Vol. 24, No. 21 Oct. 31, 2007 Good for Generics New guidelines on determining obviousness issued by the U.S. INSIDE THIS ISSUE Patent and Trademark Office (PTO) earlier this month might make itharder for brand companies to obtain and defend pharmaceutical Patents: GSK sues PTO patents, one legal expert says.
UntitledJournal of Antimicrobial Chemotherapy Advance Access published May 20, 2009
Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkp184 Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs Manju Y. Krishnan, Elizabeth J. B. Manning and Michael T. Collins* Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin – Madison, 2015 Linden Drive, Madison, WI 53706-110, USA Received 28 January 2009; returned 3 March 2009; revised 24 March 2009; accepted 27 April 2009 Objectives: To evaluate the BACTECTM MGITTM 960/MGIT Para TB (MGIT) system for drug susceptibilitytesting of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some formsof Crohn's disease.
Methods: MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, theBACTECTM460TB system (BACTEC) and conventional agar dilution methods.
Results: MICs determined by MGIT methods showed 80% – 100% agreement (+1 log2 dilution) withthose determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromy-cin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+1 log2dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at2 log2 dilution differences. For clarithromycin, the MGIT method had greater agreement with the agardilution method (70% at the same dilution) than the BACTEC method (60% at +1 log2 dilution); agree-ment increased to 100% at +2 log2 dilutions in both cases. The MGIT and agar dilution methods agreed60% and 100% for amikacin MICs at +1 log2 dilution and +2 log2 dilutions, respectively. By all methodsMICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100%agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80%agreement for rifampicin using published MIC thresholds available for M. avium complex strains.
Conclusions: This study shows that the MGIT system can be used for rapid and reliable drug suscepti-bility testing of MAP.
Keywords: agar dilution, BACTEC, MGIT, MIC More recent clinical trials have treated CD patients with combinations of macrolides, rifabutin and clofazimine based on Mycobacterium avium subsp. paratuberculosis (MAP) causes limited MAP antibiotic susceptibility data and on the assumption Johne's disease ( paratuberculosis), a chronic inflammatory that MAP should be similar in susceptibility to other members bowel disease in ruminants. In recent years there have been of the M. avium complex (MAC).8 – 14 Clinical outcomes of several reports suggesting an association between MAP and Crohn's disease (CD), a chronic granulomatous inflammatory bowel disease in humans.1 – 5 In vitro drug susceptibility data specific for human clinical The idea that MAP may contribute to some cases of CD is isolates of MAP are needed to better guide clinical trials target- not new. Previous attempts (1982 – 94) to test this theory ing MAP in CD patients. However, at present there are no stan- involved the treatment of CD patients with anti-tubercular drugs dard methods available for drug susceptibility testing of MAP.
assumed to be efficacious against MAP.6 Failure to observe sig- The few such studies available used a radiometric [BACTECTM nificant clinical improvement dampened enthusiasm for the 460 TB (BACTEC); Becton Dickinson, Sparks, MD, USA] mycobacterial aetiology theory. Only later was it recognized that broth dilution method,17 – 20 which is an accurate and reliable MAP is resistant to most first-line tuberculosis (TB) drugs.7 macrodilution method for susceptibility testing of MAC.21 *Corresponding author. Tel: þ1-608-262-8457; Fax: þ1-608-265-6463; E-mail: [email protected] # The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected] Krishnan et al.
In clinical laboratories the radiometric system has largely been 2 – 3 days. The turbidity of the culture was adjusted with Middlebrook replaced with the new fully automated BACTECTM MGIT 7H9 broth to be equivalent to that of a No. 1 McFarland standard (for (mycobacterial growth indicator tube) 960 system (MGIT) agar dilution method) or a 0.5 McFarland standard (for MGIT and (Becton Dickinson) for the isolation22,23 and susceptibility radiometric methods) using a spectrophotometer (Biomate 3; Thermo testing of pathogenic mycobacteria.24,25 The MGIT system was Fisher Scientific, Waltham, MA, USA). Cultures (8 – 10 days old) of optimized for detection of MAP with a MAP-specific culture M. avium 101 and 104 strains were adjusted to a turbidity equivalent medium, MGITTM ParaTB medium, and has been successfully to that of a 0.5 McFarland standard.
used for faster and sensitive isolation of MAP from clinicalsamples and also for quantification of MAP.26 We previously described drug stability in the MGITTM ParaTB medium27 andin vitro susceptibility of MAP to thiopurine drugs.28 In the Ciprofloxacin, levofloxacin, azithromycin, clarithromycin, amikacin, present study, the MGIT 960 system – MGITTM ParaTB medium ethambutol, clofazimine, isoniazid and dapsone were purchased was evaluated for susceptibility testing of MAP strains to differ- from Sigma-Aldrich (St Louis, MO, USA). Rifampicin and rifabutin ent classes of drugs and the results were compared with those of were purchased from USP (Rockville, MD, USA).
radiometric (BACTEC) and agar dilution methods.
Stock solutions of drugs were prepared using the most appropri- ate solvent: water (amikacin, ethambutol and isoniazid), 0.1 Nsodium hydroxide (ciprofloxacin and levofloxacin), methanol (rifam-picin and rifabutin), ethanol (azithromycin and clarithromycin) or Materials and methods methanol acidified by trace amounts of glacial acetic acid (clofazi-mine). The stock solutions were filter-sterilized, if required, and stored at 2808C for up to 2 months.
Ten clinical isolates of MAP (nine of human origin and one of Prior to testing, each drug was freshly diluted in sterile deionized bovine origin) were used (Table 1). The strains were verified as MAP water. Each drug was tested at a suitable concentration range based by detecting IS900 by PCR. All strains were grown in Middlebrook on reported Cmax values. Broadly, ciprofloxacin, levofloxacin, azi- 7H9 broth containing 10% oleic acid – albumin – dextrose – catalase thromycin, clarithromycin, rifabutin and isoniazid were tested at (OADC) supplement (Becton Dickinson) and 2 mg/mL mycobactin J doubling dilutions in the range 32 – 0.125 mg/L. Amikacin was (Allied Monitor, Fayette, MO, USA). Cultures (6 – 8 weeks old) were tested in the range 25 – 0.78 mg/L. Rifampicin was tested in the harvested, resuspended in fresh Middlebrook 7H9 medium – glycerol range 12 – 0.094 mg/L. Ethambutol, clofazimine and dapsone were (20% final concentration) and transferred to screw-capped test tested in the range 20 – 0.156 mg/L.
tubes containing 8 – 10 glass beads (3 mm). Tubes were vigorouslyvortexed, and allowed to stand for 30 min. The supernatant wasstored as aliquots at 2808C until used.
Susceptibility testing by the MGIT method The method was adapted from those for Mycobacterium tuberculosis M. avium controls and M. avium and described earlier for MAP.28 Briefly, the finalMGITTM ParaTB medium was prepared according to manufacturer's Strains 101 and 104 were gifts from Dr Venkata Reddy (Sequella Inc., instructions by adding 0.8 mL MGITTM ParaTB supplement and Rockville, MD, USA). Colonies from Middlebrook 7H10 agar plates 0.5 mL 50% egg yolk (Becton Dickinson) to 7 mL of MGITTM were inoculated into Middlebrook 7H9 broth containing 10% OADC.
ParaTB medium. Clofazimine and amikacin were tested in theabsence of egg yolk since the MICs were found to be affected byegg yolk. Each tube received 0.1 mL of inoculum (bacterial suspen- Preparation of inocula sion with a turbidity equivalent to that of a 0.5 McFarland standard) Prior to susceptibility testing, the frozen MAP were inoculated into and 0.1 mL of drug. This inoculum resulted in cfu between 5104 4 mL of fresh Middlebrook 7H9 medium and incubated at 378C for and 5105/mL, as shown by plate counts. Growth control tubes Table 1. MAP strains used in the study ATCC 43015 (Linda) human, Crohn's disease patient—ileum human, Crohn's disease patient—intestinal tissue ATCC 43545 (Dominic) human, Crohn's disease patient—intestinal tissue ATCC 49164 (Holland) human, Crohn's disease patient—intestinal tissue human, Crohn's disease patient—ileum, UCFa human, Crohn's disease patient—ileum, UCFa human, Crohn's disease patient—ileum, UCFa human, Crohn's disease patient—ileum, UCFa human, Crohn's disease patient—ileum, UCFa bovine, clinical case of paratuberculosis, JTCb aGift from Saleh Naser, University of Central Florida, Orlando, FL, USA.
bIsolated at the Johne's Testing Centre, Madison, WI, USA.
Drug susceptibility testing of M. paratuberculosis received 0.1 mL of sterile water instead of drug. The 1:100 growth minor modifications.29 Drug dilutions were placed in volumes of control tubes received 1/100 of the standard inoculum. Tubes were 0.1 mL at the centre of quadrant Petri plates and overlaid by molten, incubated in the MGITTM 960 instrument (Becton Dickinson) and cooled, Middlebrook 7H10 agar (supplemented with OADC and time to detect (TTD) data were recorded. An experiment was con- mycobactin J). Plates were swirled gently to allow mixing of drug sidered valid only when the undiluted growth control tube became with the medium before solidification of agar. An inoculum suspen- signal-positive between 2.5 and 4.5 days of incubation and the 1:100 sion equivalent in turbidity to that of a No. 1 McFarland standard inoculum control tube signalled positive between 7 and 10 days.
was diluted to 1022 and 1024. Drug-containing and control quad- MIC was defined as the lowest concentration of drug resulting in a rants were inoculated with 0.1 mL of the 1022 dilution while the TTD value greater than that of the 1:100 growth controls.
1:100 control (1% control) quadrants received 0.1 mL of the 1024dilution. Plates were sealed and incubated at 378C for 4 – 5 weeks.
Susceptibility testing by the BACTEC method The BACTECTM 12B medium (Becton Dickinson) was modified topermit MAP growth, by adding 1 mL of 50% egg yolk suspension Graph Pad Prism version 5 (GraphPad software Inc, San Diego, CA, (Becton Dickinson) and 0.1 mL of mycobactin J (80 mg/mL) to USA) was used for non-parametric correlation (Spearman) analysis.
each vial. Egg yolk was omitted for testing clofazimine. The P, 0.05 was interpreted as indicating significant correlation.
medium was alkalized while testing azithromycin by adding 0.3 mLof 3% tri-potassium phosphate.29 Drug dilutions were added involumes of 0.1 mL. Drug-containing and growth control vialsreceived 0.1 mL of MAP suspension equivalent in turbidity to that of a 0.5 McFarland standard, as recommended by the manufacturer Table 2 summarizes the agreement between MGIT and the other for susceptibility testing of M. tuberculosis. The 1:100 growth two methods in determining MICs of each of the drugs, except control vials received 0.1 mL of a 100-fold dilution of the sameMAP suspension. All vials were incubated at 378C. Growth index isoniazid and dapsone, for the 10 MAP isolates. Isoniazid and (GI) readings were obtained using a BACTECTM 460 TB instrument dapsone MICs by all methods were greater than the highest con- (Becton Dickinson). The 1:100 control vials were read on alternate centration tested. MICs determined by MGIT and BACTEC days until the GI was 30 (considered positive MAP growth above methods showed 80 – 100% agreement at +1 log2 dilution (i.e.
background30) at which time all drug-containing vials were read that one doubling dilution) for ciprofloxacin, levofloxacin, azithro- same day and again the following day. Differences in GI between mycin, rifampicin, rifabutin and clofazimine. The MICs of azi- consecutive days (DGI) were calculated for each culture. The MIC was defined as the lowest concentration of drug showing both a DGI radiometric medium were .2-fold higher than the MICs deter- and absolute GI lower than those of the 1:100 growth control.31 mined in MGITTM ParaTB medium (data not shown). MICs ofazithromycin in MGITTM ParaTB medium ( pH 6.8) correlatedbetter with those in alkalized ( pH 7.3) BACTECTM 12B Susceptibility testing by the agar dilution method medium. Clarithromycin was tested at pH 6.8 in both broth Methods recommended by the CLSI (formerly the NCCLS) for media and showed 60% agreement at +1 log2 dilution and M. tuberculosis and slow-growing mycobacteria were used with 100% agreement at +2 log2 dilutions. However, for this drug, Table 2. Agreement in MIC determinations by MGIT in comparison with BACTEC and agar dilution methods for nine antimicrobialsagainst 10 MAP strains Percentage agreement in MIC determination between methods MGIT and agar dilution +2 log2 dilutions +2 log2 dilutions CIP, ciprofloxacin; LVX, levofloxacin; AZM, azithromycin; CLR, clarithromycin; AMK, amikacin; RIF, rifampicin; RFB, rifabutin; EMB, ethambutol;CLF, clofazimine; ND, not determined.
aMICs at pH 6.8 in MGITTM ParaTB medium and pH 7.3 in BACTECTM 12B medium.
bMICs at pH 6.8.
cMICs in the absence of egg yolk.
Krishnan et al.
there was better agreement between MGIT and agar dilution The MGIT and agar dilution methods showed significant MIC (70%, 90% and 100% at the same, +1 and +2 log2 dilutions, correlation for all drugs (64% – 93%), except ethambutol (52%).
respectively). MICs of ethambutol showed 70% and 100% MIC range, MIC50 and MIC90 values determined by the agreement between MGIT and BACTEC methods at +1 and +2 MGIT method were compared with those by the BACTEC and log2 dilutions, respectively. The MICs of ethambutol were agar dilution methods (Table 3). MIC ranges of ciprofloxacin, higher by the BACTEC method for most strains. Non-parametric levofloxacin, azithromycin, clarithromycin, rifabutin, clofazi- correlation analysis of MICs obtained by MGIT and BACTEC mine (by all three methods) and amikacin (tested by two methods showed significant correlation (P, 0.05) for all 10 methods only) were either the same or differed by only one dou- drugs compared (between 80% and 98% for 9 drugs and 73% bling dilution. The presence of egg yolk in broth caused for ethambutol).
.4-fold higher MICs of clofazimine and amikacin in broth Amikacin MICs determined in MGIT medium without egg methods compared with the agar method (data not shown). All yolk in comparison with those found by the agar dilution strains tested were resistant to isoniazid and dapsone at the method showed 60% agreement at +1 log2 dilution and 100% highest concentrations tested (higher than the respective Cmax agreement at +2 log2 dilutions; generally, slightly higher MICs values) by all three methods. The upper limits of the MIC range were obtained for most strains by the agar dilution method.
of ethambutol and rifampicin were .2 log2 dilutions higher by MICs found by MGIT and agar dilution methods showed 90% – agar dilution. There was good agreement between methods with 100% agreement at +1 log2 dilution for ciprofloxacin, levofloxa- respect to MIC50 and MIC90 values (minimum concentration of cin, azithromycin, clarithromycin and clofazimine. MICs of a drug that inhibited at least 50% and 90%, respectively, of the rifampicin, ethambutol and rifabutin showed 80%, 90% and strains tested) for all drugs except ethambutol. The MIC90 of 100% agreement between the MGIT and agar dilution methods ethambutol was four times higher by the agar dilution as com- at +2 log2 dilutions. The agar dilution method yielded higher pared with the MGIT method. MGIT-derived MICs of all the MICs of ethambutol, compared with both broth culture methods.
drugs for M. avium strains 101 and 104 are shown in Table 4.
Table 3. Summary of MICs of 11 drugs for 10 MAP strains determined by three methods MIC50 and MIC90 by three methods CIP, ciprofloxacin; LVX, levofloxacin; AZM, azithromycin; CLR, clarithromycin; AMK, amikacin; RIF, rifampicin; RFB, rifabutin; EMB, ethambutol;CLF, clofazimine; DPS, dapsone; INH, isoniazid; ND, not determined.
aMICs at pH 6.8 in MGITTM ParaTB medium and pH 7.3 in BACTECTM 12B medium.
bMICs at pH 6.8.
cMICs in the absence of egg yolk.
Table 4. MICs of various drugs for M. avium strains 101 and 104, as determined by the MGIT method CIP, ciprofloxacin; LVX, levofloxacin; AZM, azithromycin; CLR, clarithromycin; AMK, amikacin; RIF, rifampicin; RFB, rifabutin; EMB, ethambutol;CLF, clofazimine; DPS, dapsone; INH, isoniazid.
Drug susceptibility testing of M. paratuberculosis Table 5. Tentative classificationa of MAP strains by the MGIT drug susceptibility method and comparison with that of the BACTEC andagar dilution methods MGIT and BACTEC methods MGIT and agar dilution methods both S both I both R S and I R and I S and R agreement (%) both S both I both R S and I R and I S and R agreement (%) S, susceptible; I, intermediate; R, resistant; AZM, azithromycin; CLR, clarithromycin; CIP, ciprofloxacin; AMK, amikacin; RIF, rifampicin; RFB, rifabutin;EMB, ethambutol; CLF, clofazimine.
aBased on interpretive criteria for MAC recommended by: bthe CLSI;29 and cHeifets.32 Currently there are no criteria for drug susceptibility MAP growth rate at the more alkaline pH. MIC determinations interpretation specific for MAP. However, for MAC organisms, for MAP by the agar dilution method required 4 – 5 weeks.
the CLSI defines clinically significant resistance to two macro-lides, azithromycin and clarithromycin, as MICs .256 mg/Land .32 mg/L, respectively, using the BACTECTM medium at pH 6.8.29 For other drugs that are active againstMAC, there are thresholds for MIC interpretation determined in The fully automated, non-radiometric MGIT 960 system in 7H12 broth.32 To evaluate the ability of the MGIT method to conjunction with the MGITTM ParaTB medium allows rapid and designate MAP strain drug susceptibility, the 10 MAP strains sensitive recovery of MAP from clinical specimens. The present tested were classified as susceptible, intermediate or resistant to study shows that the system can also be reliably used for drug eight drugs based on the two aforementioned criteria. Drug sus- susceptibility testing of MAP strains. MICs of all drugs com- ceptibility classification by the MGIT method showed 70% – pared between MGIT and BACTEC methods showed 60% – 100% agreement with the BACTEC and agar dilution methods 100% agreement at single doubling dilution or 100% agreement for ciprofloxacin, azithromycin, clarithromycin and rifampicin at two doubling dilution differences. MICs by MGIT and agar (Table 5). Except for clofazimine and ethambutol, disagreement dilution methods agreed 60% – 100% at single doubling dilution between the MGIT and the other two methods was observed or 100% at two doubling dilution differences for all drugs whenever the other methods classified a strain as susceptible except rifampicin and ethambutol. The higher MICs of rifampi- versus intermediate or resistant versus intermediate. For etham- cin determined by agar dilution could be due to decreased stab- butol, two strains, UCF3 and UCF8, were classified as suscep- ility of the drug in agar and the extended incubation time tible by the MGIT method but resistant by agar dilution. One required to obtain results, as reported in other studies.36 The strain (Linda) was classified as resistant to clofazimine by the MICs of ethambutol in Middlebrook 7H12 broth are known to MGIT method but susceptible by the other two methods. There be lower than in 7H11 agar plates and the difference is more are no cut-offs proposed for three drugs tested in this study pronounced for M. avium than M. tuberculosis.37 The present against MAC: levofloxacin, isoniazid and dapsone. For three study shows that the MIC90 of ethambutol for MAP is 2-fold MAP strains, the MICs of levofloxacin were at or above its higher by the BACTEC and 4-fold higher by the agar dilution Cmax,33 while for all 10 strains the MICs were higher than the method. However, Piersimoni et al.38 showed 2-fold lower respective Cmax for isoniazid and dapsone34,35 by all three MICs of ethambutol for MAC in BACTECTM 12B medium methods. Based on the same criteria, the MGIT-derived MICs compared with MGIT. This discrepancy could be due to differ- classified the two MAC strains 101 and 104 as susceptible ences in media composition as the MGITTM ParaTB medium is to ciprofloxacin, azithromycin, clarithromycin, rifampicin and specifically enriched for growth of MAP. Moreover, observations rifabutin. Strain 101 was found susceptible to amikacin, while with clofazimine and amikacin indicated that egg yolk in broth strain 104 showed intermediate amikacin susceptibility. Both media results in 4-fold higher MICs of these drugs for MAP strains showed intermediate susceptibility to ethambutol, but (data not shown). Decreased activity of clofazimine in the pres- resistance to clofazimine and dapsone.
ence of egg yolk in the medium was observed in a prior drug For MAP, the time required to determine MICs by the MGIT stability study as well.27 However, a similar effect of egg yolk method was 7 – 10 days, while the BACTEC method required on amikacin was not observed in that study, probably due to the 10 – 12 days, depending upon the MAP strain being tested.
test strain (Escherichia coli) used for amikacin.
However, azithromycin testing in BACTECTM 12B medium at Since the few published studies on drug susceptibility of pH 7.2 required slightly longer (12 – 14 days), due to a slower MAP report data on a limited number of strains, few drugs and a Krishnan et al.
variety of methods, comparison of studies is difficult. However, results for ciprofloxacin, ethambutol, rifampicin and dapsone in paratuberculosis in Crohn's disease. Curr Opin Gastroenterol 2008; 24: the present study are comparable to those of Zanetti et al.,39 who studied 12 MAP strains, of both human and animal origin, 3. Feller M, Huwiler K, Stephan R et al. Mycobacterium avium sub- using the MGIT system. The discrepancies for clarithromycin species paratuberculosis and Crohn's disease: a systematic review and and rifabutin (lower MICs found in the present study) could be meta-analysis. Lancet Infect Dis 2007; 7: 607 – 13.
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ocho razones por las que la juventud norteamericana Cómo se ha aplastado la resistencia juvenil en Estados Unidos Cuadernos de reflexión: La juventud y la rebeldía Nota de presentación: Dr. Bruce E. Levine es un psicólogo estadounidense, especializado en psicología clínica, muy crítico de la corriente principal de su profesión. Escribe habitualmente en diversos medios, AlterNet,