Mycopathologia (2014) 178:315–319 DOI 10.1007/s11046-014-9782-0
In Vitro Antifungal Susceptibility of Malassezia pachydermatis Strains Isolated from Dogs with Chronic and Acute Otitis Externa E. Chiavassa • P. Tizzani • A. Peano
Received: 31 July 2013 / Accepted: 26 June 2014 / Published online: 17 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa. Topical therapies with different antifungal agents, mainly azole compounds, are generally successful in controlling the yeast overgrowth, but treatment failure and rapid recurrences are common. This study compared the in vitro antifungal susceptibility of M. pachydermatis isolates obtained from chronic and acute cases of otitis externa. The aim was to assess the possible onset of resistance mechanisms in isolates involved in long-lasting episodes with poor response to treatment. We evaluated the in vitro susceptibility to miconazole (MCZ) and clotrimazole (CTZ) of 42 isolates of M. pachydermatis obtained from dogs with chronic (group A, n = 25) and acute otitis (group B, n = 17), using a modified CLSI M27-A3 microdilution method. All isolates were inhibited by the antifungal agents employed, but Malassezia isolates from group A were significantly associated with higher minimum inhibitory concentration (MIC) values for both agents (Median MIC values: MCZ group A 2 lg/ml, group B 1 lg/ml; CTZ group A 8 lg/ml, group B 4 lg/ml). These findings prove that these isolates had a reduced in vitro
E. Chiavassa (&) P. Tizzani A. Peano Settore di Parassitologia e Malattie Parassitarie, Dipartimento di Scienze Veterinarie, Universita` degli Studi di Torino, Via Leonardo da Vinci 44, Grugliasco, 10095 Turin, Italy e-mail: [email protected]
susceptibility to the antifungal agents tested. However, it is unlikely that this could have any influence on the outcome of a topical treatment. Indeed, marketed products include concentrations of the tested agents that largely exceed even the highest MICs found in this study (in most cases at least 1,000 9 the MIC, or greater). In conclusion, this study suggests that isolates of M. pachydermatis involved in chronic cases of canine external otitis and exposed to repeated antifungal treatments are unlikely to develop mechanisms of resistance of clinical relevance. Keywords Malassezia pachydermatis Antifungal susceptibility CLSI reference broth microdilution method Otitis externa
Introduction Otitis externa represents one of the most common reasons for which dogs are presented to veterinary attention [1]. Malassezia pachydermatis is the yeast isolated with the highest frequency from affected ears [2]. This organism is normally present in the external ear canal and rapidly multiplies during favourable conditions, thus becoming a secondary factor of otitis [1]. Factors that predispose dogs to Malassezia otitis externa (MOE) include anatomical anomalies or changes that create ear canal stenosis, increased cerumen secretion or retention, moisture, and
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inhibition of air circulation. Under these conditions, primary inflammatory diseases of the ear canal, which can occur as part of atopic dermatitis or adverse food reactions, can often lead to MOE. Moreover, a hypersensitivity response to the yeast itself is likely to occur in many dogs with allergies [3, 4]. In clinical practice, topical therapy appears to be the most practical and popular way to treat MOE, and different azoles, such as clotrimazole (CTZ) and miconazole (MCZ), are used in aural formulations [3]. These agents are generally successful in controlling the yeast overgrowth, but treatment failure or recurrences are common, usually when primary causes, and predisposing factors are unidentified or uncorrected [5]. Unsatisfactory responses to therapy are also often attributed to the yeast strain being ‘‘resistant’’ to the antifungal employed, with a consequent switch to another agent. This approach may be supported by previous reports of M. pachydermatis strains being resistant to various drugs, based on the results of in vitro susceptibility testing [6–10]. Moreover, it has been previously shown that multiple in vitro passages at subtherapeutic concentrations of azole agents can lead to a decrease in susceptibility of M. pachydermatis to these agents, thus suggesting that this species is capable of developing resistance mechanisms [11]. However, this possibility has not yet been confirmed in vivo. In addition, compared to the extensive research carried out for other medically relevant yeast species (e.g. Candida spp. And Cryptococcus spp.) [12–14], the value of in vitro susceptibility testing for predicting the therapy outcome of M. pachydermatis treatment has been far less comprehensively investigated. Indeed, standard parameters and guidelines specifically dedicated to this yeast are not yet available, and neither are interpretive criteria. This lack of standardization in methodology and interpretation for susceptibility testing raises the question of the reliability and clinical value of previous in vitro results, and consequently challenges the existence of clinically relevant resistance in field isolates of M. pachydermatis. In order to contribute in clarifying this matter, this study compared the in vitro antifungal susceptibility of M. pachydermatis isolates from chronic and acute cases of otitis externa, with the intent of individuating differences potentially ascribable to the onset of resistant mechanisms in the isolates exposed to treatment.
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Mycopathologia (2014) 178:315–319 Table 1 Sex, breed and age of dogs with chronic Malassezia otitis (group A) and acute Malassezia otitis (group B) Group A (n = 18)
Group B (n = 11)
Sex Male
10
3
8
8
Labrador Retriever
1
0
Golden Retriever
1
2
German Shepherd
2
0
Springer Spaniel
3
0
Beagle
1
0
Female Breed
Shi-tzu
2
0
Pug
2
2
Newfoundland
0
2
American Bulldog
3
0
Poodle
2
0
1
5
5.7
3.5
Mix breed Age Average
Materials and Methods Samples M. pachydermatis isolates were collected from 18 dogs with chronic otitis (group A) and from 11 dogs with acute otitis (group B). Background of animals included in this study is summarized in Table 1. In cases of bilateral otitis, each ear was considered separately, so that overall 25 and 17 isolates from group A and B, respectively, were available for the analyses. Otitis was considered as being chronic in cases that lasted for more than 2 months (Hensel et al. 15). All dogs with chronic MOE had been previously treated with various topical ear products containing MCZ and CTZ; conversely dogs with acute MOE had never received specific treatments for M. pachydermatis. For purpose of comparison, the control isolates were chosen from group of dogs (group B) without previous exposure to antifungal agents. Isolates were collected with sterile swabs introduced into the ear canal and cultured on Sabouraud dextrose agar (SDA) (Biolife Italiana, Milan) supplemented with gentamicin and cloramphenicol. Identification of M. pachydermatis was based on macroscopic and microscopic morphology of colonies. The use of SDA allowed
Mycopathologia (2014) 178:315–319
confirming the identification of the yeast, since M. Pachydermatis is the only representative of the genus Malassezia able to grow on lipid-free media [3, 4]. In Vitro Antifungal Susceptibility testing The Clinical and Laboratory Standards Institute (CLSI) procedure for susceptibility testing of yeasts described in the M27-A3 document [16] was used in the study, with specific adaptations for M. pachydermatis as described previously [17, 18]. In particular, Christensen’s urea broth medium with Tween 80 and Tween 40 proved to be an optimal growth support for M. Pachydermatis [17, 18] compared to the RPMI 1640 medium suggested by the CLSI. Uniform yeast suspensions were prepared in the culture medium from 4-dayold colonies subcultured at 37 °C on SDA. Inoculum suspensions were adjusted using a spectrophotometer, and viable colony counts were performed to confirm inoculum size. Final yeast cell concentrations were approximately 1–5 9 105 cells/ml. Stock suspensions of MCZ (Janssen Pharmaceuticals, Beerse, Belgium) and CTZ (Sigma Aldrich, Milan, Italy) were prepared in dimethyl sulfoxide. Twofold dilutions of the drug solutions were dispensed into 96-well plates, with final drug concentrations of 0.06–32 lg/ml. Plates were incubated at 37 °C and analysed at 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed a 50 % decrease or more of optical density compared to the growth control, as assessed by using a spectrophotometer (630-nm filter).
317 Table 2 Miconazole (MCZ) and Clotrimazole (CTZ) MICs (lg/ml) for Malassezia pachydermatis isolates from dogs with chronic otitis (group A) and with acute otitis (group B) MIC (lg/ ml)
Group A (n = 25)
Group B (n = 17)
MCZ
CTZ
MCZ
CTZ
32 16 8 4 2 1 0.5 0.25 0.125 0.06
0 0 1 (4.0 %) 5 (20.0 %) 17 (68.0 %) 2 (8.0 %) 0 0 0 0
0 3 (12.0 %) 10 (40.0 %) 11 (44.0 %) 1 (4.0 %) 0 0 0 0 0
0 0 0 1 (5.9 %) 7 (41.2 %) 9 (52.9 %) 0 0 0 0
0 0 1 (5.9 %) 10 (58.8 %) 6 (35.3 %) 0 0 0 0 0
MCZ: v2 = 11.17, p value = 0.005
p
value = 0.01;
CTZ:
v2 = 12.93,
while MICs for group B strains were 1–4 (MCZ) and 2–8 (CTZ) lg/ml (Table 2). MIC50 (MIC required to inhibit growth of at least 50 % of the isolates tested) for group A was 8 (CTZ) and 2 (MCZ) lg/ml, while for group B, it was 4 (CTZ) and 1 (MCZ) lg/ml. MIC90 (MIC required to inhibit growth of at least 90 % of the isolates tested) for group A was 16 (CTZ) and 4 (MCZ) lg/ml, while for group B, it was 4 (CTZ) and 2 (MCZ) lg/ml. Significant differences were found between MICs in group A and group B for both agents (Table 2), with Malassezia isolates of dogs with acute otitis associated with significantly lower MIC values (MCZ: Fisher = 8.37, p = 0.003, Odds Ratio = 0.077; CTZ: Fisher = 5.06, p = 0.02, Odds ratio = 0.076).
Statistical Analysis Differences of frequencies for MCZ and CTZ MICs between group A and group B were evaluated using the Chi square test. Contingency tables and the Fisher’s exact test were used to evaluate the odds ratio related to group A and group B frequencies for ‘‘low MIC values’’. A ‘‘low MIC value’’ was intended as the lowest MIC found for each agent. A p value of \ 0.05 was considered to be statistically significant.
Results The MIC range of M. pachydermatis isolates from group A were 1–8 (MCZ) and 2–16 (CTZ) lg/ml,
Discussion In the present study, we focused on the role of M. pachydermatis and, in particular, we investigated whether cases with poor responses to therapy could be due to an emerging drug resistance of the yeast. This issue is worth expanding due to the repeated reports of M. pachydermatis isolates resistant to different antifungal agents commonly used in the dog [7, 9, 10, 19–26]. Yeast isolates were thus collected from dogs with chronic Malassezia otitis poorly responding to therapy (group A). The animals with the above-mentioned characteristics were chosen since such patients are frequently
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presented for consultation after having been treated with numerous available commercial products containing different antifungals. Accordingly, these patients may have a higher probability of harbouring yeast strains that have developed resistance mechanisms due to repeated exposure to several antifungals, frequently with incorrect dosages. Through the CLSI-modified method all Malassezia isolates, from both groups, were found to be inhibited by the azole drugs employed, with MCZ showing higher absolute activity against M. pachydermatis compared with CTZ, as reported previously [17]. Regarding the comparison between the two groups of animals, the distribution of MIC values was significantly increased for isolates sampled from dogs with chronic otitis. This does not necessarily mean that the increased MICs are associated with clinical failure; instead, it demonstrates at best that these strains have a reduced in vitro susceptibility to antifungal agents, perhaps due to repeated exposure to a number of antifungal agents. Indeed, marketed products include concentrations of MCZ and CTZ that largely exceed even the highest MICs found in this study (in most cases at least 1,000 9 the MIC, or greater), and the antifungal agents are likely to reach adequate tissue levels for controlling the yeast overgrowth because such products are used topically, that is, they are instilled directly into the ear canals. Moreover, these products may be applied several times a day, thus ensuring a cumulative local effect. These conclusions provide an opportunity to briefly comment on the previous literature, which may instead suggest that lower antifungals’ potency accounts for poor responses to therapy in cases of Malassezia otitis, as several studies reported yeast strains resistant to various commonly used agents in veterinary practice [6–10, 19–27, 29]. Evaluation of these previous studies reveals that the evidence in support of the existence of clinically relevant resistance was weak. Indeed, all were based entirely on results of in vitro susceptibility tests, and in vitro results were never related to the outcome of therapy. Moreover, none of the breakpoints employed to categorize strains as being susceptible/resistant had a rationale capable of giving a predictive value of the final clinical outcome. For example, the approaches of Rougier [10] and Lyskova [9], who respectively referred to ‘‘zone interpretive standard breakpoints for veterinary pathogens’’ and ‘‘zone diameter interpretive standards for ‘‘microscopic fungi’’, appear to be too
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generic and thus of poor clinical relevance. Another way of categorizing strains, through the use of the MIC50/ MIC90 values (S if MIC of the strain B MIC50, R If MIC of the strain [ MIC90), appears to be an accepted and valid interpretive criterion as it has been employed in several studies [7, 8, 19, 20, 23–25]. However, although MIC50 and MIC90 values serve to describe and summarize the distribution of MICs for a population of organisms, they do not necessarily mirror the concentrations that the tested drugs can reach in infected tissues. Finally, in two other studies [21, 27] the breakpoints established for Candida [30] were applied to M. pachydermatis. These breakpoints have been based on a number of pharmacokinetic studies of the drugs, and the evaluation of the clinical outcome in human infections [14]. Thus, such a categorization appears to be of poor significance once applied to a different fungus/host combination (i.e. M. pachydermatis/dog). In conclusion, additional reasons (incorrect management of the underlying conditions; incorrect drug administration; poor compliance of the owner, etc.) were probably responsible for treatment failure and the consequent chronicization of Malassezia otitis in the animals studied, and this supports the view that treatment failure of Malassezia dermatitis/otitis can be attributed in most cases to poor management of conditions underlying the yeast overgrowth [5], rather than to drug resistance. Moreover, our results show that resistance mechanisms of clinical relevance are unlikely to develop, at least towards MCZ and CTZ, in strains of M. pachydermatis exposed to lasting antifungal therapies. Further analyses on an extended data set of strains are necessary to confirm these conclusions and to verify whether they also apply to other antifungal agents commonly used in the dog. Acknowledgments This work was supported by a grant of Elanco Animal Health, division of Eli Lilly Italy S.p.A. The authors express their acknowledgments to Elena Borio and Roberta Morandi for their laboratory support. Conflict of interest
None to declare.
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Mycopathologia (2014) 178:315–319 2. Crespo MJ, Abarca ML, Cabanes FJ. Occurrence of Malassezia spp. in the external ear canals of dogs and cats with and without otitis externa. Med Mycol. 2002;40:115–21. 3. Bond R, Guillot J, Cabanes J. Malassezia yeasts in animal diseases. In: Boekhout T, Gue´ho-Kellermann E, Mayser P, Velegraki A, editors. Malassezia and the skin. Berlin: Springer; 2010. p. 271–99. 4. Chen TA, Hill PB. The biology of Malassezia organisms and their ability to induce immune responses and skin disease. Vet Dermatol. 2005;16:4–26. 5. ESCCAP. Guideline 2 superficial mycoses in dogs and cats. 2011. http://www.esccap.org/uploads/docs/uh82a6e8_ esccapgl2guidelines.pdf. 6. Bernardo FM, Martins HM, Martins ML. A survey of mycotic otitis externa of dogs in Lisbon. Rev Iberoam Micol. 1998;15:163–5. 7. Coutinho SD, Paula CR. Susceptibility to antifungal agents of Malassezia pachydermatis isolates from dogs. Pol J Vet Sci. 2001;4:77–81. 8. Eichenberg M, Appelt CE, Berg V, Muschner A, Nobre M, Mata D, Alves S, Ferreiro L. Susceptibility of Malassezia pachydermatis to azole antifungal agents evaluated by a new broth microdilution method. Acta Sci Vet. 2003;31:75–80. 9. Lyskova P, Vydrzalova M, Mazurova J. Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa. J Vet Med A Physiol Pathol Clin Med. 2007;54:559–63. 10. Rougier S, Borell D, Pheulpin S, Woehrle F, Boisrame B. A comparative study of two antimicrobial/anti-inflammatory formulations in the treatment of canine otitis externa. Vet Dermatol. 2005;16:299–307. 11. Nakano Y, Wada M, Tani H, Sasai K, Baba E. Effects of beta-thujaplicin on anti-Malassezia pachydermatis remedy for canine otitis externa. J Vet Med Sci. 2005;67:1243–7. 12. Arikan S. Current status of antifungal susceptibility testing methods. Med Mycol. 2007;45:569–87. 13. Canton E, Espinel-Ingroff A, Peman J. Trends in antifungal susceptibility testing using CLSI reference and commercial methods. Expert Rev Anti Infect Ther. 2009;7:107–19. 14. Johnson EM. Issues in antifungal susceptibility testing. J Antimicrob Chemother. 2008;61:13–8. 15. Hensel P, Austel M, Wooley RE, Keys D, Ritchie BW. In vitro and in vivo evaluation of a potentiated miconazole aural solution in chronic malassezia otitis externa in dogs. Vet Dermatol. 2009;20:429–434. 16. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard, M27-A3. 3rd ed. Wayne, PA: NCCLS; 2008. 17. Peano A, Beccati M, Chiavassa E, Pasquetti M. Evaluation of the antifungal susceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabendazole using a modified CLSI M27-A3 microdilution method. Vet Dermatol. 2012;23:131–5. 18. Rincon S, Cepero de Garcia MC, Espinel-Ingroff A. A modified Christensen’s urea and CLSI broth microdilution
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In Vitro Antifungal Susceptibility of Malassezia pachydermatis Strains Isolated from Dogs with Chronic and Acute Otitis Externa E. Chiavassa • P. Tizzani • A. Peano
Received: 31 July 2013 / Accepted: 26 June 2014 / Published online: 17 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Malassezia pachydermatis is a yeast that is frequently involved as a secondary/perpetuating factor in canine otitis externa. Topical therapies with different antifungal agents, mainly azole compounds, are generally successful in controlling the yeast overgrowth, but treatment failure and rapid recurrences are common. This study compared the in vitro antifungal susceptibility of M. pachydermatis isolates obtained from chronic and acute cases of otitis externa. The aim was to assess the possible onset of resistance mechanisms in isolates involved in long-lasting episodes with poor response to treatment. We evaluated the in vitro susceptibility to miconazole (MCZ) and clotrimazole (CTZ) of 42 isolates of M. pachydermatis obtained from dogs with chronic (group A, n = 25) and acute otitis (group B, n = 17), using a modified CLSI M27-A3 microdilution method. All isolates were inhibited by the antifungal agents employed, but Malassezia isolates from group A were significantly associated with higher minimum inhibitory concentration (MIC) values for both agents (Median MIC values: MCZ group A 2 lg/ml, group B 1 lg/ml; CTZ group A 8 lg/ml, group B 4 lg/ml). These findings prove that these isolates had a reduced in vitro
E. Chiavassa (&) P. Tizzani A. Peano Settore di Parassitologia e Malattie Parassitarie, Dipartimento di Scienze Veterinarie, Universita` degli Studi di Torino, Via Leonardo da Vinci 44, Grugliasco, 10095 Turin, Italy e-mail: [email protected]
susceptibility to the antifungal agents tested. However, it is unlikely that this could have any influence on the outcome of a topical treatment. Indeed, marketed products include concentrations of the tested agents that largely exceed even the highest MICs found in this study (in most cases at least 1,000 9 the MIC, or greater). In conclusion, this study suggests that isolates of M. pachydermatis involved in chronic cases of canine external otitis and exposed to repeated antifungal treatments are unlikely to develop mechanisms of resistance of clinical relevance. Keywords Malassezia pachydermatis Antifungal susceptibility CLSI reference broth microdilution method Otitis externa
Introduction Otitis externa represents one of the most common reasons for which dogs are presented to veterinary attention [1]. Malassezia pachydermatis is the yeast isolated with the highest frequency from affected ears [2]. This organism is normally present in the external ear canal and rapidly multiplies during favourable conditions, thus becoming a secondary factor of otitis [1]. Factors that predispose dogs to Malassezia otitis externa (MOE) include anatomical anomalies or changes that create ear canal stenosis, increased cerumen secretion or retention, moisture, and
123
316
inhibition of air circulation. Under these conditions, primary inflammatory diseases of the ear canal, which can occur as part of atopic dermatitis or adverse food reactions, can often lead to MOE. Moreover, a hypersensitivity response to the yeast itself is likely to occur in many dogs with allergies [3, 4]. In clinical practice, topical therapy appears to be the most practical and popular way to treat MOE, and different azoles, such as clotrimazole (CTZ) and miconazole (MCZ), are used in aural formulations [3]. These agents are generally successful in controlling the yeast overgrowth, but treatment failure or recurrences are common, usually when primary causes, and predisposing factors are unidentified or uncorrected [5]. Unsatisfactory responses to therapy are also often attributed to the yeast strain being ‘‘resistant’’ to the antifungal employed, with a consequent switch to another agent. This approach may be supported by previous reports of M. pachydermatis strains being resistant to various drugs, based on the results of in vitro susceptibility testing [6–10]. Moreover, it has been previously shown that multiple in vitro passages at subtherapeutic concentrations of azole agents can lead to a decrease in susceptibility of M. pachydermatis to these agents, thus suggesting that this species is capable of developing resistance mechanisms [11]. However, this possibility has not yet been confirmed in vivo. In addition, compared to the extensive research carried out for other medically relevant yeast species (e.g. Candida spp. And Cryptococcus spp.) [12–14], the value of in vitro susceptibility testing for predicting the therapy outcome of M. pachydermatis treatment has been far less comprehensively investigated. Indeed, standard parameters and guidelines specifically dedicated to this yeast are not yet available, and neither are interpretive criteria. This lack of standardization in methodology and interpretation for susceptibility testing raises the question of the reliability and clinical value of previous in vitro results, and consequently challenges the existence of clinically relevant resistance in field isolates of M. pachydermatis. In order to contribute in clarifying this matter, this study compared the in vitro antifungal susceptibility of M. pachydermatis isolates from chronic and acute cases of otitis externa, with the intent of individuating differences potentially ascribable to the onset of resistant mechanisms in the isolates exposed to treatment.
123
Mycopathologia (2014) 178:315–319 Table 1 Sex, breed and age of dogs with chronic Malassezia otitis (group A) and acute Malassezia otitis (group B) Group A (n = 18)
Group B (n = 11)
Sex Male
10
3
8
8
Labrador Retriever
1
0
Golden Retriever
1
2
German Shepherd
2
0
Springer Spaniel
3
0
Beagle
1
0
Female Breed
Shi-tzu
2
0
Pug
2
2
Newfoundland
0
2
American Bulldog
3
0
Poodle
2
0
1
5
5.7
3.5
Mix breed Age Average
Materials and Methods Samples M. pachydermatis isolates were collected from 18 dogs with chronic otitis (group A) and from 11 dogs with acute otitis (group B). Background of animals included in this study is summarized in Table 1. In cases of bilateral otitis, each ear was considered separately, so that overall 25 and 17 isolates from group A and B, respectively, were available for the analyses. Otitis was considered as being chronic in cases that lasted for more than 2 months (Hensel et al. 15). All dogs with chronic MOE had been previously treated with various topical ear products containing MCZ and CTZ; conversely dogs with acute MOE had never received specific treatments for M. pachydermatis. For purpose of comparison, the control isolates were chosen from group of dogs (group B) without previous exposure to antifungal agents. Isolates were collected with sterile swabs introduced into the ear canal and cultured on Sabouraud dextrose agar (SDA) (Biolife Italiana, Milan) supplemented with gentamicin and cloramphenicol. Identification of M. pachydermatis was based on macroscopic and microscopic morphology of colonies. The use of SDA allowed
Mycopathologia (2014) 178:315–319
confirming the identification of the yeast, since M. Pachydermatis is the only representative of the genus Malassezia able to grow on lipid-free media [3, 4]. In Vitro Antifungal Susceptibility testing The Clinical and Laboratory Standards Institute (CLSI) procedure for susceptibility testing of yeasts described in the M27-A3 document [16] was used in the study, with specific adaptations for M. pachydermatis as described previously [17, 18]. In particular, Christensen’s urea broth medium with Tween 80 and Tween 40 proved to be an optimal growth support for M. Pachydermatis [17, 18] compared to the RPMI 1640 medium suggested by the CLSI. Uniform yeast suspensions were prepared in the culture medium from 4-dayold colonies subcultured at 37 °C on SDA. Inoculum suspensions were adjusted using a spectrophotometer, and viable colony counts were performed to confirm inoculum size. Final yeast cell concentrations were approximately 1–5 9 105 cells/ml. Stock suspensions of MCZ (Janssen Pharmaceuticals, Beerse, Belgium) and CTZ (Sigma Aldrich, Milan, Italy) were prepared in dimethyl sulfoxide. Twofold dilutions of the drug solutions were dispensed into 96-well plates, with final drug concentrations of 0.06–32 lg/ml. Plates were incubated at 37 °C and analysed at 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed a 50 % decrease or more of optical density compared to the growth control, as assessed by using a spectrophotometer (630-nm filter).
317 Table 2 Miconazole (MCZ) and Clotrimazole (CTZ) MICs (lg/ml) for Malassezia pachydermatis isolates from dogs with chronic otitis (group A) and with acute otitis (group B) MIC (lg/ ml)
Group A (n = 25)
Group B (n = 17)
MCZ
CTZ
MCZ
CTZ
32 16 8 4 2 1 0.5 0.25 0.125 0.06
0 0 1 (4.0 %) 5 (20.0 %) 17 (68.0 %) 2 (8.0 %) 0 0 0 0
0 3 (12.0 %) 10 (40.0 %) 11 (44.0 %) 1 (4.0 %) 0 0 0 0 0
0 0 0 1 (5.9 %) 7 (41.2 %) 9 (52.9 %) 0 0 0 0
0 0 1 (5.9 %) 10 (58.8 %) 6 (35.3 %) 0 0 0 0 0
MCZ: v2 = 11.17, p value = 0.005
p
value = 0.01;
CTZ:
v2 = 12.93,
while MICs for group B strains were 1–4 (MCZ) and 2–8 (CTZ) lg/ml (Table 2). MIC50 (MIC required to inhibit growth of at least 50 % of the isolates tested) for group A was 8 (CTZ) and 2 (MCZ) lg/ml, while for group B, it was 4 (CTZ) and 1 (MCZ) lg/ml. MIC90 (MIC required to inhibit growth of at least 90 % of the isolates tested) for group A was 16 (CTZ) and 4 (MCZ) lg/ml, while for group B, it was 4 (CTZ) and 2 (MCZ) lg/ml. Significant differences were found between MICs in group A and group B for both agents (Table 2), with Malassezia isolates of dogs with acute otitis associated with significantly lower MIC values (MCZ: Fisher = 8.37, p = 0.003, Odds Ratio = 0.077; CTZ: Fisher = 5.06, p = 0.02, Odds ratio = 0.076).
Statistical Analysis Differences of frequencies for MCZ and CTZ MICs between group A and group B were evaluated using the Chi square test. Contingency tables and the Fisher’s exact test were used to evaluate the odds ratio related to group A and group B frequencies for ‘‘low MIC values’’. A ‘‘low MIC value’’ was intended as the lowest MIC found for each agent. A p value of \ 0.05 was considered to be statistically significant.
Results The MIC range of M. pachydermatis isolates from group A were 1–8 (MCZ) and 2–16 (CTZ) lg/ml,
Discussion In the present study, we focused on the role of M. pachydermatis and, in particular, we investigated whether cases with poor responses to therapy could be due to an emerging drug resistance of the yeast. This issue is worth expanding due to the repeated reports of M. pachydermatis isolates resistant to different antifungal agents commonly used in the dog [7, 9, 10, 19–26]. Yeast isolates were thus collected from dogs with chronic Malassezia otitis poorly responding to therapy (group A). The animals with the above-mentioned characteristics were chosen since such patients are frequently
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presented for consultation after having been treated with numerous available commercial products containing different antifungals. Accordingly, these patients may have a higher probability of harbouring yeast strains that have developed resistance mechanisms due to repeated exposure to several antifungals, frequently with incorrect dosages. Through the CLSI-modified method all Malassezia isolates, from both groups, were found to be inhibited by the azole drugs employed, with MCZ showing higher absolute activity against M. pachydermatis compared with CTZ, as reported previously [17]. Regarding the comparison between the two groups of animals, the distribution of MIC values was significantly increased for isolates sampled from dogs with chronic otitis. This does not necessarily mean that the increased MICs are associated with clinical failure; instead, it demonstrates at best that these strains have a reduced in vitro susceptibility to antifungal agents, perhaps due to repeated exposure to a number of antifungal agents. Indeed, marketed products include concentrations of MCZ and CTZ that largely exceed even the highest MICs found in this study (in most cases at least 1,000 9 the MIC, or greater), and the antifungal agents are likely to reach adequate tissue levels for controlling the yeast overgrowth because such products are used topically, that is, they are instilled directly into the ear canals. Moreover, these products may be applied several times a day, thus ensuring a cumulative local effect. These conclusions provide an opportunity to briefly comment on the previous literature, which may instead suggest that lower antifungals’ potency accounts for poor responses to therapy in cases of Malassezia otitis, as several studies reported yeast strains resistant to various commonly used agents in veterinary practice [6–10, 19–27, 29]. Evaluation of these previous studies reveals that the evidence in support of the existence of clinically relevant resistance was weak. Indeed, all were based entirely on results of in vitro susceptibility tests, and in vitro results were never related to the outcome of therapy. Moreover, none of the breakpoints employed to categorize strains as being susceptible/resistant had a rationale capable of giving a predictive value of the final clinical outcome. For example, the approaches of Rougier [10] and Lyskova [9], who respectively referred to ‘‘zone interpretive standard breakpoints for veterinary pathogens’’ and ‘‘zone diameter interpretive standards for ‘‘microscopic fungi’’, appear to be too
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generic and thus of poor clinical relevance. Another way of categorizing strains, through the use of the MIC50/ MIC90 values (S if MIC of the strain B MIC50, R If MIC of the strain [ MIC90), appears to be an accepted and valid interpretive criterion as it has been employed in several studies [7, 8, 19, 20, 23–25]. However, although MIC50 and MIC90 values serve to describe and summarize the distribution of MICs for a population of organisms, they do not necessarily mirror the concentrations that the tested drugs can reach in infected tissues. Finally, in two other studies [21, 27] the breakpoints established for Candida [30] were applied to M. pachydermatis. These breakpoints have been based on a number of pharmacokinetic studies of the drugs, and the evaluation of the clinical outcome in human infections [14]. Thus, such a categorization appears to be of poor significance once applied to a different fungus/host combination (i.e. M. pachydermatis/dog). In conclusion, additional reasons (incorrect management of the underlying conditions; incorrect drug administration; poor compliance of the owner, etc.) were probably responsible for treatment failure and the consequent chronicization of Malassezia otitis in the animals studied, and this supports the view that treatment failure of Malassezia dermatitis/otitis can be attributed in most cases to poor management of conditions underlying the yeast overgrowth [5], rather than to drug resistance. Moreover, our results show that resistance mechanisms of clinical relevance are unlikely to develop, at least towards MCZ and CTZ, in strains of M. pachydermatis exposed to lasting antifungal therapies. Further analyses on an extended data set of strains are necessary to confirm these conclusions and to verify whether they also apply to other antifungal agents commonly used in the dog. Acknowledgments This work was supported by a grant of Elanco Animal Health, division of Eli Lilly Italy S.p.A. The authors express their acknowledgments to Elena Borio and Roberta Morandi for their laboratory support. Conflict of interest
None to declare.
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