Molecular and phenotypic identification of bacterial species isolated from cows with mastitis from three regions of Poland.

BACKGROUND: Bovine mastitis is a widespread disease affecting dairy cattle worldwide and it generates substantial losses for dairy farmers. Mastitis may be caused by bacteria, fungi or algae. The most common species isolated from infected milk are, among others, Streptococcus spp., Escherichia coli, Staphylococcus aureus and non-aureus staphylococci and mammaliicocci. The aim of this paper is to determine the frequency of occurrence of bacterial species in milk samples from cows with mastitis from three regions of Poland: the north-east, the south-west and the south. To this end 203 milk samples taken from cows with a clinical form (CM) of mastitis (n = 100) and healthy animals (n = 103) were examined, which included culture on an appropriate medium followed by molecular detection of E. coli, S. aureus, Streptococcus agalactiae and Streptococcus uberis, as one of the most common species isolated from mastitis milk. RESULTS: The results obtained indicated that S. uberis was the most commonly cultivated CM species (38%, n = 38), followed by S. aureus (22%, n = 22), E. coli (21%, n = 21) and S. agalactiae (18%, n = 18). Similar frequencies in molecular methods were obtained for S. uberis (35.1%) and S. aureus (28.0%). The variation of sensitivity of both methods may be responsible for the differences in the E. coli (41.0%, p = 0.002) and S. agalactiae (5.0%, p = 0.004) detection rates. Significant differences in composition of species between three regions of Poland were noted for E. coli incidence (p < 0.001), in both the culture and molecular methods, but data obtained by the PCR method indicated that this species was the least common in north-eastern Poland, while the culture method showed that in north-eastern Poland E. coli was the most common species. Significant differences for the molecular method were also observed for S. uberis (p < 0.001) and S. aureus (p < 0.001). Both species were most common in southern and south-western Poland. CONCLUSIONS: The results obtained confirm the need to introduce rapid molecular tests for veterinary diagnostics, as well as providing important epidemiological data, to the best of our knowledge data on Polish cows in selected areas of Poland is lacking.

Standardization of the protocol for oral cavity examination and collecting of the biological samples for microbiome research using the next-generation sequencing (NGS): own experience with the COVID-19 patients.

To date, publications have shown that compositions of oral microbiota differ depending on their habitats (e.g. tongue, tonsils, pharynx). The absence of set standards for the choice of the areas and conditions of material collection makes the oral microbiome one of the most difficult environments for a comparative analysis with other researchers, which is a meaningful limitation during an assessment of the potential effects of microorganisms as biomarkers in the courses of various human diseases. Therefore, standardisation of basic conditions of a dental examination and collection of material for the next generation sequencing (NGS) is worth attempting. The standardisation of the dental exam and collection of the clinical materials: saliva, swab from the tongue ridge, hard palate, palatine tonsils and oropharynx, supragingival plaque and subgingival plaque. Protocol involved the patients (n = 60), assigned to 3 groups: I-COVID-19 convalescents who received antibiotics, n = 17, II-COVID-19 convalescents, n = 23 and III-healthy individuals, n = 20. The collected biological samples were used to conduct NGS (16S rRNA). The conditions of patient preparation for collecting biological materials as well as the schedule of dental examination, were proposed. Based on the research conducted, we have indicated the dental indicators that best differentiate the group of COVID-19 patients (groups I and II) from healthy people (group III). These include the DMFT, D and BOP indices. The use of alpha and beta diversity analysis provided an overall insight into the diversity of microbial communities between specific niches and patient groups. The most different diversity between the studied group of patients (group II) and healthy people (group III) was noted in relation to the supragingival plaque. The order of activities during the dental exam as well as while collecting and securing clinical materials is particularly important to avoid technical errors and material contamination which may result in erroneous conclusions from the analyses of the results of sensitive tests such as the NGS. It has been shown that the dental indices: DMFT, D number, PI and BOP are the best prognostic parameters to assess the oral health. Based on beta diversity the most sensitive niche and susceptible to changes in the composition of the microbiota is the supragingival plaque. The procedures developed by our team can be applied as ready-to-use forms in studies conducted by other researchers.

Oral microbiota study of the patients after hospitalisation for COVID-19, considering selected dental indices and antibiotic therapy using the next generation sequencing method (NGS).

Background: Poor oral hygiene and the increased incidence and severity of periodontitis may exacerbate SARS-CoV-2 infection. The aim was to evaluate the oral microbiota of 60 participants divided into groups: COVID-19 convalescents who received antibiotics during hospitalization (I), COVID-19 convalescents without antibiotic therapy (II) and healthy individuals (III). Materials and Methods: Dental examination was conducted, and oral health status was evaluated using selected dental indexes. Clinical samples (saliva, dorsal swabs, supragingival and subgingival plaque) were collected and used for metagenomic library to the next-generation sequencing (NGS) preparation. Results: Each of the clinical materials in particular groups of patients showed a statistically significant and quantitatively different bacterial composition. Patients from group I showed significantly worse oral health, reflected by higher average values of dental indexes and also a higher percentage of Veillonella, Tannerella, Capnocytophaga and Selenomonas genera in comparison to other groups. Additionally, a statistically significant decrease in the amount of Akkermansia type in both groups with COVID-19 was observed for all materials. Conclusions: The primary factor affecting the composition of oral microbiota was not the SARS-CoV-2 infection itself, but the use of antibiotic therapy. The increased percentage of pro-inflammatory pathogens observed in COVID-19 patients underscores the importance of preventing periodontal disease and improving oral hygiene in the future.

Duodenal microbiota and weight-loss following sleeve gastrectomy and Roux-en-Y gastric bypass - a pilot study.

BACKGROUND: Bariatric surgery is the most effective method of morbid obesity treatment. Microbiota has many functions in human body and many of them remain to be unknown. The aim of this study was to establish if the composition of duodenal microbiota influences success rate of bariatric surgery. METHODS: It was a prospective cohort study. The data concerning demographics and comorbidities was collected perioperatively. The duodenal biopsies were collected prior to surgery with the gastroscope. Then DNA analysis was conducted. The data connected to the operation outcomes was gathered after 6 and 12 months after surgery. RESULTS: Overall, 32 patients were included and divided into two groups (successful - group 1 and unsuccessful - group 0) based on percentage excess weight loss after 6 months were created. The Total Actual Abundance was higher in group 0. In group 0 there was a significantly higher amount of Roseburia and Arthrobacter (p = 0.024, p = 0.027, respectively). Genus LDA effect size analysis showed Prevotella, Megasphaera and Pseudorhodobacter in group 1 to be significant. Whereas abundance of Roseburia and Arthrobacter were significant in group 0. CONCLUSIONS: Duodenal microbiota composition may be a prognostic factor for the success of the bariatric surgery but further research on the larger group is needed.

Do NAAT-Based Methods Increase the Diagnostic Sensitivity of Streptococcus agalactiae Carriage Detection in Pregnant Women?

The aim of the study was to evaluate particular polymerase chain reaction primers targeting selected representative genes and the influence of a preincubation step in a selective broth on the sensitivity of group B Streptococcus (GBS) detection by nucleic acid amplification techniques (NAAT). Research samples were vaginal and rectal swabs collected in duplicate from 97 pregnant women. They were used for enrichment broth culture-based diagnostics, bacterial DNA isolation, and amplification, using primers based on species-specific 16S rRNA, atr and cfb genes. To assess the sensitivity of GBS detection, additional isolation of samples preincubated in Todd-Hewitt broth with colistin and nalidixic acid was performed and then subjected to amplification again. The introduction of the preincubation step increased the sensitivity of GBS detection by about 33-63%. Moreover, NAAT made it possible to identify GBS DNA in an additional six samples that were negative in culture. The highest number of true positive results compared to the culture was obtained with the atr gene primers, as compared to cfb and 16S rRNA primers. Isolation of bacterial DNA after preincubation in enrichment broth significantly increases the sensitivity of NAAT-based methods applied for the detection of GBS from vaginal and rectal swabs. In the case of the cfb gene, the use of an additional gene to ensure the appropriate results should be considered.

New insights into diversity of the upper respiratory tract microbiota and its relationship with SARS-CoV-2 viral load in the nasopharyngeal epithelial cells in patients with COVID-19.

INTRODUCTION: The effects of SARS­CoV­2 infection on the composition of the upper respiratory tract (URT) microbiota are yet to be established, and more attention to this topic is needed. OBJECTIVES: The study aimed to assess the bacterial profile and the possible association between the URT microbiota composition and the SARS­CoV­2 viral load. PATIENTS AND METHODS: Nasopharyngeal swabs were taken from 60 adult patients with SARS­CoV­2 infection who were divided into 3 groups based on the quantification cycle (Cq) value in the quantitative polymerase chain reaction test: group I (n = 20), Cq lower than or equal to 31 (high replication rate); group II (n = 20), Cq greater than 31 and lower than 38 (low replication rate), and group III (n = 20), Cq higher than or equal to 38 (virus eliminated from the nasopharyngeal epithelial cells). The obtained genetic libraries of 16S rRNA were sequenced and taxonomic diversity profiling was performed to determine the α- and ß­biodiversity in each group. RESULTS: A significantly lower abundance of Prevotella species was noted in group I, as compared with groups II and III. Akkermansia muciniphila, Faecalibacterium prausnitzii, Fusicatenibacterium saccharivorans, and Bacteroides dorei abundance was characteristic of and significantly greater in group I than in groups II and III. Overall, the microbiota composition was the most diverse in group I, whereas groups II and III were more homogenous in terms of taxonomic diversity. CONCLUSIONS: The arbitrary division of patients according to the SARS­CoV­2 viral load was reflected in diverse composition of their bacterial microbiota, which implies an association between these 2 factors. The patients with a low viral replication rate and those who eliminated the virus from the epithelial cells belonged to a group with a less diverse microbiota community than the patients with a high viral replication rate.

Identifying Bacteria Species on Microscopic Polyculture Images Using Deep Learning.

Preliminary microbiological diagnosis usually relies on microscopic examination and, due to the routine culture and bacteriological examination, lasts up to 11 days. Hence, many deep learning methods based on microscopic images were recently introduced to replace the time-consuming bacteriological examination. They shorten the diagnosis by 1-2 days but still require iterative culture to obtain monoculture samples. In this work, we present a feasibility study for further shortening the diagnosis time by analyzing polyculture images. It is possible with multi-MIL, a novel multi-label classification method based on multiple instance learning. To evaluate our approach, we introduce a dataset containing microscopic images for all combinations of four considered bacteria species. We obtain ROC AUC above 0.9, proving the feasibility of the method and opening the path for future experiments with a larger number of species.

Repetitive transcranial direct current stimulation modulates the brain-gut-microbiome axis in obese rodents.

BACKGROUND: Complex interactions between the brain, gut and adipose tissue allow to recognize obesity as a neurometabolic disorder. The recent data have shown that gut microbiota can play a potential role in obesity development. Transcranial direct current stimulation (tDCS) is a safe and non-invasive technique to modulate the activity of cerebral cortex and other connected brain areas also in context of appetite control. The objective of this study was to evaluate the effects of repetitive anodal tDCS (AtDCS) of prefrontal cortex on feeding behavior, metabolic status and selected phyla of gut microbiota in rats with obesity induced by high-calorie diet (HCD). METHODS: 32 female Wistar rats were equally divided into 4 subgroups depending on diet effect (lean versus obese) and type of stimulation (active versus sham tDCS versus no stimulation). Feed intake, body weight, blood lipoproteins and leptin levels as well as Firmicutes and Bacteroidetes in intestines and stool were examined. RESULTS: HCD changed feeding behavior and metabolic parameters typically for obesity-related ranges and resulted in an abundance of Firmicutes at the expanse of Bacteroidetes in the large intestine and stool. AtDCS decreased appetite, body weight, and cholesterol levels. In addition, AtDCS reduced ratio of the average number of Firmicutes to average number of Bacteroidetes in all examined tissues. CONCLUSIONS: Repetitive AtDCS is not only effective for appetite restriction but can also modulate gut microbiome composition which demonstrates the existence of the brain-gut-microbiome axis and points at this technique as a promising complementary treatment for obesity. However, the effects should be further replicated in human studies.

Analysis of the Gut Mycobiome in Adult Patients with Type 1 and Type 2 Diabetes Using Next-Generation Sequencing (NGS) with Increased Sensitivity-Pilot Study.

The studies on microbiome in the human digestive tract indicate that fungi could also be one of the external factors affecting development of diabetes. The aim of this study was to evaluate the quantitative and qualitative mycobiome composition in the colon of the adults with type 1 (T1D), n = 26 and type 2 (T2D) diabetes, n = 24 compared to the control group, n = 26. The gut mycobiome was characterized in the stool samples using the analysis of the whole internal transcribed spacer (ITS) region of the fungal rDNA gene cluster by next-generation sequencing (NGS) with increased sensitivity. At the L2 (phylum) level, Basidiomycota fungi were predominant in all 3 study groups. Group T1D presented significantly lower number of Ascomycota compared to the T2D group, and at the L6 (genus) level, the T1D group presented significantly lower number of Saccharomyces genus compared to control and T2D groups. In the T1D group, a significant positive correlation between total cholesterol and low-density lipoprotein cholesterol (LDL-C) levels and fungi of the genus Saccharomyces, and in the T2D group, a negative correlation between the total cholesterol level and Malassezia genus was found. The obtained results seem to be a good foundation to extend the analysis of the relationship between individual genera and species of fungi and the parameters determining the metabolism of carbohydrates and lipids in the human body.

The Gut Microbiota Profile According to Glycemic Control in Type 1 Diabetes Patients Treated with Personal Insulin Pumps.

Recently, several studies explored associations between type 1 diabetes (T1DM) and microbiota. The aim of our study was to assess the colonic microbiota structure according to the metabolic control in T1DM patients treated with insulin pumps. We studied 89 T1DM patients (50.6% women) at the median age of 25 (IQR, 22-29) years. Pielou's evenness (p = 0.02), and Shannon's (p = 0.04) and Simpson's diversity indexes (p = 0.01), were higher in patients with glycosylated hemoglobin (HbA1c) ≥ 53 mmol/mol (7%). There were no differences in beta diversity between groups. A linear discriminant analysis effect size (LEfSe) algorithm showed that one family (Ruminococcaceae) was enriched in patients with HbA1c < 53 mmol/mol, whereas one family (Streptococcaceae) and four species (Ruminococcus torques, unclassified species of Lactococcus, Eubacteroim dolichum, and Coprobacillus cateniformis) were enriched in patients with HbA1c ≥ 53 mmol/mol. We found that at class level, the following pathways according to Kyoto Encyclopedia of Genes and Genomes were enriched in patients with HbA1c < 53 mmol/mol: bacterial motility proteins, secretion system, bacterial secretion system, ribosome biogenesis, translation proteins, and lipid biosynthesis, whereas in patients with HbA1c ≥ 53 mmol/mol, the galactose metabolism, oxidative phosphorylation, phosphotransferase system, fructose, and mannose metabolism were enriched. Observed differences in alpha diversity, metabolic pathways, and associations between bacteria and HbA1c in colonic flora need further investigation.

Comparison of Antigen Tests and qPCR in Rapid Diagnostics of Infections Caused by SARS-CoV-2 Virus.

Diagnostics of the coronavirus disease 2019 (COVID-19) using molecular techniques from the collected respiratory swab specimens requires well-equipped laboratory and qualified personnel, also it needs several hours of waiting for results and is expensive. Antigen tests appear to be faster and cheaper but their sensitivity and specificity are debatable. The aim of this study was to compare a selected antigen test with quantitative polymerase chain reaction (qPCR) tests results. Nasopharyngeal swabs were collected from 192 patients with COVID-19 symptoms. All samples were tested using Vitassay qPCR SARS-CoV-2 kit and the Humasis COVID-19 Ag Test (MedSun) antigen immunochromatographic test simultaneously. Ultimately, 189 samples were tested; 3 samples were excluded due to errors in taking swabs. The qPCR and antigen test results were as follows: 47 positive and 142 negative, and 45 positive and 144 negative, respectively. Calculated sensitivity of 91.5% and specificity of 98.6% for the antigen test shows differences which are not statistically significant in comparison to qPCR. Our study showed that effectiveness of the antigen tests in rapid laboratory diagnostics is high enough to be an alternative and support for nucleic acid amplification tests (NAAT) in the virus replication phase in the course of COVID-19.

Next-Generation Sequencing as a Tool to Detect Vaginal Microbiota Disturbances during Pregnancy.

The physiological microbiota of the vagina is responsible for providing a protective barrier, but Some factors can disturb the balance in its composition. At that time, the amounts of the genus Lactobacillus decrease, which may lead to the development of infection and severe complications during pregnancy. The aim of the study was the analysis of the bacterial composition of the vagina in 32 Caucasian women at each trimester of pregnancy using the next-generation sequencing method and primers targeting V3-V4 regions. In the studied group, the dominant species were Lactobacillus iners, Lactobacillus gasseri, and Lactobacillusplantarum. Statistically significant differences in the quantitative composition between trimesters were observed in relation to Lactobacillus jensenii,Streptococcus agalactiae, Lactobacillus iners, Gardnerella spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus Lactobacillus, and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: Streptococcus agalactiae, Gardnerella spp., Atopobium vaginae, and Enterococcus faecalis. The composition of the vaginal microbiota during pregnancy was subject to partial changes over trimesters. Although in one-third of the studied patients, both the qualitative and quantitative composition of microbiota was relatively constant, in the remaining patients, physiological and potentially pathogenic fluctuations were distinguished.

Deep learning approach to describe and classify fungi microscopic images.

Preliminary diagnosis of fungal infections can rely on microscopic examination. However, in many cases, it does not allow unambiguous identification of the species due to their visual similarity. Therefore, it is usually necessary to use additional biochemical tests. That involves additional costs and extends the identification process up to 10 days. Such a delay in the implementation of targeted therapy may be grave in consequence as the mortality rate for immunosuppressed patients is high. In this paper, we apply a machine learning approach based on deep neural networks and bag-of-words to classify microscopic images of various fungi species. Our approach makes the last stage of biochemical identification redundant, shortening the identification process by 2-3 days, and reducing the cost of the diagnosis.

Quantitative changes in selected bacteria in the stool during the treatment of Crohn's disease.

PURPOSE: The aim of this study was to determine quantitative changes in selected species of bacteria (Bacteroides fragilis, Lactobacillus fermentum, Lactobacillus rhamnosus, Serratia marcescens) in the stool of patients with Crohn's disease (CD) in the course of induction treatment with exclusive enteral nutrition (EEN) or anti-tumor necrosis factor alpha (Infliximab, IFX) vs. healthy controls (HC). MATERIALS/METHODS: DNA was isolated from stool samples of CD (n = 122) and HC (n = 17), and quantitative real-time Polymerase Chain Reaction (qPCR) was applied. In both treatment groups, the first stool sample was taken before the start of treatment, and the second 4 weeks after its end: in EEN (n = 48; age (mean; SD) 13.35 ± 3.09 years) and IFX groups (n = 13; age (mean; SD) 13.09 ± 3.76 years). RESULTS: The only species that showed a statistically significant difference between the two groups of patients before any therapeutic intervention was L. fermentum. Moreover, its number increased after completion of EEN and differed significantly when compared with the HC. In the IFX group the number of L. fermentum decreased during the therapy but was significantly higher than in the HC. The number of S. marcescens in the EEN group was significantly lower than in the controls both before and after EEN. CONCLUSION: The implemented treatment (EEN or IFX) modifies the microbiome in CD patients, but does not make it become the same as in HC.

Changes in the Intestinal Microbiota Are Seen Following Treatment with Infliximab in Children with Crohn's Disease.

The aim of the study was to determine the impact of biological treatment with tumor necrosis factor α antibodies (anti-TNF-α) on the intestinal microbiome of children with severe Crohn's disease (CD) and to evaluate the differences in the intestinal microbiome between patients treated with biological therapy and healthy children. Microbiota composition was analyzed by 16S next-generation sequencing (NGS) and microbial profiles were compared between studied groups. Fifty-four samples (from 18 patients before and after anti-TNF-α induction therapy and 18 healthy children) were used in the sequencing analysis. Shannon's diversity index (p = 0.003, adj. p = 0.010) and observed operational taxonomic units (OTUs) (p = 0.007, adj. p = 0.015) were different between controls and patients with prior therapy for CD. Statistically significant dissimilarities between beta diversity metrics, indicating distinct community composition across groups, were observed in patients with CD before and after therapy. We did not observe any differences between controls and patients with CD after therapy. Core microbiome analysis at species level showed that 32 species were present only in patients with CD but not in controls. The results show that biological treatment is associated with changes in the intestinal microbiome of patients with CD: these changes result in an intestinal microbiome pattern similar to that seen in healthy children. Long-term observation is necessary to determine whether treatment can lead to full restoration of a healthy-like microbiome.

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.

Differences in the intestinal microbiome of healthy children and patients with newly diagnosed Crohn's disease.

The aetiology of inflammatory bowel diseases (IBD) seems to be strongly connected to changes in the enteral microbiome. The dysbiosis pattern seen in Crohn's disease (CD) differs among published studies depending on patients' age, disease phenotype and microbiome research methods. The aims was to investigate microbiome in treatment-naive paediatric patients to get an insight into its structure at the early stage of the disease in comparison to healthy. Stool samples were obtained from controls and newly diagnosed patients prior to any intervention. Microbiota was analysed by 16SrRNAnext-generation-sequencing (NGS). Differences in the within-sample phylotype richness and evenness (alpha diversity) were detected between controls and patients. Statistically significant dissimilarities between samples were present for all used metrics. We also found a significant increase in the abundance of OTUs of the Enterococcus genus and reduction in, among others, Bifidobacterium (B. adolescentis), Roseburia (R.faecis), Faecalibacterium (F. prausnitzii), Gemmiger (G. formicilis), Ruminococcus (R. bromii) and Veillonellaceae (Dialister). Moreover, differences in alpha and beta diversities in respect to calprotectin and PCDAI were observed: patients with calprotectin <100 µg/g and with PCDAI below 10 points vs those with calprotectin >100 µg/g and mild (10-27.7 points), moderate (27.5-40 points) or severe (>40 points) CD disease activity had higher richness and diversity of gut microbiota. The results of our study highlight reduced diversity and dysbiosis at the earliest stage of the disease. Microbial imbalance and low abundance of butyrate-producing bacteria, including Bifidobacterium adolescentis, may suggest benefits of microbial modification therapy.

Dependence of Colonization of the Large Intestine by Candida on the Treatment of Crohn's Disease.

The aim of this study was to determine if there are quantitative differences in Candida fungi between pediatric patients with Crohn's disease (before and after exclusive enteral nutrition (EEN), and the biologic therapy with anti-tumor necrosis factor alpha - (IFX)), and healthy controls. DNA was isolated from fecal samples and PCR was used to determine the number of fungal cells. Both therapeutic interventions resulted in a statistically significant decrease in Pediatric Crohn's Disease Activity Index. The numbers of Candida decreased during both therapeutic intervention but the difference was statistically significant for the IFX intervention only (p = 0.045). Moreover, fungi population in both study groups declined during intervention when compared to the control group but the difference was significant before treatment only in the IFX group (p = 0.013). The total distribution of Candida with both IFX and EEN as well as in the control group differed significantly (p = 0.01) before treatment only. No correlation between the numbers of Candida and disease activity as well as the following biochemical parameters: serum iron concentration, protein or glucose level were found. It cannot be ruled out that, in combination with genetic and immunological disorders, fungi can contribute to the initiation of the disease process and perpetuation of active inflammation.The aim of this study was to determine if there are quantitative differences in Candida fungi between pediatric patients with Crohn's disease (before and after exclusive enteral nutrition (EEN), and the biologic therapy with anti-tumor necrosis factor alpha ­ (IFX)), and healthy controls. DNA was isolated from fecal samples and PCR was used to determine the number of fungal cells. Both therapeutic interventions resulted in a statistically significant decrease in Pediatric Crohn's Disease Activity Index. The numbers of Candida decreased during both therapeutic intervention but the difference was statistically significant for the IFX intervention only (p = 0.045). Moreover, fungi population in both study groups declined during intervention when compared to the control group but the difference was significant before treatment only in the IFX group (p = 0.013). The total distribution of Candida with both IFX and EEN as well as in the control group differed significantly (p = 0.01) before treatment only. No correlation between the numbers of Candida and disease activity as well as the following biochemical parameters: serum iron concentration, protein or glucose level were found. It cannot be ruled out that, in combination with genetic and immunological disorders, fungi can contribute to the initiation of the disease process and perpetuation of active inflammation.

Comparison of PCR, Fluorescent in Situ Hybridization and Blood Cultures for Detection of Bacteremia in Children and Adolescents During Antibiotic Therapy.

The gold standard in microbiological diagnostics of bacteremia is a blood culture in automated systems. This method may take several days and has low sensitivity. New screening methods that could quickly reveal the presence of bacteria would be extremely useful. The objective of this study was to estimate the effectiveness of these methods with respect to blood cultures in the context of antibiotic therapy. Blood samples from 92 children with sepsis were analyzed. Blood cultures were carried out in standard automated systems. Subsequently, FISH (Fluorescent In-Situ Hybridization) and nested multiplex-real-time-PCR (PCR) were performed. Blood cultures, FISH and PCR yielded positive results in 18%, 39.1%, and 71.7% of samples, respectively. Significant differences were found between the results obtained through culture before and after induction of antibiotherapy: 25.5% vs. 9.7%. There was no significant difference in FISH and PCR results in relation to antibiotics. The three methods employed demonstrated significant differences in detecting bacteria effectively. Time to obtain test results for FISH and PCR averaged 4-5 hours. FISH and PCR allow to detect bacteria in blood without prior culture. These methods had high sensitivity for the detection of bacteremia regardless of antibiotherapy. They provide more timely results as compared to automated blood culture, and may be useful as rapid screening tests in sepsis.The gold standard in microbiological diagnostics of bacteremia is a blood culture in automated systems. This method may take several days and has low sensitivity. New screening methods that could quickly reveal the presence of bacteria would be extremely useful. The objective of this study was to estimate the effectiveness of these methods with respect to blood cultures in the context of antibiotic therapy. Blood samples from 92 children with sepsis were analyzed. Blood cultures were carried out in standard automated systems. Subsequently, FISH (Fluorescent In-Situ Hybridization) and nested multiplex-real-time-PCR (PCR) were performed. Blood cultures, FISH and PCR yielded positive results in 18%, 39.1%, and 71.7% of samples, respectively. Significant differences were found between the results obtained through culture before and after induction of antibiotherapy: 25.5% vs. 9.7%. There was no significant difference in FISH and PCR results in relation to antibiotics. The three methods employed demonstrated significant differences in detecting bacteria effectively. Time to obtain test results for FISH and PCR averaged 4­5 hours. FISH and PCR allow to detect bacteria in blood without prior culture. These methods had high sensitivity for the detection of bacteremia regardless of antibiotherapy. They provide more timely results as compared to automated blood culture, and may be useful as rapid screening tests in sepsis.

Characteristics of gut microbiota in adult patients with type 1 and type 2 diabetes based on next­generation sequencing of the 16S rRNA gene fragment.

Introduction Scientific data indicate a possible influence of gut microbiota on the development of type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively). Sequence analysis of 16S ribosomal RNA identified several hundred bacterial species of the intestinal ecosystem, most of which cannot be cultured. Objectives We aimed to evaluate gut microbiota composition in adult patients with T1DM and T2DM and establish a link between microbiological test results and patients' clinical data. Patients and methods We examined DNA isolated from fecal samples in 3 groups: healthy volunteers (n = 23), patients with T1DM (n = 22), and patients with T2DM (n = 23). Next­generation sequencing was performed on the MiSeq platform. Results At the phylum level, the Firmicutes bacteria prevailed (>77%) in all groups. At the taxonomic levels L2 (phylum) and L6 (genus), significant differences were demonstrated in bacterial profiles, particularly in the T2DM group. A negative correlation was observed between several genera of bacteria and the percentage of glycated hemoglobin A1c in the T2DM group, while a positive correlation was revealed between bacteria belonging to the genus Bifidobacterium and high­density lipoprotein cholesterol levels in both T1DM and T2DM groups. Conclusions Our results provide grounds for conducting research in the field of gut microbiota in order to develop individualized therapy for patients with diabetes based on modifying the microbiota composition, as a new method for controlling glycemia. Next­generation sequencing allows a rapid identification of the DNA of all bacteria present in the sample and their taxonomic classification.