 I63 G RPhll/YNB* 0 162 )

Rl'hll/HR* I1 16s h ,113 0 153 4

applied onto each plate. After 72 h of incubation in a moist air incubntor, the MIC: was read as the value 011 the test strip scale where growth inhibition was complctc (100%) or nlmosl complete (95"h). To facilitdte the comparison of E-test results with those of the microdilution method, MICs determined b y the E-test were rnised to the nevt two-fold dilution level used in the mici odilution ine t h od .

Results

Crowth characteristics in diffrrent media

l 'he averaqe growth turbidity at 48 h in five media or media iombinations is presented in Tablr 1 . Of the 51 strains tested, six were slow growers and failed to reach the minimum growth level for test interpretation set at OD,,li 20.01 a t 48 11.

However growth was adequate fin- all six of these strains at 72 h.

Table 2 summarizes the in uitro susceptibility to amphotericin B of 5 I strains of C r . npoformans by a broth microdilution test read at 48 and 72 h and with the E-test read at 72 h. 'lhble 3 presents a comparison of IC:,,,s with various media and techniques for strains CNl and CN3. Overall, the use of different media or media combinations in a microdilution format appeared to have little or no influence on the results ohtained wilh 5-fluorocytosinr, fluconazole and itraconazole (Tables 4, 5, 6).

Modal IC5,,s for A'l'CC strains 901 I2 and 901 1 3 ,

-

Table 2. In d 7 u activity of amphotrricin H against C'r. nrq~!fb,nran.r determined b y broth nricroi-lilutioii and L t c s t

hletlia*l lCi,,(mg 1 - 1 ) at:

18 I1 72 I1

Range 50%t 90%t I%, agrccnicnt Range 5O'X,i- 9O%t "0 agreemellt with RPhII with KPXlI

within niir wflitliin oiie (two) dilutions: (two) dilutions:

RPR.II/RPMI 0.06-2 0.125 0 . 5 0.125 2 0.5 2 I'N I3 / Y N B 0.06 1 0.5 1 68 (95) o. I 25-2 0.5 I 715 ( 1 00) IIPlII /YNR 0.06 I 11.25 I 75 (CJ8) 0.06 ~~ 2 0.5 2 83 ( 100) l iPhll/ H R 0.06 I 0.25 1 01 (98) 0.06-2 0.5 I 100 ( 100) h 1 3 / h13 0 . O l G 0.5 0.03 0. I20 21 (45) 0.016 0.5 0.06 0. I25 I9 (.+(i) Ercsl$ - - O.O(j-4 0.25 0.5 8 2 (9-k)llTl -

* Fii.st mrdiuni used lor anitiungal clilulion (50 pl), (lie second lor inoculutn preparation (511 PI). t IC:,,s enconiljassing 50 and 90?6 of all isolates tested. : Pwcrntage of IC,,,s ol)taincd within onc o r two doul,ling dilutions of' the I C 5 + ol)raiiird by tlir rrf'rrrncc riiicrorlilutioii nirtliiid in Kl'hll broth. 4 For the purpose of comparison, the h<llCs deterinincd by tlir E-test wcre raised t n thv nrs t twJot'old diltihin levrl used i n tlir microdilutioti mrthod. 7 I:.-test results at 72 I1 comp;md 10 liPbll at 48 11.

mycoses 39, 20 1-206 ( 1996)

Table 3. Susreptil)ility tu amphotcririn B (I<:5o) of <,'r, n ~ q j i r r n m ~ strains CNI and C N 3 as detcrmiiiecl b y the microtlilution and E-test mrthnds

LIcthotIs and media hIICa (mg 1- ') f i r strains

CN I (jN3

hlicrodilution meihod RPhIl 0.500 I .om h13 o. I 25 0.500

E-lest Inclhocl RPhll agar 0. I25 2.000 I

as tested in RPMI broth and read at 48 11, were 0.25 and 0.25 mg I - ' for arnphotericin B, 1 and 128 mg I - ' for 5-Auorocytosine, 2 and 4 mg I - ' for flucoiiazole and 0.06 and 0.125 mg I - ' for itraconazole respectively. Both strains were tested on eight occasions for the first three drugs and

five times with itraconazole; results were all within one two-fold dilution from the mode.

Discussion

In 1992, the NCCLS proposed a standard method for the susceptibility testing of yeasts [ 11. This reference standard should eventually be replaced, for routine testing, by a mow cf5cient and less costly method which most likely will be in a microdilution format [4, 91. This technolo,gy allows for test solutions to hc prepared in advance and kept frozen until use. Also, endpoint determination is facilitated by the use of an automatic plate reader. This tool may prove to be useful in determining the degree of inhibition to be used as endpoint criterion in the search for correlation of in vitru testing with clinical outcome. Various micro-

- Table 4. fn oitro activity of 5-fluorocytohinc against Cr. r w j ; m n ~ i n . s determined by hroth microdilution

Mrdia* lC,,,(mg I - ') at: ~ ~~~

18 11 72 I1

Raiige 5 W t 90?/,t '%, agrrernrnt Raiigr 50%t 90"ht '%I agreement with RPMI with RPMI within one within m e (two) dilutions: (two) dilutions:

- RPMI/RPMI 0.06- 128 I 2 0.25 -128 2 8 YNB/YNB 0.125-128 I 4 138 (93) 0.5- 128 2 8 83 (91) RPMI/YNB 0.06- 128 1 2 9 3 (95) 0 . 5 128 1 4 89 (98) RPXll / H R 0.125 128 I 4 87 (98) 0.25 128 1 4 93 (98)

*First medium used for antifililgal dilution (50 PI) , the second f i r ino~~iiluni prqiaration (50 pl). t lC:J(ls encompassing 50 a i d 90"h of all isolates tested. :: l'crcentagc of IC,,,s obtained within one or two doubling dilutioiis of the IC,,s obt;tinetl ly the referencr tnirroclilution niethtrd in KPhlI broth.

~

48 I1 72 I1

Range 50%t 90%t '%I agiwmrnt R;uigc. 50'!ht 90"ht '%I agreement with IiPlcll with IiPMI within one within one (two) cliliitioiis$ (two) dilutions:

- KI'hfI/RPAII 0.06 16 2 tl 0.5 ~ I6 t f3 YNB/YNB 0.5- I 6 2 8 93 (98) 0.25- I6 2 8 96 ( loo) RPhII/YNB 0.125-8 2 8 91 (95) 0.25 8 2 4 98 ( loo) liI'M1/ H R 0.25- I6 2 1 98 (98) 0.5 - I f i 2 8 93 (100)

*First mcdium used for antiiiingal dilutioii (50 kl), the srcond for inomlum preparation (50 111).

t ICS,s enrompa~sing 50 and 90% of all isolates trstecl. $ Pcrcentagc of IC:,,.; ol,tainrd within one or two cloulding dilutioris of the ICS,s ohtainrd b y the refcrcncc. microclilution method in KPMl broth.

mycoses 39, 20 1-206 ( 19%)

SIJSC:EI'TIBILITY TESTING OF CR. NEOFORMANS 205

Table 6. In z'i/ru activity of itraconazole ;igainst C r . nw/irmnns detrrminecl by broth microdilution

RPRII/RPhlI 0.03- I 0.125 0.25 0 . 0 5 0.5 0.125 0.25 YNU/YNU 0.06-0.5 0.125 0.25 nn ( I 00) 0.03 ~ 1 0.25 1 no ( ioo) RPhlI/YNB 0.03- I 0.125 0.25 0 3 (100) 0.03-0.5 0 . 125 0.5 wj p n ) R 1 3 1 I / H R 0.03 1 I). 12.5 0.25 n9 (98) 0.05-0.5 0.125 0.5 91 ( 100)

*First medium used for aiitifiingal dilution (50 pl), the second lor inoculuni preparation (50 pl). t lC5f,s cricompassing 50 and 90%) of all isolatrs tcsted. $ Prrcmtage of IC:5r,s ol,tainrd within one or two doubling clilutiona of the 1C:,,s obtaincd by the referrircr microdilution niethod in RPMI broth.

dilution methods inspired by M27P [ I ] have already been evaluated [Y, 111.

Using a microdilution method, we studied the effect of RPMI, YNB and RPMI combined 1 : 1 with YNB and HR, on the growth and susceptibility 015 1 isolates of Cr. neof0rman.r to amphotericin B, 5-fluorocytosine, fluconazole and itraconazole. Ghannoum et al. have shown that YNB enhances the growth of this organism in comparison to RPhlI [2]. Our results concur with their obser- vations. Furthermore, growth rate was also improved when iriocula were prepared in YNB or HR, while still using antifurigals diluted in RPMI. However, the growth of Cr. neoformans in general, remains slow compared to that of Candida species. Indeed, six of our 51 isolates did not produce sufficient growth for test interpretation at 48 11 (OD,,, <0.010) in any of the media tested. Recently, Odds et al. [5] studied the effect of glucosc concentration and addition of various carbon, nitrogen and vitamin sources in RPMI 1640 and reported that none produced significant changes in growth yield. Incubation under an atmosphere of oxygen or with agitation did produce a slight increase of up to two-fold in growth turbidity. In our experience, some strains of Cr. neoformaris are particularly fastidious and remain so independently of the media and incubation conditions tested until now.

'I'hc compression of the MIC range observed with amphotericin B is an important cause for concern [2-41. The investigation of this problem with regard to Cr. neoformaiis is further complicated by the rarity of resistant isolates. To our knowl- edgr, the only detailed clinically significant description of polyene-resistance in Cr. neoformans was rcported by Powderly et (11. [7]. Strains CN1 and CN3 were isolated from the cerebrospinal

fluid of an AIDS patient before and after fluconazole and amphotericin B treatment. Strain C:N3 was shown to exhibit relative resistance to amphotericin B ( 1.6 mg 1 - ') as compared to strain CN1 (0.4 mg 1- I). Kelly et 01. described d defect in sterol isomerase in strain CN3 [8]. The differ- ence in susceptibility between these two isolates was hardly noticeable with the mcdia used in our microdilution method but was better recognized with the E-test (Table 2), the results obtained with this method rangirig four two-fold dilutions compared to only one to two with the microdilution method. The ranges of IC50s observed with amphotericin B were 3 two-fold dilutions in YNB (0.125-1 .0 mg l - ' ) , 4 in RPMI (0.125- 2.0 mg I - ' ) , 5 in RPMI+YNB (0.06-2.0mg 1 - I ) , RPMI+HR (0.06-2.0mg I - ' ) and M3 (0.016-0.5 mg I - ' ) , and 6 with the E-test performed on RPMI agar (0.03-4.0 mg 1 - ' ) . The disappointing results obtained with strains CN 1 and CN3 in M3 broth may he due to the selection of an inappropriate lot of medium. Indeed, the efficacy of this medium has been observed in other studies [ 3 ] but may vary from one manufacturer to another or from lot to lot.

Overall, the distribution of IC,,s for this collec- tion of Cr. neoformans isolates is very similar to that seen by del Poeta rt al. [ 101. IC:,,p obtained with RPMI alone and with RPMI/YNB correlated within one dilution for 78, 89, 88 and 84% and within two dilutions for 98, 95, 95 and 100% of test strains with amphotericin B, 5-fluorocytosine, fluconazole arid itraconazolc respectively. These results are similar to those observed when compar- ing RPMI and YNB alone. In conclusion, our data show that, with microdilution plates containing antifungal drugs diluted in KPMI broth, inucu- lum preparation of Cr. neojirmanr in YNB may be

mycoses 39, 20 1-206 ( 1 996)

a convenient way of obtaining an increase in growth yield with Ci. neoformnn.s without affecting trst results. However, for most isolates, sufficient growth is achie\ml for sl~ectrophotometric test interpretation at 48 h in KPMI alone. With regard to the iiarrowness of the range of ICs obtained with amphotericin B, iioiie 0 1 the test conditioiis investigated in this study appeared to have n positive effect, with the exception of the E-test.

Acknowledgments

We arc gratefill to W. G. l’owderly and R. C. Summerbell for providing some of the isolates tested in this study. We also wish to thank I,. Massicotte for the preparation of test microdilution trays. This work was supported in part by a grant from Janssen Pharinaceuticn.

References

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Haldwiii, R. C:. & Powdrrly, W’. C:. (1994) Rcsistance to amphotericin R associated with tlcfectivc sterolA”’ iso- incrasc in a CppptororcuJ nmfi.rmnn.i strain from an AIDS paticlit. t;E2ZfiS Microbial. Letten 122, 39-42, Barchiesi, F., Colombo, A. L., hlcGough, D. A. & Kinaldi, RI. G. (1994) Comparative study of broth macrodilution and inicrodilutioii techniques for in z1iti-a aiitifuiigal susceptibility testing of yeasts by using tlic Natioiial Chnmittee for Clinical 1A)oratory Standards’ proposed Standard. 7. (,‘/in. illirrohzol. 32, 2494-2500. Del Poeta, M., Barchicsi, F., L4rzc~ii, D., hlarinucci, G. & Scalise, c;. ( 1 994) ‘I’urbidinietric and visual criteria for in zitla susccptihility testing of (,’typtoroc.r.u.> rwjotbntinn.r cliiiical isolates. iZ!yc.o.se.\ 37, 41 1-416. PkillcI, hl. A,, Messrr, S. A. & Coffiiianii, S . (1995) Comparison of visual and spectrophotornetric methocls of MIC endpoint determiiiations by Lisiiig broth mierodilu- tioii metliods to test five antifiiiigal agents, includitig the iicw triazole D0870. J . U i n . hlicrohinl. 33, 10!34 1097.

mycoses 39, 201-206 (1996)

effect of media on growth rate and susceptibility testing of cryptococcus neoformans : der einfluß...

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