Biotyping of Malassezia pachydermatis strains using the killer system

The killer phenomenon has been used as epidemiological marker for Candida albicans, where hundreds of biotypes can be obtained. The objective of this study is to observe the behaviour of 30 strains of Malassezia pachydermatis isolated from dogs with otitis (15) or dermatitis (15) against 9 killer yeasts, which, when grouped in triplets produced a 3 digit code (biotype). The growth inhibition of the 30 strains of M. pachydermatis due to the effect of the killer yeasts used permitted the determination of the following biotypes: 888 (33.3%), 212 (26.7%), 111 (16.7%), 312 (6.7%), 512 (6.7%), 242 (3.3%), 311 (3.3%) and 411 (3.3%). Biotypes 888, 212 and 111 occurred most frequently in both ear canal and skin samples.     

Antifungal Susceptibility, Enzymatic Activity, PCR-Fingerprinting and ITS Sequencing of Environmental Cryptococcus laurentii Isolates from Uberaba, Minas Gerais, Brazil

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human infection have been already reported. This study aimed to evaluate the phospholipase, proteinase and hemolysins activity, the antifungal susceptibility profile, the genetic variability by M13 and (GACA)(4) fingerprinting and the internal transcribe spacer (ITS) sequencing of 38 C. laurentii isolates recovered from captive bird droppings and surrounding hospital areas. All of them exhibited phospholipase activity, while the hemolytic activity was evidenced in 34 (89.4%) isolates. None of them exhibited proteinase activity. Twenty-seven isolates (71.1%) presented susceptibility dose dependent to fluconazole. Most isolates (94.7%) were susceptible to voriconazole, while one (2.65%) was resistant to this drug. Twenty-one (55.3%) isolates showed reduced susceptibility to itraconazole while nine (23.7%) were resistant. Three (7.9%) and five (13.1%) isolates exhibited resistance to ketoconazole and amphotericin B, respectively. Most C. laurentii fingerprinting obtained with M13 and (GACA)(4) showed high heterogeneity. By using the two primers, seven (18.4%) isolates grouped as A (CL2, CL7, and CL8), B (CL35, CL38) and C (CL29, CL30) with 100% similarity. Different from most variable surrounding hospital isolates, all but one of the pet shops strains clustered with the two primers, although they had been recovered from different neighborhoods. All isolates were identified as C. laurentii phylogenetic group I by ITS sequencing. Thus, the presence of virulence factors, a decreased antifungal susceptibility and a heterogeneous molecular pattern of the C. laurentii isolates here described suggests this species can be a potential pathogen in the context of the immunocompromised population.     

Influence of pesticides on yeasts colonizing leaves

The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides.     

Comparison of various media and various methods for the growth and isolation of Pityrosporum pachydermatis

The pathogenic role of Pityrosporum pachydermatis in otitis externa of dogs and the related diagnostic problems are emphasized. We report results related to isolation, cultivation and identification of yeast. Agar nutritive glucosate with 1,5% of yeast extract has been showed as the best medium permitting identification in 24 hrs, associated with morfological test. Tween 80 integration (1%) to the medium permits to isolate lipolitic yeasts also.     

Antimicrobial Susceptibility and rRNA Gene Restriction Patterns among Staphylococcus intermedins from Healthy Dogs and from Dogs Suffering from Pyoderma or Otitis Externa

A total of 60 Staphylococcus intermedins strains from dogs were investigated by their sensitivity to various antibiotics (50 strains) and by their rRNA gene restriction patterns (ribotyping) (60 strains). Fifteen isolates were from healthy dogs, 9 with otitis externa, and 36 with pyoderma, including 10 strains from a previous study. Sixty per cent of the 50 strains tested for antibiotic susceptibility demonstrated resistance to penicillin, 24% to spiramycin, 20% to tetracycline, 16% to chloramphenicol, and 2% to fucidic acid. All isolates were susceptible to amoxycillin with clavulanic acid, enrofloxacin, and sulphonamides with trimethoprim. There were no significant differences in antimicrobial susceptibility patterns observed among isolates from pyoderma, otitis externa or healthy dogs. Among the 60 strains studied by ribotyping, 10 different ribotypes were identified: 6 different ribotypes among isolates from otitis externa, 8 among isolates from pyoderma, and 5 among isolates from healthy dogs. One ribotype (profile C) was dominant among the isolates from healthy dogs while another ribotype (profile A) was dominant among strains from dogs suffering from pyoderma. This profile was not demonstrated in any of the strains from healthy dogs. From 5 different dogs suffering from pyoderma, 2 different clones were demonstrated based on their plasmid profile and antibiogram. In these dogs 1 of the clones always belonged to ribotype A. The results concerning strains of S. intermedins isolated from furunculosis suggest the existence of distinct subpopulations with different pathogenicity to dogs.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)

The aim of this study was to investigate the frequency and antifungal susceptibility of Candida clinical isolations coming from patients with candidiasis in six health care centers of Caracas, Venezuela metropolitan area. The laboratory reports were retrospectively revised from January 2003 through August 2005. The isolated yeasts identification was carried out by conventional methods and antifungal susceptibility was evaluated by ATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden). One thousand nine hundred seventy seven (1.977) yeasts were studied and their susceptibility testing were carried out only in 1,414 of them. C. albicans was the most isolated yeast (46.7%) and none-albicans Candida-species represented more than half of the isolations (53.4%). All the isolated yeasts evaluated presented CMIs<1 microg/ml to anfotericina B and showed variable susceptibility percentages to fluconazole (91.5%), itraconazole (80%) and voriconazole (98.6%).     

Subculture on potato dextrose agar as a complement to the broth microdilution assay for Malassezia pachydermatis

The main aim of this study was to verify the efficacy of subculture on potato dextrose agar (PDA) as a complement to the in vitro susceptibility test for Malassezia pachydermatis strains by a broth microdilution method, as well as to determine the MIC and MFC of azole derivatives, amphotericin B and caspofungin. The microdilution assay was performed in 96-well plates using a modified RPMI 1640 medium. The M. pachydermatis strains were resistant to caspofungin. All strains (n=50) had shown MIC values of <0.03, <0.03, 2.0, 4.0 and 4.0 mug/ml for itraconazole, ketoconazole, voriconazole, fluconazole and amphotericin B, respectively. Thus, the subculture on PDA improved the analysis of the in vitro antifungal susceptibility of M. pachydermatis.     

In vitro resistance to fluconazole and itraconazole in clinical isolates of Candida spp and Cryptococcus neoformans

An in vitro susceptibility testing of 181 strains of six species of Candida and 21 strains of Cryptococcus neoformans was carried out in order to investigate the resistance to new antifungal drugs. We have studied clinical isolates from 200 different patients of Hospital del Mar (Barcelona) and Hospital La Inmaculada (Almería). An agar diffusion method (NeoSensitabs, Rosco, Taastrup, Denmark), was employed with fluconazole, itraconazole, and reference drugs amphotericin B, flucytosine, tioconazole and ketoconazole. A high level of susceptibility was found for amphotericin B in C. neoformans strains while 19% of them were resistant to flucytosine. All the strains of C. neoformans and Candida guilliermondii were susceptible to the new azoles derivatives and also Candida parapsilosis and Candida albicans had a great susceptibility to this antifungals. A greater level of resistance was found for Candida krusei, Candida tropicalis and Candida glabrata to fluconazole, itraconazole and ketoconazole, but resistance to fluconazole and itraconazole is not always linked because ten resistant strains for fluconazole were susceptible to itraconazole, and two other resistant to itraconazole were susceptible to fluconazole.     

Identification of atypical strains of Malassezia spp. from cattle and dog

Yeast species in the genus Malassezia are lipophilic with the exception of Malassezia pachydermatis. During a study of the occurrence of Malassezia species in the external ear of 964 cattle and 6 dogs in Minas Gerais, Brazil, six lipid-dependent isolates could not be identified to known species. Four isolates came from healthy cows, one from a cow with otitis, and one from a healthy dog. When tested with Tweens and Cremophor EL as single sources of lipids, the strains grew on all sources except Cremophor EL. None of the six strains hydrolyzed esculin, and all produced catalase. Pigment production from tryptophan was variable. Partial large subunit rRNA sequences were obtained for two isolates that remained viable in culture. The strain from the cow with otitis was identified as a lipid-dependent variant of M. pachydermatis, and the strain from the dog was an atypical variant of Malassezia furfur.     

Occurrence and Population Size of <Emphasis Type="Italic">Malassezia</Emphasis> spp. in the External Ear Canal of Dogs and Cats Both Healthy and with Otitis

Malassezia yeasts are commensal organisms of human and animal skin that occasionally act as pathogens; lipid-dependent species are associated with human skin disorders. The aim of this work was to evaluate the occurrence, risk factors and population size of different species of Malassezia in the external ear canal of dogs and cats with and without otitis externa. Between 2001 and 2003, 107 healthy animals (25 cats and 82 dogs) and 123 animals with chronic otitis exteirna (48 cats and 75 dogs) were clinically examined. Sterile cotton swabs were used to collect specimens from the external ear canal and culture tests and cytological examinations were performed to detect the presence and population size of Malassezia yeasts. Malassezia yeasts were isolated from 72.9% and 40% of the cats and from 57.3% and 28.0% of the dogs, with and without otitis externa, respectively. Malassezia was frequently isolated from animals under 5 years of age. The highest prevalence of positive samples was reported in winter for cats and in autumn for dogs. Dogs with pendulous ears showed a higher incidence of infection than dogs with erect ears. More Malassezia yeasts were recovered from animals with otitis (i.e. 59.6 colony forming units – CFUs – for cats and 66.0 CFUs for dogs) than healthy animals. Out of the 413 isolates obtained from animals with and without otitis, 403 (97.6%) were identified as M. pachydermatis and 10 (2.4%) as M. globosa. A statistical evaluation of the occurrence of Malassezia yeasts in dogs and cats revealed that predisposing factors for Malassezia infections are sampling period for cats, and type of ear for dogs. The largest population of Malassezia yeasts was detected in animals with otitis, suggesting a role in the occurrence of lesions.     

院内150例真菌感染及其耐药性分析

目的研究院内真菌感染的特点及耐药状况。方法对临床标本分离的真菌,用TAB Expression鉴定仪鉴定,E—test抗真菌药敏试纸条进行药敏试验。结果150株真菌共有14个种,以白色念珠菌为主．占45．3％。各种真菌对6种抗真菌药物均有不同程度的耐药。结论院内真菌感染呈上升趋势,并且产生一定的耐药株。     

Phenotypic aspects of oral strains of Candida albicans in children with Down's syndrome.

The aim of this article is to characterize the biological aspects of oral strains of C. albicans in children with Down's syndrome. These yeasts were analyzed as to their macromorphological and enzymatic aspects and were tested as to their in vitro susceptibility to antifungal drugs using broth microdilution to determine the minimum inhibitory concentration (MIC). The morphotyping revealed that all oral C. albicans isolates from children with Down's syndrome promoted the formation of fringes regardless of size, while the control group presented smaller fringes. All oral C. albicans strains produced proteinase, but those with phospholipolytic activity showed greater enzyme capacity in the test group. In vitro susceptibility showed that all oral C. albicans isolates were sensitive to the drugs used.     

Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs

Samples were obtained from 65 unmedicated adult dogs, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, tetracycline, trimethoprim-sulphamethoxazole, streptomycin, ampicillin and rifampin. Forty-four isolates were obtained, which represents 67.7% of samples. Coagulase-negative species were most commonly found, and the most frequently isolated staphylococcus species were Staph. epidermidis and Staph. aureus. Other species, such as Staph. simulans, Staph. haemolyticus, Staph. saprophyticus and Staph. intermedius were also isolated. Resistance to antibiotics was frequently observed, with 90.9% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin. Multidrug resistance was a common finding, and one strain of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Resistance to three or more different drugs was a common finding, observed in 16 strains (36.4%) of both coagulase-positive and coagulase-negative staphylococci. This study highlights the emergence of cases of otitis externa determined by coagulase-negative staphylococcus strains and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.     

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

Forty Candida albicans strains isolated from patient's mouth with fixed orthodontic appliances were analyzed to their susceptibilities to antifungal agents, mouth rinses and essential oils. Susceptibility to fluconazole, econazole, miconazole and ketoconazole, amphotericin B and nystatin was assessed by the disk diffusion (DD) method based on the Clinical and Laboratory Standards Institute M44-A protocol, and by Etest (fluconazole and amphotericin B). The susceptibilities to mouth rinses and essential oils were also determined by the DD technique. All isolates tested were susceptible (S) to amphotericin B, nystatin and fluconazole. The overall concordance between the DD and the Etest was 100% for amphotericin and fluconazole. One isolate was resistant to econazole (2.5%) and the other to ketoconazole (2.5%). Econazole and ketoconazole had the highest percentages of susceptible dose dependent (SDD), 55 and 95%, respectively. Regarding to the susceptibility isolates profile, seven phenotypes were detected, and the 3 more represented (90% of the isolates) of them were SDD to one, two or three azoles. The study of mouth rinses showed a high variability of efficacy against C. albicans. The results showed that the isolates susceptibility to essential oils differed (P < 0.05). The profile activity was: cinnamon > laurel > mint > eucalyptus > rosemary > lemon > myrrh > tangerine. The main finding was that the susceptibility to cinnamon and laurel varied among the three more representative antifungal phenotypes (P < 0.05). The susceptibility of econazole-SDD isolates to cinnamon and lemon was higher than those of the econazole-S yeasts (P < 0.05). In contrast, econazole-SDD isolates were less affected by laurel than econazole-S counterparts (P < 0.05).     

醇化烟叶酵母菌分离鉴定及其生物学特性研究

采用涂布培养法和富集培养法对醇化烟叶样品中酵母菌进行了分离,富集培养法分离到23株,涂布培养法分离到6株。29株酵母菌的生物学特性研究表明,其中果胶酶、蛋白酶、淀粉酶、脂酶、纤维素酶、酚类利用、木质素利用、尼古丁降解、发酵产物气味等试验呈阳性的菌株数分别是1、10、0、3、10、22、1、21和29株。利用ATB系统,将ATY1、ATY8和ATY27等3株酵母菌分别鉴定为黏红酵母(Rhodotorula glutinis)、罗伦隐球酵母(Cryptococcus laurentii)和粗壮假丝酵母(Candida valida),各菌株鉴定结果的符合率分别为99.4%、98.2%和89.9%。研究结果表明,醇化烟叶中酵母菌种群数量较小,但酵母菌的生物学特性有利于烟叶醇化,值得进一步深入研究。     

Ergosterol biosynthesis pathway in Aspergillus fumigatus

The sterol composition of Aspergillus fumigatus for the biosynthesis of ergosterol is of interest since this pathway is the target for many antifungal drugs in clinical use. The sterol composition of this fungal species was analyzed by gas chromatography-mass spectrometry in different strains (susceptible and resistant to azole drugs). Also, sterols were analyzed in several A. fumigatus mutant strains deficient in enzymatic steps of the ergosterol biosynthesis pathway such as 14-alpha sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A, Erg3B and Erg3C). All sterols identified from azole-resistant A. fumigatus strains were qualitatively and quantitatively similar to the susceptible strain (CM-237). However, sterol composition of mutants strains were different depending on the lacking enzyme. The analysis of the sterol composition in these mutant strains led to a better understanding of the ergosterol biosynthesis pathway in this important fungus.     

吸毒人群口腔念珠菌的药物敏感性分析

目的了解吸毒人群口腔念珠菌对常用抗真菌药物的敏感性,为临床治疗念珠菌病提供参考资料。方法采用美国CLSI推荐的微量稀释法测定75株念珠菌对4种常用抗真菌药物两性霉素B、5-氟胞嘧啶、氟康唑和酮康唑的药物敏感性。结果 75株吸毒人群口腔念珠菌对两性霉素B、5-氟胞嘧啶、氟康唑和酮康唑的耐药率分别为0%、4%、8%和13.3%(P0.01),对氟康唑和酮康唑的交叉耐药率为8%;非白念珠菌对氟康唑和酮康唑的耐药率高于白念珠菌(P0.05)。结论吸毒人群口腔念珠菌对两性霉素B和5-氟胞嘧啶的敏感性高于对氟康唑和酮康唑的敏感性。吸毒人群口腔念珠菌存在着对氟康唑、酮康唑和5-氟胞嘧啶的天然耐药株和对唑类药物的天然交叉耐药株,且非白念珠菌对氟康唑和酮康唑的耐药率及交叉耐药率高于白念珠菌。     

Prevalent Drug Resistance Among Oral Yeasts from Asymptomatic Patients in Hainan,China

The oral cavity is a significant niche of the human microbiome and a gateway for the microbiota in many other human body sites. As a result, understanding the oral microbiota has broad implications for the prevention and management of human infectious diseases. Opportunistic yeast infections are among the most prevalent fungal infections of humans, and most opportunistic yeast pathogens are common residents of the oral mucosa. However, relatively little is known about the drug susceptibility profiles of oral yeasts. Here, we report the species distribution and patterns of antifungal susceptibility profiles among 313 yeasts isolated from the oral cavities of 301 asymptomatic hospitalized patients in Hainan Province in southern China. These yeasts were tested for their susceptibilities to the following five drugs: amphotericin B, fluconazole, itraconazole, ketoconazole, and fluorocytosine. Since none of the sampled hosts had taken any antifungal drugs at least 3 months before samples were taken, we hypothesized that little or no drug resistance should be observed. Contrary to our expectations, our analyses identified that 29 % (91/313) of the isolates were resistant to at least one drug and 14.3 % (45/313) were resistant to two or more of the five common drugs. The potential sources of the observed resistance were discussed.     

Potential therapeutic effect of yeast killer toxin

Experimental infections were produced in guinea pigs, rabbits and dogs with lesions similar to those seen in human seborrheic dermatitis and otitis externa by cutaneous application of cultures of Malassezia furfur and M. pachydermatis. Infected animals were treated by topical application of a concentrated yeast killer toxin (Hansenula anomala UCSC 25F). Clinical recovery as well as negative mycological test cultures of infected animals proved to the clearly associated with the treatment by the killer toxin.