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In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum Sabrina R. Itaqui,a Camila M. Verdi,a Juliana S. M. Tondolo,a Thaisa S. da Luz,a Sydney H. Alves,b Janio M. Santurio,a Érico S. Loretoa Programa de Pós-Graduação em Farmacologia, Centro de Ciências da Saúde, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Rio Grande do Sul, Brazila; Programa de Pós-Graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Rio Grande do Sul, Brazilb
We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 g⁄ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.
P
ythium insidiosum is the main oomycete pathogen associated with severe diseases in humans and animals, particularly horses and dogs. In humans, clinical presentations include ocular, cutaneous⁄subcutaneous, vascular, and disseminated pythiosis. While underlying diseases, such as thalassemia and hemoglobinopathy syndrome, have been observed in cutaneous, vascular, and disseminated forms of disease, ocular pythiosis can affect otherwise healthy individuals. Moreover, no underlying diseases have been observed with animal pythiosis, for which most cases are associated with pyogranulomatous disease in subcutaneous tissue or in a gastrointestinal form (1, 2). Treating pythiosis remains a challenge because this microorganism produces hyaline hyphae similar to those of true fungi but is unable to synthesize ergosterol, which is the direct or indirect main target of most antifungal drugs. Consequently, there are no definitive treatment protocols for this disease, and despite some contradictory results, P. insidiosum can be considered intrinsically resistant to most antifungal drugs (2–4). The most commonly used effective clinical management of pythiosis is aggressive surgical resection, such as amputation, and surgical debridement of skin lesions. Unfortunately, surgical interventions are not always possible, and a high rate of recurrence has been observed. Additionally, immunotherapy cure rates can reach approximately 55% and 80% in humans and horses, respectively (2, 3, 5). Independent of the use of single or combination therapies, the best cure rates are associated with rapid diagnosis and early treatment (1, 2). Regardless of the therapy of choice, little attention has been given to the specific topical treatment of pythiosis lesions. Considering the high tissue concentrations achieved by azithromycin and terbinafine and the above context, this study aimed to assess the in vitro activity for combinations of azithromycin or terbinafine (systemic therapeutic options) and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate (topical therapeutic options). We evaluated the susceptibility of 20 isolates of P. insidiosum from equine pythiosis cases and the reference strain ATCC 58637. Clinical isolates were previously identified by a nested PCR assay (6). The drugs (final concentrations tested in the wells) azithromycin (0.06 to 32 g⁄ml), benzalkonium chloride (0.25 to 32 g⁄ml), alkyltrimethylammonium bromide (cetrimide) (0.5 to 32 g⁄ml), cetylpyridinium chloride (1 to 64 g⁄ml), mupirocin (0.5
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to 32 g⁄ml), potassium permanganate (1 to 64 g⁄ml), terbinafine hydrochloride (1 to 64 g⁄ml), and triclosan (8 to 0.125 g⁄ml) were purchased as standard powders from Sigma-Aldrich (St. Louis, MO). Broth microdilution susceptibility tests were performed following the CLSI M38-A2 protocol (7) as previously described (8, 9). The MICs were determined by visual observation and represented the inhibition of 100% of mycelium growth after 48 h of incubation at 37°C. The interactions between azithromycin or terbinafine with benzalkonium, cetrimide, cetylpyridinium, mupirocin, potassium permanganate, and triclosan were evaluated using a checkerboard test (10). The lowest fractional inhibitory concentration index (FICI) was determined from the nonturbid wells along with the turbidity-nonturbidity growth interface after 48 h of incubation at 37°C and interpreted as follows: FICI ⱕ 0.5, synergism; FICI ⬎ 0.05 to ⱕ4, indifference; FICI ⬎ 4, antagonism. The results of the in vitro susceptibility and combination tests are shown in Table 1. The growth of P. insidiosum isolates was not inhibited by potassium permanganate alone (MIC ⬎ 64 g⁄ml). MIC values (geometric means) (in g⁄ml) ranged from 2 to 16 (4.68) for azithromycin and 4 to 32 (14.56) for terbinafine. Topical antimicrobial drug MICs (in g/ml) ranged from 0.5 to 2 (1.25) for triclosan, 1 to 8 (2.49) for mupirocin, 2 to 8 (3.53) for cetylpyridinium, 4 to 8 (5.31) for benzalkonium, and 4 to 16 (7.75) for cetrimide. The combinations of antimicrobial drugs showed a predominance of indifferent interactions (⬎80%) when azithromycin was associated with topical antimicrobials. Similarly, when terbinafine was combined with topical antimicrobials, there was a predominance of indifference (⬎60%), except for its combination with
Received 18 January 2016 Returned for modification 12 March 2016 Accepted 15 May 2016 Accepted manuscript posted online 23 May 2016 Citation Itaqui SR, Verdi CM, Tondolo JSM, da Luz TS, Alves SH, Santurio JM, Loreto ES. 2016. In vitro synergism between azithromycin or terbinafine and topical antimicrobial agents against Pythium insidiosum. Antimicrob Agents Chemother 60:5023–5025. doi:10.1128/AAC.00154-16. Address correspondence to Érico S. Loreto, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 In vitro activities of azithromycin and terbinafine, alone or in combination with topical antimicrobial drugs, against 21 Pythium insidiosum isolates Individual drugs
Drug combinations
Interpretation (%)
Antimicrobial agenta
MIC90 MIC range (GM)b (in g/ml) (g/ml) Drugs
Systemic drugs AZT TRB
8 32
2–16 (4.68) 4–32 (14.56)
AZT ⫹ BAC AZT ⫹ CET AZT ⫹ CPC
0.25–4 (1.07) ⫹ 2–4 (2.57) 1–8 (2.13) ⫹ 2–8 (3.53) 0.25–8 (1.25) ⫹ 1–4 (1.66)
0.38–1 (0.75) 0.50–1 (0.91) 0.50–1 (0.76)
4.8 14.3 10
95.2 85.7 90
Topical drugs BAC CET CPC MUP Potassium permanganatec TCS
8 16 8 4 ⬎64 2
4–8 (5.31) 4–16 (7.75) 2–8 (3.53) 1–8 (2.49) ⬎64 0.5–2 (1.25)
AZT ⫹ MUP AZT ⫹ TCS TRB ⫹ BAC TRB ⫹ CET TRB ⫹ CPC TRB ⫹ MUP TRB ⫹ TCS
1–4 (2.06) ⫹ 0.25–2 (1.10) 0.06–8 (0.60) ⫹ 0.25–1 (0.53) 0.25–8 (2.34) ⫹ 0.5–4 (1.82) 0.125–8 (1.76) ⫹ 0.5–4 (1.94) 1–8 (2.27) ⫹ 1–4 (1.71) 0.06–8 (3.01) ⫹ 0.5–4 (1.0) 0.25–16 (4.13) ⫹ 0.25–1 (0.55)
0.50–1.5 (0.91) 0.38–1 (0.62) 0.19–1 (0.52) 0.19–1 (0.38) 0.5–1 (0.67) 0.38–1 (0.64) 0.50–1 (0.77)
19 19 38.1 71.4 4.8 33.3 19
81 81 61.9 28.6 95.2 66.7 81
Associated MIC ranges (GMs) (g/ml)
Mean FICI range (GM) (g/ml) Synergistic Indifferent
a
AZT, azithromycin; BAC, benzalkonium; CET, cetrimide; CPC, cetylpyridinium; MUP, mupirocin; TCS, triclosan; TRB, terbinafine. GM, geometric mean. c 100% of indifferent interactions when combined with azithromycin or terbinafine. b
cetylpyridinium, for which a 71.4% predominance of synergistic interactions was observed. The interaction of azithromycin or terbinafine with potassium permanganate resulted in 100% indifferent interactions (data not shown). Antagonistic interactions were not observed. In this study, we described systemic and topical antimicrobial drugs that individually inhibit the in vitro growth of P. insidiosum. Almost none of the evaluated antimicrobials has a very low MIC, and MIC values were relatively high for some of them, such as cetrimide and terbinafine. However, the use of these compounds is favored by the high concentration allowed by the topical mode of application. Additionally, although cetylpyridinium and triclosan are widely used as oral antiseptics, they also have potential for the treatment of skin infections (11, 12). Previous studies demonstrated the in vitro and in vivo susceptibility of P. insidiosum to azithromycin, mupirocin, and terbinafine (8, 13–19) with in vitro results similar to those of our study. Although all other evaluated antimicrobials have a wide range of known antimicrobial activities against bacteria, some fungi, and parasites (20, 21), this study was the first to evaluate the antimicrobial activities of benzalkonium, cetrimide, cetylpyridinium, potassium permanganate, and triclosan against clinically isolated P. insidiosum based on broth microdilution susceptibility tests. Toxicity data for triclosan from mammalian studies, including those of humans, were been reviewed by Rodricks et al. (12). Triclosan products are not expected to cause adverse health effects, and there has been little indication of toxicity or sensitization. Pharmaceutical formulations containing quaternary ammonium compounds (benzalkonium, cetrimide, and cetylpyridinium) are nontoxic when applied to skin or mucous membranes and are generally well tolerated (22). Azithromycin, mupirocin, and terbinafine have known pharmacology, are known to be safe, and are widely used to treat infections. Azithromycin (23) and terbinafine (24) are generally safe and well tolerated after oral administration, and most cases of toxicity are related to higher doses. Mupirocin is well tolerated with topical treatment, and adverse effects are observed in less than 2% of users (25). Potassium permanganate should always be thoroughly diluted in water (e.g., 1:10,000), as higher concentrations can cause necrosis of the skin (26).
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It is important to note that the treatment of pythiosis is complex and, in most cases, requires surgical intervention, immunotherapy, and systemic antimicrobial chemotherapy. Therefore, the use of topical antimicrobials for lesions should be a complementary treatment to the main therapies. Larger in vitro and in vivo susceptibility studies are needed to elucidate the potential of the topical antimicrobials evaluated in this study for the treatment of pythiosis. In conclusion, the in vitro susceptibility data reported in this study show that the growth of P. insidiosum clinical isolates was inhibited by all antimicrobials evaluated, with the exception of potassium permanganate. Although there was a predominance of indifferent interactions between the combinations of the evaluated antimicrobials, the absence of antagonism and the lack of information regarding the topical therapy of pythiosis suggest that the antimicrobials evaluated have potential as new supplementary topical therapeutic options for the treatment of pythiosis. ACKNOWLEDGMENTS This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES-AUX PE-PNPD 743/2012). E.S.L. is a Ph.D. fellow of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (PNPD-CAPES). We declare no conflicts of interest.
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5. Santos CEP, Ubiali DG, Pescador CA, Zanette RA, Santurio JM, Marques LC. 2014. Epidemiological survey of equine pythiosis in the Brazilian Pantanal and nearby areas: results of 76 cases. J Equine Vet Sci 34:270 –274. http://dx.doi.org/10.1016/j.jevs.2013.06.003. 6. Botton SA, Pereira DIB, Costa MM, Azevedo MI, Argenta JS, Jesus FPK, Alves SH, Santurio JM. 2011. Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits. Curr Microbiol 62:1225–1229. http://dx.doi.org/10.1007/s00284-010-9781-4. 7. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. CLSI M38-A2, 2nd ed. Clinical and Laboratory Standards Institute, Wayne, PA. 8. Loreto ÉS, Tondolo JSM, Pilotto MB, Alves SA, Santurio JM. 2014. New insights into the in vitro susceptibility of Pythium insidiosum. Antimicrob Agents Chemother 58:7534 –7537. http://dx.doi.org/10.1128 /AAC.02680-13. 9. Pereira DIB, Santurio JM, Alves SH, Argenta JS, Potter L, Spanamberg A, Ferreiro L. 2007. Caspofungin in vitro and in vivo activity against Brazilian Pythium insidiosum strains isolated from animals. J Antimicrob Chemother 60:1168 –1171. http://dx.doi.org/10.1093/jac/dkm332. 10. Moody J. 2007. Synergism testing: broth microdilution checkerboard and broth macrodilution methods, p 1–23. In Garcia LS, Isenberg HD (ed), Clinical microbiology procedures handbook, 2nd ed. ASM Press, Washington, DC. 11. Fromm-Dornieden C, Rembe JD, Schafer N, Bohm J, Stuermer EK. 2015. Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management—prospects and limitations. J Med Microbiol 64:407– 414. http://dx.doi.org/10.1099/jmm.0.000034. 12. Rodricks JV, Swenberg JA, Borzelleca JF, Maronpot RR, Shipp AM. 2010. Triclosan: a critical review of the experimental data and development of margins of safety for consumer products. Crit Rev Toxicol 40: 422– 484. http://dx.doi.org/10.3109/10408441003667514. 13. Argenta JS, Alves SH, Silveira F, Maboni G, Zanette RA, Cavalheiro AS, Pereira PL, Pereira DIB, Sallis ESV, Potter L, Santurio JM, Ferreiro L. 2012. In vitro and in vivo susceptibility of two-drug and three-drug combinations of terbinafine, itraconazole, caspofungin, ibuprofen and fluvastatin against Pythium insidiosum. Vet Microbiol 157:137–142. http://dx .doi.org/10.1016/j.vetmic.2011.12.003. 14. Argenta JS, Santurio JM, Alves SH, Pereira DIB, Cavalheiro AS, Spanamberg A, Ferreiro L. 2008. In vitro activities of voriconazole, itraconazole, and terbinafine alone or in combination against Pythium insidiosum isolates from Brazil. Antimicrob Agents Chemother 52:767–769. http://dx .doi.org/10.1128/AAC.01075-07. 15. Brown TA, Grooters AM, Hosgood GL. 2008. In vitro susceptibility of Pythium insidiosum and a Lagenidium sp to itraconazole, posaconazole, voriconazole, terbinafine, caspofungin, and mefenoxam. Am J Vet Res 69:1463–1468. http://dx.doi.org/10.2460/ajvr.69.11.1463.
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16. Cavalheiro AS, Maboni G, de Azevedo MI, Argenta JS, Pereira DIB, Spader TB, Alves SH, Santurio JM. 2009. In vitro activity of terbinafine combined with caspofungin and azoles against Pythium insidiosum. Antimicrob Agents Chemother 53:2136 –2138. http://dx.doi.org/10 .1128/AAC.01506-08. 17. Heath JA, Kiehn TE, Brown AE, LaQuaglia MP, Steinherz LJ, Bearman G, Wong M, Steinherz PG. 2002. Pythium insidiosum pleuropericarditis complicating pneumonia in a child with leukemia. Clin Infect Dis 35:e60 – e64. http://dx.doi.org/10.1086/342303. 18. Jesus FP, Loreto ES, Ferreiro L, Alves SH, Driemeier D, Souza SO, Franca RT, Lopes ST, Pilotto MB, Ludwig A, Azevedo MI, Ribeiro TC, Tondolo JS, Santurio JM. 2016. In vitro and in vivo antimicrobial activities of minocycline in combination with azithromycin, clarithromycin, or tigecycline against Pythium insidiosum. Antimicrob Agents Chemother 60:87–91. http://dx.doi.org/10.1128/AAC.01480-15. 19. Loreto ES, Mario DAN, Denardi LB, Alves SH, Santurio JM. 2011. In vitro susceptibility of Pythium insidiosum to macrolides and tetracycline antibiotics. Antimicrob Agents Chemother 55:3588 –3590. http://dx.doi .org/10.1128/AAC.01586-10. 20. Al-Adham I, Haddadin R, Collier P. 2013. Types of microbicidal and microbistatic agents, p 5–70. In Fraise AP, Maillard JY, Sattar S (ed), Principles and practice of disinfection, preservation, and sterilization, 5th ed. John Wiley & Sons, Chichester, West Sussex, United Kingdom. 21. Porras-Luque JI. 2007. Topical antimicrobial agents in dermatology. Actas Dermosifiliogr 98:29 –39. http://dx.doi.org/10.1016/S0001-7310(07) 70179-5. 22. Gorman SP, Gilmore BF. 2011. Chemical disinfectants, antiseptics and preservatives, p 312–333. In Denyer SP, Hodges N, Gorman SP, Gilmore BF (ed), Hugo and Russell’s pharmaceutical microbiology, 8th ed. WileyBlackwell, Oxford, United Kingdom. 23. Gordon C, Bambeke FV. 2010. Azithromycin, p 801– 818. In Grayson ML, Kucers A (ed), Kucers’ the use of antibiotics: a clinical review of antibacterial, antifungal, antiparasitic and antiviral drugs, 6th ed. Hodder Arnold, London, United Kingdom. 24. Traboulsi R, Ghannoum M. 2010. Terbinafine, p 1763–1771. In Grayson ML, Kucers A (ed), Kucers’ the use of antibiotics: a clinical review of antibacterial, antifungal, antiparasitic and antiviral drugs, 6th ed. Hodder Arnold, London, United Kingdom. 25. Kluytmans J, Murk JL. 2010. Mupirocin, p 980 –986. In Grayson ML, Kucers A (ed), Kucers’ the use of antibiotics: a clinical review of antibacterial, antifungal, antiparasitic and antiviral drugs, 6th ed. Hodder Arnold, London, United Kingdom. 26. Schnopp C, Ring J, Mempel M. 2010. The role of antibacterial therapy in atopic eczema. Expert Opin Pharmacother 11:929 –936. http://dx.doi.org /10.1517/14656561003659992.
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In Vitro Synergism between Azithromycin or Terbinafine and Topical Antimicrobial Agents against Pythium insidiosum Sabrina R. Itaqui,a Camila M. Verdi,a Juliana S. M. Tondolo,a Thaisa S. da Luz,a Sydney H. Alves,b Janio M. Santurio,a Érico S. Loretoa Programa de Pós-Graduação em Farmacologia, Centro de Ciências da Saúde, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Rio Grande do Sul, Brazila; Programa de Pós-Graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde, Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Rio Grande do Sul, Brazilb
We describe here in vitro activity for the combination of azithromycin or terbinafine and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate. With the exception of potassium permanganate, the remaining antimicrobial drugs were active and had an MIC90 between 2 and 32 g⁄ml. The greatest synergism was observed for the combination of terbinafine and cetrimide (71.4%). In vivo experimental evaluations will clarify the potential of these drugs for the topical treatment of lesions caused by Pythium insidiosum.
P
ythium insidiosum is the main oomycete pathogen associated with severe diseases in humans and animals, particularly horses and dogs. In humans, clinical presentations include ocular, cutaneous⁄subcutaneous, vascular, and disseminated pythiosis. While underlying diseases, such as thalassemia and hemoglobinopathy syndrome, have been observed in cutaneous, vascular, and disseminated forms of disease, ocular pythiosis can affect otherwise healthy individuals. Moreover, no underlying diseases have been observed with animal pythiosis, for which most cases are associated with pyogranulomatous disease in subcutaneous tissue or in a gastrointestinal form (1, 2). Treating pythiosis remains a challenge because this microorganism produces hyaline hyphae similar to those of true fungi but is unable to synthesize ergosterol, which is the direct or indirect main target of most antifungal drugs. Consequently, there are no definitive treatment protocols for this disease, and despite some contradictory results, P. insidiosum can be considered intrinsically resistant to most antifungal drugs (2–4). The most commonly used effective clinical management of pythiosis is aggressive surgical resection, such as amputation, and surgical debridement of skin lesions. Unfortunately, surgical interventions are not always possible, and a high rate of recurrence has been observed. Additionally, immunotherapy cure rates can reach approximately 55% and 80% in humans and horses, respectively (2, 3, 5). Independent of the use of single or combination therapies, the best cure rates are associated with rapid diagnosis and early treatment (1, 2). Regardless of the therapy of choice, little attention has been given to the specific topical treatment of pythiosis lesions. Considering the high tissue concentrations achieved by azithromycin and terbinafine and the above context, this study aimed to assess the in vitro activity for combinations of azithromycin or terbinafine (systemic therapeutic options) and benzalkonium, cetrimide, cetylpyridinium, mupirocin, triclosan, or potassium permanganate (topical therapeutic options). We evaluated the susceptibility of 20 isolates of P. insidiosum from equine pythiosis cases and the reference strain ATCC 58637. Clinical isolates were previously identified by a nested PCR assay (6). The drugs (final concentrations tested in the wells) azithromycin (0.06 to 32 g⁄ml), benzalkonium chloride (0.25 to 32 g⁄ml), alkyltrimethylammonium bromide (cetrimide) (0.5 to 32 g⁄ml), cetylpyridinium chloride (1 to 64 g⁄ml), mupirocin (0.5
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to 32 g⁄ml), potassium permanganate (1 to 64 g⁄ml), terbinafine hydrochloride (1 to 64 g⁄ml), and triclosan (8 to 0.125 g⁄ml) were purchased as standard powders from Sigma-Aldrich (St. Louis, MO). Broth microdilution susceptibility tests were performed following the CLSI M38-A2 protocol (7) as previously described (8, 9). The MICs were determined by visual observation and represented the inhibition of 100% of mycelium growth after 48 h of incubation at 37°C. The interactions between azithromycin or terbinafine with benzalkonium, cetrimide, cetylpyridinium, mupirocin, potassium permanganate, and triclosan were evaluated using a checkerboard test (10). The lowest fractional inhibitory concentration index (FICI) was determined from the nonturbid wells along with the turbidity-nonturbidity growth interface after 48 h of incubation at 37°C and interpreted as follows: FICI ⱕ 0.5, synergism; FICI ⬎ 0.05 to ⱕ4, indifference; FICI ⬎ 4, antagonism. The results of the in vitro susceptibility and combination tests are shown in Table 1. The growth of P. insidiosum isolates was not inhibited by potassium permanganate alone (MIC ⬎ 64 g⁄ml). MIC values (geometric means) (in g⁄ml) ranged from 2 to 16 (4.68) for azithromycin and 4 to 32 (14.56) for terbinafine. Topical antimicrobial drug MICs (in g/ml) ranged from 0.5 to 2 (1.25) for triclosan, 1 to 8 (2.49) for mupirocin, 2 to 8 (3.53) for cetylpyridinium, 4 to 8 (5.31) for benzalkonium, and 4 to 16 (7.75) for cetrimide. The combinations of antimicrobial drugs showed a predominance of indifferent interactions (⬎80%) when azithromycin was associated with topical antimicrobials. Similarly, when terbinafine was combined with topical antimicrobials, there was a predominance of indifference (⬎60%), except for its combination with
Received 18 January 2016 Returned for modification 12 March 2016 Accepted 15 May 2016 Accepted manuscript posted online 23 May 2016 Citation Itaqui SR, Verdi CM, Tondolo JSM, da Luz TS, Alves SH, Santurio JM, Loreto ES. 2016. In vitro synergism between azithromycin or terbinafine and topical antimicrobial agents against Pythium insidiosum. Antimicrob Agents Chemother 60:5023–5025. doi:10.1128/AAC.00154-16. Address correspondence to Érico S. Loreto, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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TABLE 1 In vitro activities of azithromycin and terbinafine, alone or in combination with topical antimicrobial drugs, against 21 Pythium insidiosum isolates Individual drugs
Drug combinations
Interpretation (%)
Antimicrobial agenta
MIC90 MIC range (GM)b (in g/ml) (g/ml) Drugs
Systemic drugs AZT TRB
8 32
2–16 (4.68) 4–32 (14.56)
AZT ⫹ BAC AZT ⫹ CET AZT ⫹ CPC
0.25–4 (1.07) ⫹ 2–4 (2.57) 1–8 (2.13) ⫹ 2–8 (3.53) 0.25–8 (1.25) ⫹ 1–4 (1.66)
0.38–1 (0.75) 0.50–1 (0.91) 0.50–1 (0.76)
4.8 14.3 10
95.2 85.7 90
Topical drugs BAC CET CPC MUP Potassium permanganatec TCS
8 16 8 4 ⬎64 2
4–8 (5.31) 4–16 (7.75) 2–8 (3.53) 1–8 (2.49) ⬎64 0.5–2 (1.25)
AZT ⫹ MUP AZT ⫹ TCS TRB ⫹ BAC TRB ⫹ CET TRB ⫹ CPC TRB ⫹ MUP TRB ⫹ TCS
1–4 (2.06) ⫹ 0.25–2 (1.10) 0.06–8 (0.60) ⫹ 0.25–1 (0.53) 0.25–8 (2.34) ⫹ 0.5–4 (1.82) 0.125–8 (1.76) ⫹ 0.5–4 (1.94) 1–8 (2.27) ⫹ 1–4 (1.71) 0.06–8 (3.01) ⫹ 0.5–4 (1.0) 0.25–16 (4.13) ⫹ 0.25–1 (0.55)
0.50–1.5 (0.91) 0.38–1 (0.62) 0.19–1 (0.52) 0.19–1 (0.38) 0.5–1 (0.67) 0.38–1 (0.64) 0.50–1 (0.77)
19 19 38.1 71.4 4.8 33.3 19
81 81 61.9 28.6 95.2 66.7 81
Associated MIC ranges (GMs) (g/ml)
Mean FICI range (GM) (g/ml) Synergistic Indifferent
a
AZT, azithromycin; BAC, benzalkonium; CET, cetrimide; CPC, cetylpyridinium; MUP, mupirocin; TCS, triclosan; TRB, terbinafine. GM, geometric mean. c 100% of indifferent interactions when combined with azithromycin or terbinafine. b
cetylpyridinium, for which a 71.4% predominance of synergistic interactions was observed. The interaction of azithromycin or terbinafine with potassium permanganate resulted in 100% indifferent interactions (data not shown). Antagonistic interactions were not observed. In this study, we described systemic and topical antimicrobial drugs that individually inhibit the in vitro growth of P. insidiosum. Almost none of the evaluated antimicrobials has a very low MIC, and MIC values were relatively high for some of them, such as cetrimide and terbinafine. However, the use of these compounds is favored by the high concentration allowed by the topical mode of application. Additionally, although cetylpyridinium and triclosan are widely used as oral antiseptics, they also have potential for the treatment of skin infections (11, 12). Previous studies demonstrated the in vitro and in vivo susceptibility of P. insidiosum to azithromycin, mupirocin, and terbinafine (8, 13–19) with in vitro results similar to those of our study. Although all other evaluated antimicrobials have a wide range of known antimicrobial activities against bacteria, some fungi, and parasites (20, 21), this study was the first to evaluate the antimicrobial activities of benzalkonium, cetrimide, cetylpyridinium, potassium permanganate, and triclosan against clinically isolated P. insidiosum based on broth microdilution susceptibility tests. Toxicity data for triclosan from mammalian studies, including those of humans, were been reviewed by Rodricks et al. (12). Triclosan products are not expected to cause adverse health effects, and there has been little indication of toxicity or sensitization. Pharmaceutical formulations containing quaternary ammonium compounds (benzalkonium, cetrimide, and cetylpyridinium) are nontoxic when applied to skin or mucous membranes and are generally well tolerated (22). Azithromycin, mupirocin, and terbinafine have known pharmacology, are known to be safe, and are widely used to treat infections. Azithromycin (23) and terbinafine (24) are generally safe and well tolerated after oral administration, and most cases of toxicity are related to higher doses. Mupirocin is well tolerated with topical treatment, and adverse effects are observed in less than 2% of users (25). Potassium permanganate should always be thoroughly diluted in water (e.g., 1:10,000), as higher concentrations can cause necrosis of the skin (26).
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It is important to note that the treatment of pythiosis is complex and, in most cases, requires surgical intervention, immunotherapy, and systemic antimicrobial chemotherapy. Therefore, the use of topical antimicrobials for lesions should be a complementary treatment to the main therapies. Larger in vitro and in vivo susceptibility studies are needed to elucidate the potential of the topical antimicrobials evaluated in this study for the treatment of pythiosis. In conclusion, the in vitro susceptibility data reported in this study show that the growth of P. insidiosum clinical isolates was inhibited by all antimicrobials evaluated, with the exception of potassium permanganate. Although there was a predominance of indifferent interactions between the combinations of the evaluated antimicrobials, the absence of antagonism and the lack of information regarding the topical therapy of pythiosis suggest that the antimicrobials evaluated have potential as new supplementary topical therapeutic options for the treatment of pythiosis. ACKNOWLEDGMENTS This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES-AUX PE-PNPD 743/2012). E.S.L. is a Ph.D. fellow of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (PNPD-CAPES). We declare no conflicts of interest.
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