Comparison of the yeast microbiota of different varieties of cool-climate grapes by PCR-RAPD

The yeast microbiota occurring on different varieties of grapes grown in cool-climate is not completely researched. Therefore, its identification is important to research. On the other hand, yeasts occurring in these fruits can be potentially used as starter cultures to obtain particularly demanded features in the production of wine. In addition, rapid methods for yeast identification allow to eliminate the contamination with pathogenic yeasts, which could cause the loss of wine production. The aim of the study was to isolate and identify the yeasts occurring on the surface of the different varieties of white and red grapes, grown in cool-climate of Poland. Also, the aim was to compare the qualitative and quantitative composition of yeasts on the tested grapes. The 84 cultures of yeasts were isolated, that were initially macroscopic and microscopic analyzed and the purity of cultures was rated on the WL medium. Identification of yeasts by PCR-RAPD was carried using the M13 primer. In the PCR-RFLP method ITS1 and ITS4 primers, as well as restriction enzymes HhaI, HinfI, HaeIII, were used. Preliminary identification of yeasts by standard methods produced results very different from the results obtained by molecular methods. Among the isolated microorganisms yeasts were dominating, but bacteria and molds were also present. Using the PCR-RAPD method most strains of yeasts were identified. Yeast microflora of different varieties of white and red grapes was very similar as the same species of yeasts were identified. Yeasts of the genus Saccharomyces were present in all varieties of grapes. The Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Metschnikowia pulcherrima, Rhodotorula minuta, Pichia kluyveri, Hanseniaspora uvarum and Rhodotorula mucilaginosa were identified by PCR-RAPD. 4 of the 33 tested strains of yeasts were identified by PCR-RFLP. By PCR-RAPD only Hanseniaspora uvarum was identified. The quantity and quality of microorganisms living on the surface of grape fruits is very important for the process of winemaking. Yeasts influence the course of alcoholic fermentation, the flavor, aroma, and thus the quality of the produced wine. To a large extent their presence depends on the condition of the surface of the fruit. Many researchers reported significant differences between yeast microflora in grapes of Mediterranean and cool-climate vineyards. As they are expected to affect the final wine properties precise researching of the microflora of cool-climate grapes may lead to the isolation of new species of yeasts and thus the wines with unique characteristics can be obtained

Wstępna identyfikacja zaburzeń mowy - przesiewy logopedyczne w praktyce nauczyciela

The subject of the article is the role of speech disorder screening in speech and language therapy (SLT) prevention and intervention. The authors presented a detailed account of the screening test Testing instrument for kindergarten aged children screening, supplementing it with a classification of norms according to which a child is assessed, as well as the examples of various speech aspects tests. The article emphasizes the possibilities of preventive impact in teacher work

Quantitative evaluation of fungi of the genus Candida in the feces of adult patients with type 1 and 2 diabetes : a pilot study

BACKGROUND: Gastrointestinal tract microbiota, particularly bacterial microflora, seem to have a different qualitative and quantitative composition in both type 1 (T1DM) and type 2 diabetes (T2DM) mellitus cases as compared to non-diabetic individuals. So far, there are no data from diabetes research concerning the prevalence of fungi, particularly the most common genus, i.e. Candida, which are important components of human colon microflora. We aimed to examine whether there are quantitative changes of Candida fungi in the feces of patients with T1DM and T2DM as compared to healthy controls. FINDINGS: Overall, we included 44 diabetic patients (27 patients with T1DM and 17 with T2DM) as well as 17 healthy, non-diabetic controls. Feces and blood samples were collected from all study individuals. DNA was isolated from fecal samples and quantitative real time PCR (qPCR) was applied in order to determine the number of fungal cells. Statistical association with selected clinical and biochemical features was examined. There was a difference in the amount of Candida in the feces among the three examined groups (p = 0.007). Candida spp. populations in T1DM and T2DM subjects were larger as compared to controls (p = 0.017 and p = 0.037, respectively). However, no difference was found between T1DM and T2DM. No association was identified between the quantity of fungi and examined patients’ characteristics, except for negative correlation with blood lipid parameters in T2DM group. CONCLUSIONS: Candida fungi appear to be more prevalent in the feces of patients with T1DM and T2DM. Their amount seems to be associated with serum lipids in T2DM patients. This initial finding requires further confirmation

Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood

BACKGROUND: The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR . RESULTS: Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 10(1) CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi. CONCLUSIONS: Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours

The effect of linagliptin treatment on gut microbiota in patients with HNF1A-MODY or type 2 diabetes : a preliminary cohort study

Wstęp: W wielu dotychczas przeprowadzonych badaniach oceniano związek między cukrzycą a mikroflorą jelitową. Obserwowano zmianę mikroflory jelitowej pod wpływem inhibitorów dipeptydylopeptydazy-4 w modelach zwierzęcych. Celem niniejszego badania była ocena wpływu linagliptyny na florę bakteryjną okrężnicy u ludzi. Materiał i metody: W prospektywnym badaniu kohortowym wzięło udział 24 pacjentów: 5 chorych na cukrzycę monogenową, związaną z mutacją HNF1A, i 19 chorych na cukrzycę typu 2. Próbki kału pobrano przed i 4 tygodnie po intensyfikacji aktualnego leczenia za pomocą linagliptyny lub pochodnej sulfonylomocznika. Z próbek kału wyizolowano rRNA, a następnie wykonano sekwencjonowanie 16S nowej generacji. Wyniki: U 9 pacjentów do leczenia dołączono linagliptynę, a u 15 pacjentów dołączono sulfonylomocznik lub zwiększono jego dawkę. Po leczeniu linagliptyną nie zaobserwowano zmian na poziomach taksonomicznych L2–L7 na podstawie analizy składu mikrobiomów (ANCOM). To samo odnosiło się do różnorodności alfa [wskaźnik różnorodności Shannona, p = 0,59; wskaźnik równomierności Pielou, p = 0,68; obserwowane operacyjne jednostki taksonomiczne (OTU), p = 0,77] i różnorodności beta (nieważony wskaźnik UniFrac, p = 0,99; ważony wskaźnik UniFrac, p = 0,93; wskaźnik Braya–Curtisa, p = 0,98; wskaźnik Jaccarda, p = 0,99). Również po intensyfikacji leczenia pochodną sulfonylomocznika nie zaobserwowano zmian na poziomach taksonomicznych L2–L7 w ANCOM ani zmian w różnorodności alfa (wskaźnik różnorodności Shannona, p = 0,19; wskaźnik równomierności Pielou, p = 0,21; obserwowane OTU, p = 0,42) i różnorodności beta (nieważony wskaźnik UniFrac, p = 0,99; ważony wskaźnik UniFrac, p = 0,99; wskaźnik Braya–Curtisa, p = 1; wskaźnik Jaccarda, p = 0,99). Wnioski: Po 4 tygodniach od dołączenia linagliptyny do aktualnego leczenia cukrzycy nie zaobserwowano zmian w mikroflorze okrężnicy. Konieczne są dalsze badania w celu ustalenia, czy linagliptyna wpływa na mikroflorę okrężnicy u ludzi.Introduction. Many studies have evaluated the relationship between diabetes and microbiota. In animal models, the dipeptidyl peptidase-4 inhibitors altered the gut microbiota. We investigated whether linagliptin alters the gastrointestinal flora in humans. Materials and methods. This prospective cohort study enrolled 24 patients: 5 patients with maturity onset diabetes of the young associated with HNF1A mutation and 19 patients with type 2 diabetes mellitus. Stool samples were collected at baseline and 4 weeks after treatment intensification with either linagliptin or a sulphonylurea alongside current treatment. Faecal 16S rRNA was analysed by next-generation sequencing. Results. Nine patients initiated linagliptin whereas 15 patients initiated or increased the dose of a sulphonylurea. After linagliptin treatment, we did not observe changes in taxa in L2–L7 based on analysis of composition of microbiomes (ANCOM). The same held true for pairwise alpha diversity (Shannon diversity, p = 0.59; Pielou’s measure of evenness, p = 0.68; and observed operational taxonomic units [OTUs], p = 0.77) and beta diversity distances (unweighted UniFrac, p = 0.99; weighted UniFrac, p = 0.93; Bray-Curtis, p = 0.98; and Jaccard, p = 0.99). Similarly, after sulphonylurea intensification, we did not observe changes in taxa in L2–L7 in ANCOM, nor were there changes in alpha diversity (Shannon diversity, p = 0.19; Pielou’s measure of evenness, p = 0.21; and observed OTUs, p = 0.42) or beta diversity distances (unweighted UniFrac, p = 0.99; weighted UniFrac, p = 0.99; Bray-Curtis, p = 1; and Jaccard, p = 0.99). Conclusion. We did not observe changes in colonic microbiota 4 weeks after addition of linagliptin to current diabetes treatment. Further studies are required to determine whether linagliptin influences the colonic microbiota in human

Metagenomic analysis of duodenal microbiota reveals a potential biomarker of dysbiosis in the course of obesity and type 2 diabetes : a pilot study

Numerous scientific studies confirm that, apart from environmental and genetic factors, a significant role is played by gastrointestinal microbiota in the aetiology of type 2 diabetes and obesity. Currently, scientists mainly focus on the distal intestinal microbiota, while the equally important proximal parts of the intestine are overlooked. The aim of the study was a qualitative analysis of the structure of the duodenal mucosa microbiota in groups of patients with obesity and with type 2 diabetes and where obesity qualified for bariatric surgery: sleeve gastrectomy. The microbiological results obtained were compared with some clinical parameters. As a result, it was possible to determine the microbiological core that the treatment and control groups had in common, including phyla: Firmicutes, Proteobacteria, and Actinobacteria. The patients with obesity and with type 2 diabetes and obesity presented a significantly lower number of genus Bifidobacterium compared to healthy subjects. Furthermore, the numbers of Bifidobacterium were positively correlated with the high density lipoprotein (HDL) concentration in the groups under study. The obtained results indicate that bacteria of the genus Bifidobacterium should be considered in the future in the context of a potential biomarker in the progress of type 2 diabetes and obesity

Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

BACKGROUND: Microbiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit. RESULTS: Using qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR. CONCLUSIONS: The qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture

Antimicrobial properties of selected copper alloys on Staphylococcus aureus and Escherichia coli in different simulations of environmental conditions : with vs. without organic contamination

Background: Hospital equipment made from copper alloys can play an important role in complementing traditional methods of disinfection. Aims of the study: The aim of this study was to assess the dynamics of the antimicrobial properties of selected copper alloys in different simulations of environmental conditions (with organic contamination vs. without organic contamination), and to test alternatives to the currently used testing methods. Materials and Methods: A modification of Japanese standard JIS Z 2801 as well as Staphylococcus aureus (SA) and Escherichia coli (EC) suspended in NaCl vs. tryptic soy broth (TSB) were used in tests performed on seven commonly used copper alloys, copper, and stainless steel. Results: A much faster reduction of the bacterial suspension was observed for the inoculum prepared in NaCl than in TSB. A faster reduction for EC than for SA was observed in the inoculum prepared in NaCl. The opposite results were found for the inoculum based on TSB. A significant correlation between the copper concentration in the copper alloys and the time and degree of bacterial suspension reduction was only observed in the case of EC. Conclusions: This study confirmed the antimicrobial properties of copper alloys, and additionally showed that Staphylococcus aureus was more resistant than Escherichia coli in the variant of the experiment without organic contamination. However, even for SA, a total reduction of the bacterial inoculum’s density took no longer than 2 h. Under conditions simulating organic contamination, all of the tested alloys were shown to have bactericidal or bacteriostatic properties, which was contrary to the results from stainless steel