Medical honey: activity towards antibiotic resistant Staphylococcus pseudintermedius and Malassezia pachydermatis in canine folliculitis and otitis
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2019-12-19
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O mel de grau médico é considerado uma opção eficaz e económica no tratamento de feridas infetadas. O mel manuka (MH) e um gel à base de mel (HBO) encontram-se disponíveis no mercado permitindo a prescrição de produtos seguros, sem antibióticos, eficazes e padronizados. Neste trabalho, o primeiro objetivo consistiu em determinar a metodologia de diagnóstico e tratamento das doenças dermatológicas causadas por Staphylococcus pseudintermedius e Malassezia pachydermatis em clínica de pequenos animais em Portugal. Inicialmente, demonstrámos que ambos os microrganismos causam frequentemente várias doenças, nomeadamente, a foliculite bacteriana superficial (SBF), a dermatite das pregas de pele (FD), a dermatite generalizada por Malassezia spp. (MD) e otite externa (OE). Os antibióticos orais foram largamente prescritos para o tratamento de SBF, particularmente amoxicilina-ácido clavulânico (100%), cefalexina (94%), enrofloxacina (67%) e marbofloxacina (60%). Para o tratamento de FD e OE, os antibióticos orais foram administrados em 88% e 82% dos casos, respetivamente. Os antifúngicos orais foram prescritos para o tratamento da MD (85%), FD (70%) e OE (59%). Todas as doenças foram tratadas topicamente com antibióticos, antifúngicos e glucocorticóides. Alternativas, como produtos à base de mel, não foram frequentemente prescritas pelos clínicos portugueses. Casos clíncos causados por S. pseudintermedius multirresistentes (MDR) são frequentemente observados em clínica o que reduz dramaticamente as opções de antibioterapia. O segundo trabalho avaliou a incidência de multirresistência em S. pseudintermedius sensíveis (MSSP) e resistentes à meticilina (MRSP), previamente recolhidos de cães com SBF. Observou-se que todos os MRSP eram resistentes à amoxicilinaácido clavulânico, clindamicina e eritromicina. Foram observados elevados níveis de resistência ao trimetoprim-sulfametoxazol (97%), tetraciclina e gentamicina (87%), cefalotina (83%), enrofloxacina (83%), pradofloxacina (80%) e minociclina (50%). Observou-se um baixo nível de resistência ao cloranfenicol (17%), amicacina (7%) e rifampicina (7%). A maioria dos isolados eram MDR (38/60). Todos os isolados não-MDR eram MSSP. A resistência à meticilina foi associada à multirresistência a outras classes de antibióticos. O terceiro trabalho apresentava como objetivos determinar a eficácia in vitro do MH contra S. pseudintermedius e M. pachydermatis e o tempo necessário para obter o efeito bactericida ou fungicida. Sessenta isolados de S. pseudintermedius recolhidos de cães com SBF, foram divididos em grupos MRSP/MSSP e MDR/não-MDR. Testaram-se também vinte isolados de M. pachydermatis recolhidos de cães com OE. O MH foi testado não diluído e diluído a 80%, 40%, 30%, 20%, 15%, 10%, 7,5%, 5%, 3,7% e 2,5% p/v. A concentração mínima bactericida (MBC) de S. pseudintermedius foi de 20% p/v, sem diferença entre MSSP/MRSP ou MDR/não-MDR. Para M. pachydermatis a concentração mínima fungicida (MFC) foi de 40% p/v. Realizou-se um ensaio de “time-kill” em períodos distintos. O MH diluído eliminou ambos os microrganismos após 4 horas. O MH diluído a 40% p/v manteve atividade contra S. pseudintermedius mas demorou mais tempo a eliminar os isolados de M. pachydermatis. Finalmente, determinou-se a eficácia in vitro do HBO contra os isolados de S. pseudintermedius e M. pachydermatis. Avaliou-se a eficácia da componente de mel (HO) do produto. Os isolados de S. pseudintermedius e M. pachydermatis foram testados contra diluições seriadas de HBO (40%, 20%, 10%, 5% e 2,5% p/v). O HBO contém HO na concentração de 40%. O HO foi testado puro e em diluições seriadas (40%, 20%, 10%, 5% e 2,5% p/v). Aplicou-se o mesmo protocolo após exposição à catalase para avaliar a presença de peróxido de hidrogénio. Realizou-se um ensaio de “time-kill” para determinar tempo de exposição para obter efeito bactericida/fungicida. O MBC para S. pseudintermedius foi de 20% p/v (5-20% p/v) para HBO e HO. O HBO demonstrou valores de MBC inferiores em comparação com o HO (P=0,003). Não houve diferença entre MSSP/MRSP (HBO P=0,757; HO P=0,743). Apenas o HO foi afetado pela catalase (P=0,015). O MFC para o HBO foi de 10% p/v (5-10% p/v) e 40% p/v para o HO (20-≥40% p/v). Todos os isolados foram eliminados após 4 horas de exposição ao HBO. Resumindo, este trabalho contribui para melhorar o nosso conhecimento sobre o efeito do mel sobre o S. pseudintermedius e a M. pachydermatis. Demonstra especificamente que ambos microrganismos são sensíveis ao MH, HBO e ao mel que compõem este produto. Tratamentos à base de mel não são, no entanto, prescritos frequentemente pelos clínicos portugueses e, os antibióticos orais, são frequentemente utilizados na prática clínica, apesar da propagação do S. pseudintermedius resistente à meticilina. Finalmente, este trabalho acrescenta evidências de que S. pseudintermedius resistente à meticilina pode ser multirresistente, reforçando a necessidade de opções não-antibióticas. Os resultados poderão ser usados futuramente em ensaios clínicos que tenham como objetivo tratar infecções cutâneas e de canal auditivo causados por S. pseudintermedius e/ou M. pachydermatis.
Medical honey is considered an effective economic option for the treatment for infected wounds. Medical grade Manuka honey (MH) and a honey-based gel (HBO) are now available, allowing for the prescrition of safe, non-antibiotic, effective and standardised products. In the present doctoral thesis, we aimed to determine how clinicians diagnosed and treated dermatological conditions caused by Staphylococcus pseudintermedius and Malassezia pachydermatis in Portugal. We demonstrated to both pathogens often cause superficial bacterial folliculitis (SBF), fold dermatitis (FD), Malassezia dermatitis (MD) and otitis externa (OE). Oral antibiotics were widely prescribed for treatment of SBF, particularly amoxicillin-clavulanic acid (100%), cephalexin (94%), enrofloxacin (67%), or marbofloxacin (60%). Fold dermatitis and OE, were also treated with oral antibiotics in 88% and 82% of cases, respectively. Oral antifungals were often prescribed for MD (85%), FD (70%), and OE (59%). All the diseases were frequently treated topically with antibacterials, antifungals, and glucocorticoids. Alternative options such as honey-based products were not frequently used by clinicians. Multidrug resistant (MDR) S. pseudintermedius isolates are frequently observed, a fact that dramatically decreases antimicrobial treatment options. The second part of this work evaluated the incidence of multidrug resistance in methicillin-susceptible and resistant S. pseudintermedius isolates (MSSP/MRSP) previously collected from dogs with SBF. All MRSP exhibited resistance to amoxicillin-clavulanic acid, clindamycin and erythromycin. High resistance levels were observed to trimethoprimsulfamethoxazole (97%), tetracycline and gentamicin (87%), cefalothin (83%), enrofloxacin (83%), pradofloxacin (80%) and minocycline (50%). Low resistance level was observed for chloramphenicol (17%), amikacin (7%) and rifampicin (7%). Most isolates were multidrug resistant (MDR, 38/60). All nonMDR isolates were MSSP. Methicillin resistance was associated with MDR to other classes of antibiotics. The third part of this work, aimed to determine the in vitro efficacy of MH against S. pseudintermedius and M. pachydermatis and necessary exposure time for a bactericidal or fungicidal effect. Sixty S. pseudintermedius, previously isolated from dogs with canine SBF, were divided into MRSP/MSSP and MDR/non-MDR groups. Twenty M. pachydermatis isolates, also previously isolated from dogs with OE were tested. Manuka honey was tested undiluted and diluted at 80%, 40%, 30%, 20%, 15%, 10%, 7.5%, 5%, 3.7% and 2.5% w/v. For S. pseudintermedius the minimal bactericidal concentration (MBC) was 20% w/v with no difference between MSSP/ MRSP or MDR/non-MDR. For M. pachydermatis the minimal fungicidal concentration (MFC) was 40% w/v. A time-kill assay was performed at different times. Undiluted MH killed both microorganisms after 4 hours of exposure. Diluted MH at 40% w/v maintained activity against S. pseudintermedius but took longer time to kill M. pachydermatis isolates. Finally, we determined the in vitro efficacy of HBO, against isolates of S. pseudintermedius and M. pachydermatis. The efficacy of the product’s honey component (HO) was also evaluated. All isolates were tested against serial dilutions of HBO (40%, 20%, 10%, 5% and 2.5% w/v). HBO contains HO at 40%. HO was tested pure and after serial dilutions (40%, 20%, 10%, 5% and 2.5% w/v). The same protocol was applied after exposure to catalase to determine the influence of hydrogen peroxide. A time-kill assay was performed determine the period of time necessary for a bactericidal/fungicidal effect. MBC for S. pseudintermedius was 20% w/v (5-20% w/v) for HBO and HO. HBO had lower MBC values when compared to HO (P=0.003). No difference was observed between MSSP/MRSP isolates (HBO P=0.757, HO P=0.743). Only HO was affected by catalase (P=0.015). MFC for HBO was 10% w/v (5-10% w/v) and 40% w/v for HO (20-≥40% w/v). All isolates were killed after 4 h of exposure to HBO. In summary, our work indicates that honey was effective against S. pseudintermedius and M. pachydermatis. We could demonstrate that both microorganisms are susceptible to MH, HBO and the honey that composes this product. Nevertheless, honey treatments are not frequently prescribed by Portuguese clinicians and oral antibiotics are overused in small practice, despite the spread of antibiotic resistant S. pseudintermedius. Finally, the work adds further evidence that methicillin resistant S. pseudintermedius may be multidrug resistant which reinforces the need for non-antibiotic options. The results obtained can be used in future clinical trials aiming to treat skin and ears infections caused by S. pseudintermedius and M. pachydermatis.
Medical honey is considered an effective economic option for the treatment for infected wounds. Medical grade Manuka honey (MH) and a honey-based gel (HBO) are now available, allowing for the prescrition of safe, non-antibiotic, effective and standardised products. In the present doctoral thesis, we aimed to determine how clinicians diagnosed and treated dermatological conditions caused by Staphylococcus pseudintermedius and Malassezia pachydermatis in Portugal. We demonstrated to both pathogens often cause superficial bacterial folliculitis (SBF), fold dermatitis (FD), Malassezia dermatitis (MD) and otitis externa (OE). Oral antibiotics were widely prescribed for treatment of SBF, particularly amoxicillin-clavulanic acid (100%), cephalexin (94%), enrofloxacin (67%), or marbofloxacin (60%). Fold dermatitis and OE, were also treated with oral antibiotics in 88% and 82% of cases, respectively. Oral antifungals were often prescribed for MD (85%), FD (70%), and OE (59%). All the diseases were frequently treated topically with antibacterials, antifungals, and glucocorticoids. Alternative options such as honey-based products were not frequently used by clinicians. Multidrug resistant (MDR) S. pseudintermedius isolates are frequently observed, a fact that dramatically decreases antimicrobial treatment options. The second part of this work evaluated the incidence of multidrug resistance in methicillin-susceptible and resistant S. pseudintermedius isolates (MSSP/MRSP) previously collected from dogs with SBF. All MRSP exhibited resistance to amoxicillin-clavulanic acid, clindamycin and erythromycin. High resistance levels were observed to trimethoprimsulfamethoxazole (97%), tetracycline and gentamicin (87%), cefalothin (83%), enrofloxacin (83%), pradofloxacin (80%) and minocycline (50%). Low resistance level was observed for chloramphenicol (17%), amikacin (7%) and rifampicin (7%). Most isolates were multidrug resistant (MDR, 38/60). All nonMDR isolates were MSSP. Methicillin resistance was associated with MDR to other classes of antibiotics. The third part of this work, aimed to determine the in vitro efficacy of MH against S. pseudintermedius and M. pachydermatis and necessary exposure time for a bactericidal or fungicidal effect. Sixty S. pseudintermedius, previously isolated from dogs with canine SBF, were divided into MRSP/MSSP and MDR/non-MDR groups. Twenty M. pachydermatis isolates, also previously isolated from dogs with OE were tested. Manuka honey was tested undiluted and diluted at 80%, 40%, 30%, 20%, 15%, 10%, 7.5%, 5%, 3.7% and 2.5% w/v. For S. pseudintermedius the minimal bactericidal concentration (MBC) was 20% w/v with no difference between MSSP/ MRSP or MDR/non-MDR. For M. pachydermatis the minimal fungicidal concentration (MFC) was 40% w/v. A time-kill assay was performed at different times. Undiluted MH killed both microorganisms after 4 hours of exposure. Diluted MH at 40% w/v maintained activity against S. pseudintermedius but took longer time to kill M. pachydermatis isolates. Finally, we determined the in vitro efficacy of HBO, against isolates of S. pseudintermedius and M. pachydermatis. The efficacy of the product’s honey component (HO) was also evaluated. All isolates were tested against serial dilutions of HBO (40%, 20%, 10%, 5% and 2.5% w/v). HBO contains HO at 40%. HO was tested pure and after serial dilutions (40%, 20%, 10%, 5% and 2.5% w/v). The same protocol was applied after exposure to catalase to determine the influence of hydrogen peroxide. A time-kill assay was performed determine the period of time necessary for a bactericidal/fungicidal effect. MBC for S. pseudintermedius was 20% w/v (5-20% w/v) for HBO and HO. HBO had lower MBC values when compared to HO (P=0.003). No difference was observed between MSSP/MRSP isolates (HBO P=0.757, HO P=0.743). Only HO was affected by catalase (P=0.015). MFC for HBO was 10% w/v (5-10% w/v) and 40% w/v for HO (20-≥40% w/v). All isolates were killed after 4 h of exposure to HBO. In summary, our work indicates that honey was effective against S. pseudintermedius and M. pachydermatis. We could demonstrate that both microorganisms are susceptible to MH, HBO and the honey that composes this product. Nevertheless, honey treatments are not frequently prescribed by Portuguese clinicians and oral antibiotics are overused in small practice, despite the spread of antibiotic resistant S. pseudintermedius. Finally, the work adds further evidence that methicillin resistant S. pseudintermedius may be multidrug resistant which reinforces the need for non-antibiotic options. The results obtained can be used in future clinical trials aiming to treat skin and ears infections caused by S. pseudintermedius and M. pachydermatis.
Descrição
Doctoral Thesis in Veterinary Sciences - Clinic
Palavras-chave
antibiótico , antifúngico