Proposal of a pathway for enteric virus groups detection as indicators of faecal contamination to enhance the evaluation of microbiological quality in freshwater in Argentina.

An environmental survey was conducted in order to assess the frequency of detection of picobirnavirus (PBV), human adenovirus (HAdV) and infective enterovirus (iEV) as indicators of faecal contamination in freshwater, and to determine their potential as reporters of the presence of other enteric viruses, such as group A rotavirus (RVA). The study was carried out over a three-year period (2013-2015) in the San Roque Dam, Córdoba, Argentina. The overall frequency detection was 62.9% for PBV, 64.2% for HAdV and 70.4% for iEV. No significant differences were observed in the rates of detection for any of these viruses through the years studied, and a seasonal pattern was not present. Whenever there was RVA detection in the samples analyzed, there was also detection of iEV and/or HAdV and/or PBV. At least one of the viral groups analyzed was demonstrated in the 100% of the samples with faecal coliforms values within the guideline limits. In this setting, especially in those samples which reveal faecal indicator bacteria within the guideline limit, we propose to carry out a pathway, involving PBV, HAdV and iEV detection in order to enhance the evaluation of microbiological quality in freshwater in Argentina. The proposed methodological strategy could report faecal contamination in water, mainly of human origin, and the condition of the matrix to maintain viral viability. In addition, the viral groups selected could report the presence of RV.

Enteric Viruses in Surface Waters from Argentina: Molecular and Viable-Virus Detection.

Water resources contaminated with wastewater are an important source for the dissemination of enteric viruses with an impact on the health of the population. The aim of the study was to assess the viral contamination of freshwater from a dam in Argentina by using infectious enterovirus detection, viral RNA amplification, and a genetic characterization of five enteric viruses associated with diarrhea and hepatitis. Enterovirus infectivity (iEV) was evaluated by cell culture and direct immunofluorescence. The detection of the viral genome of rotavirus (RV), human astrovirus (HAstV), norovirus (NoV), hepatitis A virus (HAV), and hepatitis E virus (HEV) was performed by reverse transcriptase PCR (RT-PCR). A total of 48 water samples from 4 monitoring points on the body of the dam from January to December 2012 and 66 water samples from 3 tourist beaches on the edge of the dam from October 2013 to October 2015 were collected monthly. During the first period, the overall viral frequency detection was 52.1% for group A RV, 50% for HAstV, 60.4% for NoV, 22.9% for HAV, 2.1% for HEV, and 64.6% for iEV. The overall frequency detection for the second sampling was 18.2% for RV and HAstV, 31.8% for NoV, 7.57% for HEV, and 66.7% for iEV. There was no detection of HAV during this period. The genotypes and genogroups detected through the study correlated with the most common genomic variants associated with human gastrointestinal and hepatitis illnesses. The results obtained could alert the health systems and environmental sanitation to make decisions for viral control and prevention in our environment.IMPORTANCE The study shows the impact of anthropic contamination of one of the most important tourist water resources in Argentina. This course of recreational water would be a favorable scenario for infection, as well as a reservoir for the enteric viruses, creating a risk for the population exposed to these waters. The results obtained could alert the health systems and environmental sanitation to make decisions for the control and prevention of viral diseases in this environment.

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.

Lipid components and water soluble metabolites in salted and dried tuna (Thunnus thynnus L.) roes.

The salted and dried product of tuna roe (bottarga) is a seafood characteristic of the Mediterranean area and exported all over the world. Samples of bottarga from bluefin tunas (Thunnus thynnus, L.) caught in the southwest Mediterranean sea were analysed. The samples were characterised by high content of marine wax esters (55-67 mol% of lipid classes), of docosahexaenoic (22:6 n-3, 25 w%) and oleic (18:1 n-9, 19 w%) fatty acids. Cholesterol was detected as 7-9 w% of lipids. Free fatty acids, index of lipid hydrolysis, represented 32-39 mol% over total fatty acids. Among metabolites, nutrients as taurine, nicotinamide and ß-alanine, were found. The microflora comprised staphylococci, enterococci (2.2 log(10)CFU/g) and lactic acid bacteria (3 log(10) CFU/g). The food-borne pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. were not detected. These findings indicate tuna bottarga as valuable source of nutrients.

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.

Cocirculation of Rio Negro Virus (RNV) and Pixuna Virus (PIXV) in Tucumán province, Argentina.

Venezuelan equine encephalitis complex includes viruses considered emerging pathogens for humans and animals in the Americas. Two members of this complex have been detected previously in Argentina: Rio Negro Virus (RNV), detected in mosquitoes from Chaco province and rodents from Formosa province, and Pixuna Virus (PIXV), detected in mosquitoes from Chaco province. To carry out surveillance studies in other parts of the country, detection of a 195-bp fragment of alphaviruses by RT-nested PCR was performed in mosquito samples from San Miguel de Tucumán city. Four pools resulted positive and three were sequenced. Two amplicons grouped with RNV and one with PIXV. This is the first report of viral activity of members of the Venezuelan equine encephalitis complex in north-eastern Argentina.

[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.

[Microbial environmental surveillance in a bone marrow transplant unit]. / Sorveglianza microbiologica ambientale in un centro per il trapianto di midollo osseo.

The aim of this study was to evaluate, after several years of application, in a bone marrow transplant (BMT) center of a Cagliari Hospital, the effectiveness of the disinfection protocol in minimizing the risk of environmentally transmitted infections. Microbial contamination of the air was evaluated every two months during normal activity using an SAS sampler. The contamination of surfaces was determined weekly, 2hrs after sanitation, using disposable surface contact plates. The results of environmental monitoring generally showed low values of microbial contamination of air and surfaces. Only two service rooms and two patient's rooms without own bathroom showed levels of microbial contamination slightly exceeding, in a few samplings, the values considered acceptable for environments at high risk of infections. From a qualitative point of view, the microrganisms isolated generally belonged to environmental species. In conclusion our study confirms the importance of microbial monitoring in the control and prevention of outbreaks of infections in BMT Units. This approach allows significant reduction in the level of contamination not only by improving cleaning procedures, but also by motivating the cleaning staff trough making them aware of their responsibilities.

[Microbial circulation and control in two Intensive Care Units]. / Circolazione microbica e controllo microbiologico in due Unit di Terapia Intensiva.

A microbiological survey was carried out in two medical Intensive Care Units from January to June 2000. The patients, staff (hands and upper respiratory tract) and environment were monitored. The results obtained in both Care Units give cause for concern. They showed particularly high cultural positivities in bronchoaspirates collected from artificially ventilated patients, a high percentage of positive environmental samples, and frequently contaminated hands in hospital staff, conditions which may facilitate microbial circulation in the medical Intensive Care Units. It would therefore seem necessary to promptly apply specific preventive measures for both the environment and patients.