Primjena disk difuzijskog postupka u ispitivanju osjetljivosti sojeva gljivice Malassezia Pachydermatis izdvojenih iz pasa s kroničnom upalom zvukovoda

master's thesis

• Title (english): The Performance of Disk Diffusion Method for Susceptibility Testing of Malassezia Pachydermatis Strains Isolated from Dogs with Chronic Otitis Externa
• Author: Iva Benvin
• Mentor: Suzana Hađina (mentor)
• Committee member: Ljiljana Pinter (predsjednik povjerenstva)
• Committee member: Zrinka Štritof (član povjerenstva)
• Committee member: Suzana Hađina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Veterinary Medicine; (Department of Microbiology and Infectious Diseases with Clinic); Zagreb
• Defense date and country: 2020-01-24, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Veterinary Clinical Sciences

Abstract

Gljivica Malassezia pachydermatis predstavlja najčešćeg uzročnika upale sluznice vanjskog
zvukovoda u pasa koja vrlo često rezultira kroničnim tijekom i recidivirajućim terapijama
antimikoticima, najčešće iz skupine azola. Posljednjih se godina spominje njezina smanjena
osjetljivost na antimikotike ne samo u veterinarskoj, nego i u humanoj medicini. S obzirom na
to da ne postoji standardizirani postupak za istraživanje osjetljivosti vrste M. pachydermatis,
postavlja se pitanje postojanja stvarne klinički značajne otpornosti ove gljivice. Opći cilj ovog
istraživanja stoga je bio prilagoditi disk difuzijski postupak za istraživanje osjetljivosti
gljivice M. pachydermatis na osnovi standardiziranog postupka za kvasac Candida sp.
propisanog dokumentom CLSI M44-A2. Specifičan cilj bio je istražiti osjetljivost 60 sojeva
M. pachydermatis izdvojenih iz pasa s kroničnom upalom sluznice vanjskog zvukovoda na tri
različita antimikotika iz skupine azola: mikonazol, klotrimazol i itrakonazol. Suspenzija
inokuluma ovim istraživanjem optimizirana je na optičku gustoću od 3,5 McF s prosječnom
srednjom vrijednosti od 1,27x107 CFU/mL. Mueller-Hintonov agar s dodatkom 2% glukoze i
0,5 μg/l metilenskog modrila pogodovao je rastu gljivice kada je suspenzija inokuluma
pripremljena s dodatkom 0,04% Tween-a 80 kao izvorom lipida. Istodobno granice zone
inhibicije bile su jasno vidljive, a optimalno vrijeme očitavanja zona inhibicije bilo je nakon
72 sata. Istraživanjem osjetljivosti ustanovljeno je da je jedan soj gljivice M. pachydermatis
bio umjereno osjetljiv na mikonazol, dok su svi preostali sojevi bili osjetljivi na mikonazol,
klotrimazol i itrakonazol. Najveća je aktivnost uočena u primjeni klotrimazola, zatim
itrakonazola, a najmanja u mikonazola. Rezultatima ovih istraživanja optimizirali smo
postupak koji je primijenjiv u rutinskoj mikološkoj dijagnostici istraživanja osjetljivosti
gljivice M. pachydermatis.

Abstract (english)

Malassezia pachydermatis is the most common causative agent of canine otitis externa which
often evolves into chronic inflammation and long-term treatments with various antifungal
drugs, mainly azoles. In recent years, resistance to different antifungals has been increasingly
reported, not only in veterinary but also in human medicine. Since there is no standardized
method for antifungal susceptibility testing of M. pachydermatis, it is often questioned
whether a real clinical resistance to this yeast infection exist. The first aim of this study was to
modify antifungal disk diffusion method M44-A2 standardized by the Clinical and Laboratory
Standards Institute for Candida sp. in order to test antifungal susceptibility of M.
pachydermatis. The second aim was to investigate the susceptibility of 60 strains of M.
pachydermatis isolated from dogs with chronic otitis externa to three different azole
derivatives: miconazole, clotrimazole and itraconazole. Inoculum suspensions adjusted to 3.5
McF standard corresponded to the average of 1.27x107 CFU/mL. Mueller-Hinton agar
supplemented with 2% glucose and 0.5 μg/l methylene blue supported the growth of M.
pachydermatis when the yeast inoculum was prepared with 0.04% Tween 80, as the lipid
source. At the same time, it provided clearly visible zone edges. Optimal time for reading
inhibition zone diameters was at 72 hours. One tested M. pachydermatis strain was
intermediately susceptible to miconazole, while all remaining strains were susceptible to
miconazole, clotrimazole and itraconazole. Clotrimazole showed the highest antifungal
activity, followed with itraconazole, while miconazole demonstrated the lowest activity. On
the basis of reported results, disk diffusion method was optimized for the routine diagnostic
purposes of susceptibility testing of M. pachydermatis.

• Keywords: Malassezia pachydermatis; psi; kronična upala sluznice zvukovoda; disk difuzijski postupak; antimikotik
• Keywords (english): Malassezia pachydermatis; dogs; chronic otitis externa; disk diffusion method; antifungal agents
• Language: croatian
• URN:NBN: urn:nbn:hr:178:923377
• Study programme: Title: Veterinary Medicine; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica veterinarske medicine (doktor/doktorica veterinarske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2021-05-01
• Created on: 2020-11-12 08:52:14