Molecular characterization of Malassezia sympodialis and Malassezia furfur from cattle with and without otitis / Caracterização molecular de isolados de Malassezia sympodialis e Malassezia furfur provenientes de bovinos com e sem otite externa

A molecular study of Malassezia strains isolated from cattle with or without otitis was carried out by random amplified polymorphic DNA analysis (RAPD). DNA was extracted and purified from nine strains of Malassezia sympodialis and fourteen of Malassezia furfur. These microorganisms were collected from eight different bovine herds in Minas Gerais state, Brazil. The RAPD analysis and phenograms did not show the formation of genetically distinct groups among the strain isolated from cattle with or without otitis raised in the same herds. Genetic heterogeneity was observed among Malassezia strains from different geographic origins. These data suggest that genetically similar M. sympodialis and M. furfur strains found as members of the normal ear microbiota could become opportunistically active in the inflammatory process in cattle.

A caracterização molecular de amostras de Malassezia spp., isoladas de bovinos com e sem otite, foi realizada por meio da técnica do DNA polimórfico amplificado ao acaso (RAPD). DNAs de nove amostras de Malassezia sympodialis e quatorze de M. furfur foram extraídos e purificados. Essas amostras foram provenientes de oito diferentes rebanhos bovinos no estado de Minas Gerais, Brasil. A análise de RAPD e os fenogramas não revelaram a formação de grupos geneticamente distintos entre amostras isoladas de bovinos, criados no mesmo rebanho, com ou sem otite. Heterogeneidade genética foi observada entre amostras de diferentes origens geográficas. Os dados sugerem que isolados geneticamente semelhantes e membros da microbiota normal do ouvido podem participar, como oportunistas, no processo inflamatório do conduto auditivo externo de bovinos.

In vitro activities of four antifungal drugs against Trichophyton rubrum isolates exhibiting resistance to fluconazole.

Thirty-two clinical isolates of Trichophyton rubrum exhibiting resistance to fluconazole [minimum inhibitory concentrations (MICs) > or = 64 microg ml(-1)] were selected to test the antifungal activity of ketoconazole, itraconazole, griseofulvin and terbinafine. We followed the guidelines of the National Committee for Clinical Laboratory Standards for testing filamentous fungi. The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051) and Trichophyton mentagrophytes (ATCC 40004) were included for quality control. The microdilution plates were incubated at 28 degrees C and were read visually after 7 days of incubation and endpoint determination readings were performed visually. The MIC ranges for the four antifungals were: 0.0625-2 microg ml(-1) for ketoconazole, 0.25-2.0 microg ml(-1) for griseofulvin, < or =0.031-1.0 microg ml(-1) for itraconazole and < or =0.031 microg ml(-1) for terbinafine (for all tested isolates). Terbinafine was the most potent drug against T. rubrum, in vitro, followed by itraconazole, ketoconazole and griseofulvin. Much work is still needed to correlate the MICs of these drugs with clinical outcomes to develop interpretative breakpoints for T. rubrum and other dermatophytes.

In vitro antifungal oral drug and drug-combination activity against onychomycosis causative dermatophytes.

We present the results of studies of the in vitro susceptibility of 52 isolates of Trichophyton rubrum and 40 of Trichophyton mentagrophytes to griseofulvin, terbinafine, itraconazole, ketoconazole, fluconazole and cyclopiroxolamine. All test strains were recovered from patients with toe nail onychomycosis and the minimum inhibitory concentration (MIC) of each antifungal against both species was individually assessed. In addition, we investigated the MIC of the combination of cyclopiroxolamine and itraconazole and cyclopiroxolamine and ketoconazole. The NCCLS approved procedure M38-A as modified by Santos and Hamdan was employed. The studies of the two drug combinations were conducted with a checkerboard design. Analysis of the data revealed that terbinafine was the most effective in vitro against all isolates, followed in order by itraconazole, cyclopiroxolamine, ketoconazole and fluconazole. We observed no significant difference in the in vitro susceptibility profiles between either species to any of the antifungals (P<0.05). Our in vitro results confirm that terbinafine is the most effective of the antifungals included in this study. Furthermore, synergistic interactions were found in the two drug combinations with all of the dermatophyte test isolates. The latter results are in agreement with clinical data that show synergism between oral and topical antifungals in the treatment of onychomycosis.

Susceptibility of yeast isolates from cattle with otitis to aqueous solution of povidone iodine and to alcohol-ether solution.

Lipid-dependent Malassezia species, Candida spp. and Rhodotorula mucilaginosa have been associated with bovine parasitic otitis. This paper evaluated the susceptibility of 63 yeast isolates from cattle with otitis to a povidone iodine aqueous solution (1% and 0.5% v/v) and to an alcohol-ether solution (1:1 v/v). The effectiveness of these antiseptics was assessed using the European suspension test. Products achieving equal to or greater than 5-log reduction in numbers of the challenge organism after 5 min contact are considered to have as acceptable microbicidal effect (ME). The two antiseptic solutions achieved ME greater than 5, when tested at 1 and 5 min contact time, against the majority of yeast strains. The exceptions were alcohol and ether solution against two Candida tropicalis strains. Urea broth macrodilution method was used to determine the minimum inhibitory concentration (MIC), defined as the lowest concentration that resulted in a visually negative urease test or, in the case of Candida spp., turbidity inhibition when compared with that produced by the growth control. Analysis of the results for all 63 isolates showed Malassezia sympodialis and Rhodotorula mucilaginosa to be more susceptible to povidone iodine and Malassezia furfur strains to be less susceptible. Malassezia sympodialis was significantly more susceptible to alcohol-ether solution than other species. This study showed the in vitro efficacy of alcohol-ether solution and povidone iodine and proposes the need for clinical evaluation of the topical treatment and control of bovine otitis with these antiseptics and their effects on the ear microbiota and the ear canal.

Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes.

A total of 92 clinical isolates of dermatophytes (52 of Trichophyton rubrum and 40 of Trichophyton mentagrophytes) were selected for testing with six antifungal drugs (terbinafine, griseofulvin, clotrimazole, miconazole, isoconazole, and fluconazole) and two pairs of drug combinations (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine). Two methods of inoculum preparation for susceptibility testing were evaluated that used (i) inocula consisting only of microconidia of dermatophytes filtered in Whatman filter model 40 and (ii) unfiltered inocula consisting of hyphae and microconidia. We followed the recommendations of approved document M38-A of CLSI (formerly NCCLS) with some adaptations, including an incubation period of 7 days and an incubation temperature of 28 degrees C. Reference strains of Candida parapsilosis, Candida krusei, Trichophyton rubrum, and Trichophyton mentagrophytes were included as quality-control strains. MICs were consistently higher (usually 1 to 2 dilutions for drugs tested individually) when nonfiltered inocula were tested (P < 0.01) except for terbinafine. Larger MICs were seen when testing drugs with nonfiltered inocula. The curves of drug interaction were used to analyze the reproducibility of the test, and it was shown that high levels of reproducibility were achieved using the methodology that included the filtration step. The standardization of methodologies is the first step to yield reliability of susceptibility testing and to proceed with clinical laboratory studies to correlate MICs with clinical outcomes.

Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum.

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35 degrees C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35 degrees C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.

Otitis in cattle, an aetiological review.

Otitis externa in cattle has a significant impact in tropical and subtropical regions, and the aetiological agents are predominantly rhabditiform nematodes and mites of the genus Raillietia. Its prevalence is higher in mature and Zebu cattle. In advanced clinical cases there can be irreversible and fatal neural lesions. Ear infection in calves has been associated with concurrent respiratory diseases and mixed infection. The principal reported agents of otitis in calves are bacteria such as Actinomyces spp., Corynebacterium pseudotuberculosis, Escherichia coli, Haemophilus somnus, Pasteurella multocida, Mannheimia haemolytica, Pseudomonas spp., Streptococcus spp. and Mycoplasma bovis. The control and treatment of bovine otitis is not standardized and there is little evidence-based support for the diverse treatments available in the literature.

Factors associated with the prevalence of Malassezia species in the external ears of cattle from the state of Minas Gerais, Brazil.

Swabs collected from the external ears of 942 cattle, including both European and zebu breeds, were used to evaluate the epidemiology of Malassezia species. High numbers of Malassezia isolates were significantly associated with maturity of the animals and with diagnosed otitis. In healthy animals, a particularly low prevalence of the genus was found in Holstein cows, especially in the summer months, a finding perhaps correlating with the open, air-exposed ears of this breed. Pendutlous-eared zebu breeds and hybrids had higher levels of colonization, although this effect was more pronounced in humid regions, and was least in the dry north and west of the state. The most common species seen was M. sympodialis, which accounted for 47.1% of isolates from healthy cattle and 39.1% from cattle with otitis. In cattle with otitis sampled in summer, we found that the relatively thermotolerant M. sympodialis made up 80.8% of isolates. By contrast, during the winter the less thermotolerant M. globosa was the most common species from otitis, making up 56% of the isolates. Humidity and temperature may be the main factors explaining the epidemiology of Malassezia diseases and colonizations in both animals and humans.

Microbiologic profile of intra-abdominal infections at Belo Horizonte, Brazil.

Intra-abdominal infections (IAIs) represent one of the most common clinical problems in hospital practice, especially in surgical areas and centers of intensive care. The treatment of IAIs generally involves the draining of abscesses and empirical antimicrobial therapy. In this study, among 150 patients suffering from IAI, 106 (70.7%) yielded samples that presented microbial growth. Polyinfection was detected in 51.9% of the cases and varied from 2 to 9 distinct microbes per specimen. The overall mean number of micro-organisms isolated per patient was 2.17. Aerobic bacteria (as strict aerobes and facultative anaerobes), strict anaerobic bacteria, and fungi of the genus Candida represented 93.4%, 30.2%, and 13.2% of the cases positive for micro-organisms, respectively. The most common aerobic bacteria were those of the genera Staphylococcus, Escherichia, Proteus, and Streptococcus. Despite the frequent prior use (52%) with antimicrobials of recognized action against strict anaerobes, these micro-organisms constituted 30.9% of the total isolates, and the most frequently found were of the Bacteroides fragilis group and Prevotella species. The high prevalence of anaerobes in the specimens obtained from IAI demonstrates the need to give greater importance to these micro-organisms by making available material and human resources to carry out culture of the anaerobes as part of routine hospital procedures.

In vitro comparison of activities of terbinafine and itraconazole against Paracoccidioides brasiliensis.

In vitro, terbinafine is highly active against a broad spectrum of pathogenic fungi. We evaluated the activities of terbinafine and itraconazole against 31 isolates of Paracoccidioides brasiliensis. The tests were conducted by using a broth macrodilution procedure. MICs, in micrograms per milliliter, were as follows: terbinafine, 0.015 to 1.0 (geometric mean, 0.1188); itraconazole, 0.007 to 0.5 (geometric mean, 0.03165). The usual therapy for paracoccidioidomycosis is sulfonamides, amphotericin B, and azole derivatives (ketoconazole, itraconazole, and fluconazole). In comparison to amphotericin B, azole derivatives allow shorter treatment courses, can be administered orally, and are equally effective. Itraconazole has as high efficacy as ketoconazole, but with superior tolerance. It is the current drug of choice for treatment of paracoccidioidomycosis. The data obtained in this study indicate that terbinafine is active against P. brasiliensis in vitro and suggest that this allylamine can be considered a new option as drug therapy for paracoccidioidomycosis.

Prevalence of yeasts and mycelial fungi in bovine parasitic otitis in the State of Minas Gerais, Brazil.

Infestations by rhabditiform nematodes and acarids of the genus Raillietia are considered the primary causes of external otitis in cattle in tropical regions. Recently, yeasts of the genus Malassezia have been associated with a relatively high percentage of otitis cases, but the occurrence of other yeasts and mycelial fungi has not yet been reported in the literature. This work studied the presence of fungi in the ear canal of 45 cattle with external parasitic otitis. The results were positive for yeasts of the genus Malassezia in 31 (68.9%) of the 45 cultures in Mycosel medium supplemented with olive oil. The 45 cultures in Sabouraud dextrose medium revealed the growth of seven (15.5%) yeasts of the genus Candida, five (11.1%) Rhodotorula mucilaginosa, two (4.4%) fungi of the genus Aspergillus and eight 'Micelia sterilia'. Future studies may confirm and elucidate the importance of these agents in the aetiology of bovine otitis.

Epidemiological aspects of bovine parasitic otitis caused by Rhabditis spp. and/or Raillietia spp. in the state of Minas Gerais, Brazil.

The occurrence of bovine external otitis in tropical regions is predominantly assigned to parasitic infections by rhabditiform nematodes and infestations by mites of the genus Raillietia. This research had the purpose of investigating the prevalence of parasitic otitis in the dry and rainy seasons of Minas Gerais and correlating it with variables age, breed and the presence of horns. The secretion or cerumen of 981 cattle was collected with sterile swabs, placed in test tubes and exposed to sunlight for the identification of clinical infections caused by rhabditiform nematodes. The ear canals of 109 animals were flushed to study the occurrence of mites of the genus Raillietia. Clinical otitis caused by rhabditiform nematodes was observed only in animals of the Gyr breed, affecting 169 (60.1%) of the total (278) of these cattle. The prevalence of otitis in this breed was significantly higher for mature adult cows (P<0.001) and in cows with horns (P<0.001). In a total of nine animals suspected of raillietiosis, the examinations revealed the presence of Raillietia spp. in two Dutch cows. These results demonstrate that treatment must be focused on adult Gyr cattle with horns. This and future studies with an emphasis on epidemiological aspects may contribute to development of alternative approaches to reduce disease.

Microorganisms isolated from root canals presenting necrotic pulp and their drug susceptibility in vitro.

The knowledge about causative agents involved in endodontic infections is increasing, especially due to the improvement of culture techniques for anaerobic bacteria, showing that these microorganisms are predominant in this pathology. In this study, 31 canals with pulp necrosis were microbiologically analyzed before and after manipulation. Obligate and facultative anaerobes, microaerophilic bacteria and yeasts were recovered from 24, 14, 5 and 2 clinical specimens, respectively. The most frequent genera were Prevotella, Fusobacterium, Lactobacillus, Streptococcus, Clostridium and Peptostreptococcus for bacteria and Candida and Saccharomyces for yeasts. Strong positive associations, using an odds ratio system, were found between Clostridium and Prevotella and between Peptostreptococcus and Fusobacterium. Even after the instrumentation and the use of Ca(OH)2, facultative anaerobes were detected in two root canals and yeasts in three. Microorganisms were isolated from seven canals at the end of the endodontic treatment: facultative anaerobes from five and yeasts from one. The microbiological evaluation of root canals with pulp necrosis suggests the presence of polymicrobial infections, mainly involving obligate anaerobes, and shows that the infection may persist after treatment.

Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis.

Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC.

In vitro susceptibilities of Paracoccidioides brasiliensis yeast form to antifungal drugs.

A total of 12 Paracoccidioides brasiliensis yeast form strains, including clinical and environmental isolates, were tested for their susceptibilities to amphotericin B, ketoconazole, fluconazole, and itraconazole. The tests were determined using a broth macrodilution procedure. In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates.

Prevalence of Malassezia spp. in the ears of asymptomatic cattle and cattle with otitis in Brazil.

Yeasts of the genus Malassezia are lipophilic microorganisms that are saprophytes that can act as opportunistic pathogens in animals. Malassezia pachydermatis is commonly isolated from the ear canal and skin of healthy dogs, or in association with seborrheic dermatitis and otitis externa conditions. The objective of the present study was to determine the occurrence of Malassezia spp. in the ears of healthy bovines and bovines with otitis. Specimens (secretion or cerumen) were collected with sterile swabs, inoculated onto Mycosel medium, supplemented with olive oil, and incubated at 35 degrees C for 1 week. Yeasts were identified according to morphological characteristics, growth in Dixon medium at 32 degrees C and Sabouraud glucose medium modified by the addition of Tween 20, 40 or 80. The results showed that 54.7% of the cultures were positive in bovines with otitis (75) and 34.6% were positive in healthy bovines (378). Analysis of the positive cultures (41) from animals with otitis allowed presumptive identification of 24 strains corresponding to M. globosa (12), M. slooffiae (5), M. furfur (5) and M. sympodialis (2). Further studies on a larger number of animals may confirm the trend verified thus far, i.e. a higher frequency of isolation of Malassezia spp. from animals with otitis than from healthy animals (P<0.01) and a predominance of the species M. globosa.

Microevolution of a standard strain of Cryptococcus neoformans resulting in differences in virulence and other phenotypes.

Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.

Molecular epidemiology of Cryptococcus neoformans in Brazil and the United States: evidence for both local genetic differences and a global clonal population structure.

Little is known about the global molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans. We studied 51 clinical and environmental (pigeon excreta) isolates from two cities in Brazil (Belo Horizonte and Rio de Janeiro) by analyzing their carbon assimilation patterns, electrophoretic karyotypes, restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element-1 (CNRE-1), and URA5 sequences. Results were compared to those previously obtained for isolates from New York City by the same DNA typing methods. Computer-assisted analysis of RFLPs and contour-clamped homogeneous electrophoresis (CHEF) patterns and URA5 sequences was performed to generate dendrograms. Some environmental and clinical isolates were found to be indistinguishable by CHEF, CNRE-1 RFLP, and URA5 sequence analyses. Similarly, some isolates from Rio de Janeiro and Belo Horizonte were indistinguishable by the three DNA typing techniques. Overall, Brazilian isolates appeared to be less heterogeneous by DNA analysis than isolates from other regions. Several Brazilian isolates were highly related to New York City isolates. Phylogenetic analysis of the sequences obtained for the Brazilian isolates and those obtained for New York City isolates was congruent with the dendrogram generated from the CNRE-1 RFLP data. In summary our results indicate (i) that the discriminatory power of the DNA typing method differs for Brazilian and New York City strains, with the order being CNRE-1 RFLP analysis > URA5 sequence analysis > CHEF analysis and CHEF analysis > URA5 sequence analysis > CNRE-1 RFLP analysis, respectively; (ii) that there are differences in local genetic diversity for Brazilian and New York City isolates; (iii) that there is additional evidence linking clinical isolates to those in pigeon excreta; and (iv) that some isolates from Brazil and New York City are closely related, consistent with the global dispersal of certain pathogenic strains.

Effects of three azole derivatives on the lipids of different strains of Sporothrix schenckii.

The comparative effects of ketoconazole, itraconazole, and fluconazole on the lipids of five Sporothrix schenckii strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols, by thin-layer chromatography and ultraviolet spectrophotometry. Growth of the S. schenckii isolates in the presence of azole derivative concentrations below the minimum inhibitory concentration (MIC) resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol. Concerning the MIC values, fluconazole was found to be the least effective drug. On the other hand, as determined from a comparison of the effects of the three azoles on the sterol content of the strains studied, no significant differences in efficacy were found among the tested drugs.

Ocular and concomitant cutaneous sporotrichosis.

We report here the case of a 12-year-old girl with concomitant cutaneous and ocular sporotrichosis. Sporothrix schenckii was isolated from skin lesions and aqueous humour. The difficulty in the diagnosis and treatment of this form of mycosis is discussed and the data are compared with those published in the few reports available in the literature.