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Journal of Antimicrobial Chemotherapy (2009) 64, 1018 – 1023doi:10.1093/jac/dkp339Advance Access publication 16 September 2009 Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis 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 9 July 2009; returned 18 August 2009; revised 24 August 2009; accepted 24 August 2009 Objectives: Mycobacterium avium subspecies paratuberculosis (MAP) has been targeted for treatmentwith clarithromycin and rifamycin derivatives in numerous cases of Crohn’s disease (CD).
6-Mercaptopurine and its pro-drug azathioprine are widely used as immunomodulators in the treatmentof CD and have recently been shown to have anti-MAP activity in vitro. The objectives of the studywere to evaluate the in vitro effects on MAP of (i) 6-mercaptopurine when combined with each of eight conventional antibacterial agents with in vitro anti-MAP activity and (ii) antibacterial combinations con-sisting of two drugs (clarithromycin combined with amikacin, rifampicin, ciprofloxacin or ethambutol)and three drugs (clarithromycin, rifabutin and clofazimine).
Methods: The drug interaction effects on nine human isolates of MAP were determined by the chequer-board method adapted for the BACTECTMMGITTM960 culture system and by calculation of the fractionalinhibitory concentration index (FICI) for drug combinations.
Results: Synergism (FICI 0.5) was observed between 6-mercaptopurine and azithromycin (sevenisolates), clarithromycin, rifampicin, rifabutin (four isolates each) and ethambutol (two isolates).
6-Mercaptopurine was not antagonistic with any of the antibacterial agents tested. Among thecombinations of two and three antibacterials tested, the clarithromycin/rifampicin combination wassynergistic against four isolates, while all other combinations showed no interaction.
Conclusions: This in vitro study suggests that 6-mercaptopurine may be synergistic with macrolidesand rifamycin derivatives against MAP. The activity of clarithromycin against MAP seems to beenhanced by rifampicin.
Keywords: drug combination, FIC, immunomodulator, antibiotics, susceptibility, paratuberculosis,Crohn’s disease Crohn’s disease (CD), a chronic, debilitating inflammatory boweldisease of humans.3,4 Owing to the strong MAP– CD association,5,6 Treatment of mycobacterial diseases requires a combination of potential anti-MAP agents (macrolides, rifamycin derivatives and drugs to limit antimicrobial drug resistance during long-term clofazimine) were used in clinical trials and case studies with therapy and to deal with the innate susceptibility differences promising outcomes in most reports,7 –12 but not in all.13,14 among mycobacteria in different physiological states, e.g.
Earlier in vitro studies have shown that macrolides, especially dormant in vivo.1 In vitro interaction studies on antimicrobial clarithromycin, are highly effective against MAP.15,16 This agents are necessary to select synergistic and avoid antagonistic observation is consistent with antimicrobial susceptibility data for M. avium complex (MAC).17,18 Presently, the recommended Mycobacterium avium subspecies paratuberculosis (MAP) is treatment and prophylactic regimens for disease due to MAC are the causative agent of Johne’s disease, a chronic inflammatory combinations of two or three drugs: a macrolide (clarithromycin bowel disease affecting a broad range of animals including pri- or azithromycin) in combination with rifampicin or rifabutin mates.2 It is also the most investigated potential causative agent of and/or ethambutol.19 For CD, a combination of antimicrobials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*Corresponding author. Tel: þ1-608-262-8457; Fax: þ1-608-265-6463; E-mail: mcollin5@wisc.edu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
# 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: journals.permissions@oxfordjournals.org Effect of drug combinations on M. avium subspecies paratuberculosis along with immunosuppressive agent(s) was recommended as methanol (rifampicin and rifabutin); ethanol (azithromycin and clarithromycin); or methanol acidified by trace amounts of glacial Azathioprine is an immunomodulator that induces and acetic acid (clofazimine). Drug stock solutions were filter sterilized, if maintains remission in CD.21 Azathioprine and its derivative required, and stored at 2808C for up to 2 months, except for 6-mercaptopurine are eventually metabolized in vivo forming 6-mercaptopurine, which was freshly made each time it was used. Prior thioguanine nucleotides (TGNs), which induce cytotoxicity and to testing, each drug was freshly diluted in sterile deionized water.
recently shown to have anti-MAP activity in vitro.23,24 The objectives of this work were to evaluate the actions of clarithro-mycin or 6-mercaptopurine with other anti-MAP agents and The method described for mycobacteria was adapted for the with each other on the in vitro growth of MAP.
BACTECTMMGITTM960 system (Becton Dickinson).26 Briefly, eachdrug in a combination (two-drug and three-drug combinations) wastested at their respective MICs and at up to five doubling dilutions(sub-MICs) for the strain being tested. Anticipating one doubling dilution difference from previously determined MICs, two more sets of combinations each containing one of the drugs at 2Â the MIC and doubling dilutions thereof were tested. MICs of each individual Nine human (CD cases) isolates of MAP were tested. They included drug were re-determined in the same experiment in which drug five clinical strains (UCF3, UCF4, UCF5, UCF7 and UCF8) kindly combinations were tested. All drug dilutions as either individual donated by Saleh Naser (University of Central Florida, Orlando, FL, drugs or combinations were added to the culture medium in a total USA) and four ATCC strains (43015, 43544, 43545 and 49164). All the isolates were verified as MAP by assay for insertion sequence Tubes were incubated in the MGITTM960 instrument and growth (IS)900 by PCR,25 as well as by in vitro growth characteristics. The was recorded as a TTD value. The TTDs for the strains ranged from isolates were grown in Middlebrook 7H9 broth (Becton Dickinson, 2.5 to 4.4 days (average 3.56+0.69) in control tubes and from 7 to Sparks, MD, USA) supplemented with 10% oleic acid/albumin/dex- 12.7 days (average 9.87+1.86) in 1% control tubes. The MIC of indi- trose/catalase (OADC) and 2 mg/L mycobactin J (Allied Monitor, vidual drugs was defined as the lowest drug concentration required to Fayette, MO, USA). Eight-week-old cultures were harvested and suppress MAP growth resulting in a TTD greater than that of the 1% resuspended in fresh medium containing glycerol (final concen- growth control. The MIC of a drug in a combination (MICcomb) was tration of 20%, v/v). The bacterial suspension was declumped by defined as the lowest concentration of that drug in the mixture that vortexing in screw-capped glass test tubes containing 3 mm glass resulted in a TTD greater than the 1% growth control.
beads. The suspension was then allowed to stand for 30 min. The The fractional inhibitory concentration (FIC) of each drug in a supernatant of MAP obtained by this method was found to contain mixture was calculated as MICcomb/MIC. Drug interaction was bacilli evenly spread out as single cells on a Ziehl – Neelsen-stained determined based on the FIC index (FICI) for the specific drug com- smear. The harvested supernatant was stored as aliquots at 2808C.
FICI ¼ ðMICdrug A comb=MICdrug A aloneÞ Preparation of inocula for in vitro drug susceptibility testing Frozen stock cultures were thawed and added to 4 mL of PBS inscrew-capped test tubes containing 3 mm glass beads and declumped For two-drug combinations, drug interactions were considered once again as described above. The resulting supernatant was har- synergistic if the FICI was 0.5 and antagonistic if the FICI was vested and its turbidity adjusted using PBS to an optical density at600 mm (OD600) of $0.13 using a spectrophotometer (Biomate 3, Table 1. Effects of 6-mercaptopurine in combination with other Thermo Fisher Scientific, Waltham, MA, USA) to match a 0.5 anti-MAP agents on the in vitro growth of nine MAP isolates McFarland turbidity standard. MGITTM ParaTB medium tubes(Becton Dickinson) were inoculated with 0.1 mL of the MAP suspen-sion (drug testing and growth control tubes) or a 100-fold dilution of the suspension (1:100 growth control). The organism was added to the culture medium 24 h prior to adding drugs in order to permit the bacilli to adjust to the growth conditions and prepare for log-phase growth. Tubes of MGITTM ParaTB medium so inoculated were found to contain 1Â105 – 5Â105 cfu/mL prior to adding drugs. Minor butconsistent differences in growth rates were observed between strains as reflected in the time to detection (TTD) values in the MGIT Azithromycin, clarithromycin, amikacin, ciprofloxacin, ethambutol, clofazimine and 6-mercaptopurine were purchased from Sigma-Aldrich (St Louis, MO, USA). Rifampicin and rifabutin were purchased fromUSP (Rockville, MD, USA). Stock solutions of drugs were prepared 6-MP, 6-mercaptopurine; AZM, azithromycin; CLR, clarithromycin; AMK, using the most appropriate solvent: water (amikacin and ethambutol); amikacin; CIP, ciprofloxacin; RIF, rifampicin; RFB, rifabutin; EMB, 0.1 N sodium hydroxide (ciprofloxacin); DMSO (6-mercaptopurine); Table 2. MIC and FICI data of individual MAP strains for two-drug combinations containing 6-mercaptopurine MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI MICalone MICcomb MICalone MICcomb FICI 6-MP, 6-mercaptopurine; CIP, ciprofloxacin; AZM, azithromycin; CLR, clarithromycin; AMK, amikacin; RIF, rifampicin; RFB, rifabutin; EMB, ethambutol; CLF, clofazimine.
Grey shading indicates synergy (FICI 0.5).
Downloaded from http://jac.oxfordjournals.org at Health Sciences Libraries, University of Wisconsin-Madison on July 8, 2010 Effect of drug combinations on M. avium subspecies paratuberculosis .4.0; there was considered to be no interaction if the FICI was .0.5 6-Mercaptopurine – antibiotic combinations It was recently shown that the immunosuppressive drugs azathiopr- activity.23,24 These drugs could potentially affect the activity of antimicrobials when used together for treatment of CD. We assessed drug interactions in vitro by determining the FICI values of two-drug (6-mercaptopurine plus antimicrobial) combinations containing 6-mercaptopurine and one of the eight antibiotics that showed in vitro anti-MAP activity in our previous study.16 MICs of 6-mercaptopurine for the MAP isolates used in the presentstudy ranged from 1 to 128 mg/L. We have previously observed that the MBCs of 6-mercaptopurine for all the nine strains were .128 mg/L, the highest concentration tested. Even for a strain having an MIC of 1 mg/L, there was only a 0.4 log10 reduction in cfu at 64 mg/L and there was no concentration-dependent killing.
These data suggest that the drug is bacteriostatic to MAP.
Antimicrobial drugs either showed synergy or no interaction with 6-mercaptopurine, and none of the combinations was antagonistic for MAP growth inhibition in vitro (Table 1). Azithromycin showed a synergistic interaction with 6-mercaptopurine for seven out of the nine MAP strains, while clarithromycin, rifampicin and rifabutin showed synergy with 6-mercaptopurine for four MAP strains each. Ethambutol was synergistic with 6-mercaptopurine for only two MAP strains. Anti-MAP activities of amikacin, cipro- floxacin and clofazimine were not affected by 6-mercaptopurine for any of the nine MAP isolates tested. Individual MICs and FICI values for all nine strains are shown in Table 2.
This is the first in vitro drug combination study on 6-mercaptopurine and antibiotics as anti-MAP agents and is relevant for the treatment of CD cases that are associated with MAP. In humans, azathioprine is rapidly converted into 6-mercaptopurine, which in turn is metabolized to TGNs—the active metabolites responsible for immunosuppression.22 The low serum levels (,0.1 mg/L) and short half-lives (1 – 3 h) of 6-mercaptopurine are attributed to its rapid conversion into TGNs.28,29 The concentrations of active metabolites and toxic Table 3. Effect of antibiotic combinations containing clarithromycin on the in vitro growth of MAP isolates CLR, clarithromycin; AMK, amikacin; RIF, rifampicin; CIP, ciprofloxacin; Nine isolates were tested except for the three-drug combination.
metabolites of 6-mercaptopurine are known to vary among and within individuals due to the complexity of the metabolic path-ways involved and genetic polymorphisms in the metabolizing M. T. C. was a paid consultant to Becton Dickinson. Other enzymes.30 TGN concentrations approaching 250 pmol/8Â108 red blood cells are reported to be associated with disease remis-sion.22 Currently it is not known how 6-mercaptopurine inhibitsthe growth of MAP or whether the bacterium actively converts6-mercaptopurine into TGNs. In this scenario, even though the in vitro data presented here are not sufficient to predict the clini-cal efficacy of synergistic 6-mercaptopurine – antibiotic combi- 1. Mitchison DA. Combined antimicrobial treatment in tuberculosis nations on MAP, it is encouraging to find that these combinations and in non tuberculosis infections. In: Brumfitt W, Curcio L, Silvestri L,eds.
can be synergistic or neutral in their effects, but not antagonistic.
2. Chacon O, Bermudez LE, Barletta RG. Johne’s disease, inflam- Drug combinations containing clarithromycin matory bowel disease, and Mycobacterium paratuberculosis. Annu Rev Two-drug combinations, containing clarithromycin and one of the four anti-MAP agents amikacin, rifampicin, ciprofloxacin or paratuberculosis in Crohn’s disease. Curr Opin Gastroenterol 2008; 24: ethambutol (each belonging to different antimicrobial drug classes), were tested against nine MAP isolates. In addition, a 4. Pineton de Chambrun G, Colombel JF, Poulain D et al.
three-drug combination containing clarithromycin, rifabutin and Pathogenic agents in inflammatory bowel diseases. Curr Opin clofazimine, a common combination used in CD trials targeting MAP, was tested against three MAP strains. Clarithromycin 5. Abubakar I, Myhill D, Aliyu SH et al. Detection of Mycobacterium when combined with rifampicin showed either synergism (four avium subspecies paratuberculosis from patients with Crohn’s disease isolates) or no effect (five isolates) on inhibition of MAP growth using nucleic acid-based techniques: a systematic review and in vitro (Table 3). The other three drugs, amikacin, ciprofloxacin meta-analysis. Inflamm Bowel Dis 2008; 14: 401 – 10.
and ethambutol, did not show any interaction with clarithromycin 6. Feller M, Huwiler K, Stephan R et al. Mycobacterium avium sub- for any of the nine MAP isolates tested. The three MAP isolates species paratuberculosis and Crohn’s disease: a systematic review and tested with the three-drug combination, clarithromycin, clofazi- meta-analysis. Lancet Infect Dis 2007; 7: 607 – 13.
mine and rifabutin, did not show any drug interaction. Individual 7. Gui GP, Thomas PR, Tizard ML et al. Two-year-outcomes MIC and FICI values for all nine strains for two-drug combi- analysis of Crohn’s disease treated with rifabutin and macrolide anti- nations containing clarithromycin are shown in Table 4.
biotics. J Antimicrob Chemother 1997; 39: 393 – 400.
Clarithromycin in our earlier in vitro study was highly active 8. Graham DY, Al-Assi MT, Robinson M. Prolonged remission in against MAP, with MICs ranging between 0.125 and 1.0 mg/L.16 Crohn’s disease following therapy for Mycobacterium paratuberculosisinfection. Gastroenterol 1995; 108: A826.
Similar observations have been made in the past by others.15 The 9. Hermon-Taylor J, Barnes N, Clarke C et al. Mycobacterium drug has also been tested as an anti-MAP agent in clinical trials paratuberculosis cervical lymphadenitis, followed five years later by against CD.12,14,20 In the only prior report on the effect of drug terminal ileitis similar to Crohn’s disease. BMJ 1998; 316: 449 –53.
combinations on in vitro growth of MAP, the combination of 10. Shafran I, Kugler L, El-Zaatari FA et al. Open clinical trial of rifa- clarithromycin and ethambutol showed synergy for three of four butin and clarithromycin therapy in Crohn’s disease. Dig Liver Dis MAP strains.15 More drug interaction studies have been reported on M. avium isolates. Piersimoni et al.31 demonstrated synergistic 11. Borody TJ, Bilkey S, Wettstein AR et al. Anti-mycobacterial activity of clarithromycin with rifabutin, ethambutol and cipro- therapy in Crohn’s disease heals mucosa with longitudinal scars. Dig floxacin in 54%, 16% and 8% of 37 clinical M. avium isolates, respectively. In another study on M. avium clinical isolates, clari- 12. Leiper K, Morris AI, Rhodes JM. Open label trial of oral clarithro- thromycin activity was enhanced by ethambutol and rifampicin in mycin in active Crohn’s disease. Aliment Pharmacol Ther 2000; 14: eight and three of nine strains tested, respectively.32 The study demonstrates the utility of a rapid (8 – 10 days) 13. Goodgame RW, Kimball K, Akram S et al. Randomized con- screening method for study of anti-MAP drug interactions in a trolled trial of clarithromycin and ethambutol in the treatment of Crohn’s research setting prior to ex vivo and in vivo experiments. This disease. Aliment Pharmacol Ther 2001; 15: 1861– 6.
in vitro study also demonstrates, for the first time, potential 14. Selby W, Pavli P, Crotty B et al. Two-year combination antibiotic synergistic anti-MAP effects of 6-mercaptopurine – antibacterial therapy with clarithromycin, rifabutin, and clofazimine for Crohn’s drug and clarithromycin – rifampicin drug combinations.
disease. Gastroenterology 2007; 132: 2313– 9.
15. Rastogi N, Goh KS, Labrousse V. Activity of clarithromycin paratuberculosis and further enhancement of its extracellular and intra-cellular activities by ethambutol. Antimicrob Agents Chemother 1992; We thank Kelly Anklam for her valuable technical assistance.
16. Krishnan MY, Manning EJB, Collins MT. Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. para- tuberculosis to 11 antimicrobial drugs. J Antimicrob Chemother 2009;64: 310 – 6.
This study was supported by funding from The Broad Medical 17. Bermudez LE, Yamazaki Y. Effects of macrolides and ketolides Research Programme of the Broad Foundation.
on mycobacterial infections. Curr Pharm Des 2004; 10: 3221 – 8.
Effect of drug combinations on M. avium subspecies paratuberculosis 26. Heifets LB, Iseman MD, Lindholm-Levy PJ. Combinations of rifampin or rifabutine plus ethambutol against Mycobacterium avium complex. Bactericidal synergistic, and bacteriostatic additive or syner- gistic effects. Am Rev Respir Dis 1988; 137: 711 – 5.
19. Griffith DE. Therapy of nontuberculous mycobacterial disease.
27. Odds FC. Synergy, antagonism, and what the chequerboard Curr Opin Infect Dis 2007; 20: 198 –203.
puts between them. J Antimicrob Chemother 2003; 52: 1.
20. Hulte´n K, Almashhrawi A, El-Zaatari FA et al. Antibacterial 28. Chan GL, Erdmann GR, Gruber SA et al. Azathioprine metab- therapy for Crohn’s disease: a review emphasizing therapy directed olism: pharmacokinetics of 6-mercaptopurine, 6-thiouric acid and against mycobacteria. Dig Dis Sci 2000; 45: 445 – 56.
6-thioguanine nucleotides in renal transplant patients. J Clin Pharmacol 21. Akobeng AK. Crohn’s disease: current treatment options. Arch 29. Van Os EC, Zins BJ, Sandborn WJ et al. Azathioprine pharma- 22. Wright S, Sanders DS, Lobo AJ et al. Clinical significance of cokinetics after intravenous, oral, delayed release oral and rectal foam azathioprine active metabolite concentrations in inflammatory bowel administration. Gut 1996; 39: 63– 8.
30. Gearry RB, Barclay ML. Azathioprine and 6-mercaptopurine 23. Greenstein RJ, Su L, Haroutunian V et al. On the action of meth- pharmacogenetics and metabolite monitoring in inflammatory bowel otrexate and 6-mercaptopurine on M. avium subspecies paratuberculo- disease. J Gastroenterol Hepatol 2005; 20: 1149– 57.
31. Piersimoni C, Tortoli E, Mascellino MT et al. Activity of seven 24. Shin SJ, Collins MT. Thiopurine drugs azathioprine and 6-mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrob Agents Chemother 2008; 52: 418 – 26.
J Antimicrob Chemother 1995; 36: 497 – 502.
25. Collins MT, Lisby G, Moser C et al. Results of multiple diagnos- 32. Rastogi N, Labrousse V. Extracellular and intracellular activities tic tests for Mycobacterium avium subsp. paratuberculosis in patients of clarithromycin used alone and in association with ethambutol and with inflammatory bowel disease and in controls. J Clin Microbiol 2000; rifampin against Mycobacterium avium complex. Antimicrob Agents

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