Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.

Looking for Candida nivariensis and C. bracarensis among a large Italian collection of C. glabrata isolates: results of the FIMUA working group.

Two recently described pathogenic Candida species, C. nivariensis and C. bracarensis, share many phenotypic characteristics with C. glabrata and are easily misidentified as such. The aim of this study was to determine the occurrence of these cryptic species in Italy. One thousand yeast isolates collected in 14 Italian regions and identified as C. glabrata by phenotypic and biochemical methods were included in this study: 928 were screened on CHROMagar and 72 were analysed by a multiplex PCR. None of these cryptic species was identified despite the nationwide distribution and the variety of biological origin of the isolates.

Molecular detection of virulence factors and antibiotic resistance pattern in clinical Enterococcus faecalis strains in Sardinia.

In this study, the antibiotic resistance pattern and the presence of genes encoding several virulence factors in 91 Enterococcus faecalis strains isolated from different human clinical sources in Sardinia were investigated. Genotypic determination of virulence genes (gelE, esp, agg, ace, cylA,B,M,L(L),L(S), efaA, fsrB) was carried out by PCR. The production of gelatinase and haemolytic activity were also determined. Antimicrobial susceptibility tests were performed by an automated microdilution test (Vitek). The strains examined in this study contained at least one and up to as many as all virulence genes investigated. Examining the distribution of these factors in the different groups of clinical strains, we found that all but one virulence determinant were detected more frequently among urinary isolates. The detection of some factors by PCR did not always correlate with its phenotypic expression. Antibiotic susceptibilities among the Enterococcus faecalis strains investigated in our study were typical for the species, with expected levels of acquired resistance. Faecal isolates had the highest percentage of resistance, especially to high level-gentamicin, ciprofloxacin and norfloxacin. In summary, a wide variety of genes encoding virulence factors have been detected among our clinical Enterococcus faecalis strains, and those isolated from UTI were characterized by a higher virulence potency compared with strains from other clinical sources. Silent virulence genes (cyl or gelE) were frequently detected, therefore both the genotypic and phenotypic assays seem necessary for a better characterization of the strains. Our results may serve as a basis for additional surveillance studies of infections caused by this microorganism.

[Phenotypic and molecular characterization of Candida species in ICU]. / Caratterizzazione fenotipica e molecolare di Candida isolate in un reparto di Terapia Intensiva.

Two hundred sixty three Candida isolates were obtained from specimens of patients hospitalized in a Intensive Care Unit. Candida albicans was the predominant species, followed by C. tropicalis, C. krusei, C. glabrata e C. parapsilosis. For C. albicans isolates, amphotericin B was the more efficient antifungal (2.3% of resistant strains), while voriconazole was the more efficient for C. krusei and C. glabrata, known for their lower susceptibility to fluconazole. RAPD-PCR technique with CDU primer was used for the molecular characterization of 48 C. albicans strains isolated from 10 patients. Genetic similarity at 90% level was observed for some Candida strains isolated from the same patient, indicating a possible colonization from the original strain. Moreover the high similarity coefficient observed between isolates from different patients may indicate an exogenous colonization originating from hospital-endemic strains or inadequate manipulation by health care workers.

Characterization of yeast population and molecular fingerprinting of Candida zeylanoides isolated from goat's milk collected in Sardinia.

The occurrence of yeast microflora in raw goat's milk collected from 62 dairy farms located in different areas of Sardinia was evaluated. Candida zeylanoides was the most frequently occurring species followed by different Basidiomycetous species. In the strains isolated some biochemical characteristics of technological interest were investigated and a predominance of lipolytic yeast species was found. We employed a simple method of DNA extraction that in a minimal time and with low-cost provided a high quality of DNA for RAPD analysis of 32 isolates of C. zeylanoides. The primers M13 and CDU were used and at 40% of similarity, two distinct clusters were observed. The presence of C. krissii species was supposed but further molecular studies are needed to exclude the presence of an as-yet-undescribed species.

Prevalence of Candida species in different hospital wards and their susceptibility to antifungal agents: results of a three year survey.

Over a three years period, 472 Candida isolates were obtained from specimens of patients hospitalized either in "at risk", Bone Marrow Transplant Unit and Intensive Care Unit, or in conventional wards, Pneumological Divisions of the "Binaghi" Hospital of Cagliari (Italy). Antifungal susceptibility profile to amphotericin B, voriconazole, fluconazole and ketoconazole was determined. Candida albicans was the predominant species while Candida krusei was the most frequent non-albicans species. C. krusei was significantly more common among Bone Marrow Transplant Unit and Intensive Care Unit than Pneumological Divisions patients (17.9% and 14.1% vs. 6.0%; p < 0.05). No significant differences were observed when the same distribution was analysed with regard to the other Candida species or when Bone Marrow Transplant Unit and Intensive Care Unit were compared. The profiles of susceptibility to the antifungal drugs among isolates from the different hospital wards showed no significant differences, even though most of MIC values were higher for Intensive Care Unit isolates compared to those for Bone Marrow Transplant Unit and Pneumological Divisions. For C. albicans isolates, amphotericin B was the more efficient antifungal (97.7% S), while fluconazole (6.1% R [Resistant] and 2.6% SDD [Susceptible Dose Dependent]) and ketoconazole (4.1% R and 3.2% SDD) showed the lowest activity. Voriconazole was the more efficient antimycotic for C. krusei (96.7% S) and Candida glabrata (100% S [Sensible]) isolates. This study has shown a significantly higher presence of non-albicans Candida in at risk wards as well as a decreased susceptibility to the older azoles (ketoconazole and fluconazole) among C. albicans isolates.

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese.

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.

Yeast populations in Sardinian feta cheese.

In this study, the yeast populations in feta cheese from two different Sardinian dairies were examined. Samples of good quality feta (32) and samples of feta with a slimy surface defect (10) were examined from Dairy A. Similar, samples of good quality feta (23), feta with slimy surface defects (14) and samples with swelling defects (6) were examined from Dairy B. Kluyveromyces lactis was the dominating species in feta from Dairy A (95.2% of samples) followed by Debaryomyces hansenii (76.2%), Dekkera anomala (28.6%) and Dek. bruxellensis (19%). D. hansenii was dominant in samples from Dairy B (93%), followed by K. lactis (23.3%), Geotrichum candidum (23.3%) and Dek. anomala (18.6%). No significant difference was observed between the occurrence of yeast species in feta of good quality and in feta with slimy surface defects, thus confirming that slimy production is not associated with yeast contaminations. The swelling of samples observed in Dairy B seems to be caused by Dek. anomala. In fact, this strong fermenting species was present in all swelled samples in numbers exceeding 10(6) CFU g(-1), while it was isolated in very low concentration in only 5.4% of good samples.

Yeasts associated with Sardinian ewe's dairy products.

In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.

[Pollution by mites and/or mold spores in work environments and the risk of occupational respiratory pathology]. / Inquinamento da acari e/o micofiti in ambienti di lavoro e rischio di patologia respiratoria professionale.

In recent years the measurement of mold spore concentrations in working environments where workers spend long periods of time has acquired considerable importance because of the diffusion of diseases caused by fungi. On this basis, the aim of the research was to evaluate the level of environmental fungal pollution and contamination by mites in three factories producing seasoned foods, i.e., hams, sausages and cheeses. We also studied the occurrence of fungal species in the upper respiratory tract of workers operating in the washing and brushing areas, which was compared with the environmental concentrations in these areas, where contamination with fungal spores was visibly high. The results showed high and widespread fungal pollution in most of the working environments sampled and a significant presence of the same species in the upper respiratory tract of the workers. The data obtained indicate that the environments sampled can constitute a possible health risk factor for workers.

Pollen and mold allergy: aerobiologic survey in the atmosphere of Cagliari, Italy (1986-1988).

The occurrence of pollens and molds in the atmosphere of Cagliari, Italy over a 3-year period (1986-88) is reported. Pollen grains are present in the air from February to July, with a maximum in May. Urticaceae and Cupressaceae were the major contributors to the total pollen airspora, followed by Graminaceae, Oleaceae, Pinaceae, Cheno-Amaranthaceae, and Fagaceae. Fungal spores showed two periods of high sporulation, from April to June and in September-October, and two marked decreases, during winter and August. Cladosporium was by far the most abundant spore identified, followed by Basidiomycetes, Alternaria, and Fusarium.

[Hygienic and microbiological quality of starters and coagulants used in the production of sheep's milk cheese]. / Qualita' igienica e microbiologica di cagli e innesti utilizzati nella produzione di formaggio di pecora.

The hygienic level of the raw materials is prevailing in the determination of microbial quality of finished product in food production. Not all alimentary products have the same possibilities of being contaminated. They develop, with a series of intermediate passages, from products that can not be contaminated to products that can be easily attached from micro-organisms. It is essential to locate the points of possible pollution and monitor them with appropriate modalities for a good quality of finished products. The "starters" and "coagulants" of dairy industry may be carrier of microbial pollution if they are improperly handled during the processing and storage. 202 samples of starters and 128 of coagulants, produced by large and small industries have been examined in this work. Total viable count, total Coliforms, anaerobic bacteria and Yeasts have been determined. A quantitative valuation has been also carried out above the lactic bacteria in all samples of starters. The starters results have shown a clean quantitative difference between the industrial products and natural cultures about the lactic microflora value, as well as more high frequency of pollution in samples in which lactic acid bacteria were found below standard. Among the coagulants, no microbial pollution has been detected in the commercial enzymes, whereas serious hygienic problems have shown up in the "rennets"; they have also presented an high anaerobic bacteria number/gr. of product. According to the related data, the AA. think starters and coagulants can have not unfavourable influence on dairy production only is some hygienic operations requirements are followed in their preparation.

[Results of an airborne spore study in various regions of southern Sardinia]. / Risultati di una indagine aerosporologica in diverse località della Sardegna meridionale.

Fungal air spores can play a significant role in several allergic manifestations. Therefore, the identification of geographic areas of mould distribution could be helpful to the clinician, especially if associated with fungal air spore recording in homes or working environments of sensitized subjects, in determining the real clinical importance of sensitization to fungi. On this basis, we studied the occurrence of airborne fungi at two urban sites and at two rural sites in the South of Sardinia, from May 1987 to April 1988, using the gravity plate method. Our survey has pointed out a significant difference about the occurrence of airborne spores in the areas sampled. Spore concentrations were lower at the urban sites during all the survey period. On the whole 6319 fungal colonies belonging to 28 different genera have been found. Cladosporium, Alternaria, Penicillium and Aspergillus, represented by a range of species, were the most common fungi identified in all sites examined. Remarkable the incidence of Yeasts, represented by the genera Candida, Saccharomyces, Rhodotorula and Sporobolomyces. Aureobasidium, Stemphilium, Botrytis, Chaetomium, Mucor and Rhizopus have been found in all sites but they have not been steadily isolated during the survey. Several other genera have been found only sporadically. Our results seem to confirm that fungal air spores, because of its quantity and variety, can represent a serious problem for human health in Sardinia.