Publications by authors named "Tomasz Gosiewski"

53 Publications

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.

Nutrients 2021 Mar 25;13(4). Epub 2021 Mar 25.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 18 Czysta Street, 31-121 Krakow, Poland.

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), = 26 and type 2 (T2D) diabetes, = 24 compared to the control group, = 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 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 and in the T2D group, a negative correlation between the total cholesterol level and 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.
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http://dx.doi.org/10.3390/nu13041066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064496PMC
March 2021

Challenging the gold standard: methods of sampling for microbial culture in patients with chronic rhinosinusitis.

Eur Arch Otorhinolaryngol 2021 Mar 27. Epub 2021 Mar 27.

Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, 30-239, Krakow, Poland.

Purpose: Chronic rhinosinusitis (CRS) is a highly prevalent multifactorial disorder. Culture-directed antibiotics are frequently prescribed to patients with CRS and the middle nasal meatus (MM) is traditionally believed to be a representative sampling site of the sinuses as a whole. The purpose of our study was to reevaluate the reliability of the MM as a sampling site in patients with CRS who suffer from impaired drainage from the sinuses to the MM.

Methods: Swabs and tissue biopsies were collected from the MM, maxillary sinus and frontal sinus from 50 patients with CRS. The results of bacterial culture were compared between sampling methods and sites in relation to the patency of the sinus ostia.

Results: 782 bacterial isolates were cultured from the samples. Concordant results between the MM and the sinus cavity were noted in 80% of patients for the maxillary sinus, but only 66% for the frontal sinus and 76% for the sinuses a whole. The differences were similarly prevalent in patients with open and occluded sinus ostia. Notably, swabs from all three sites provided representative information in 92% of patients and tissue biopsies did not provide additional information compared to multiple swabs.

Conclusion: The traditional method of sampling from the middle meatus provides inadequate information in 24% of patients with CRS, which may result in inadequate antibiotic therapy and contribute to increasing antibiotic resistance. Additional sampling from the sinuses should be recommended whenever possible, while invasive sampling is not necessary.
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http://dx.doi.org/10.1007/s00405-021-06747-zDOI Listing
March 2021

Relationship between bariatric surgery outcomes and the preoperative gastrointestinal microbiota: a cohort study.

Surg Obes Relat Dis 2021 May 22;17(5):889-899. Epub 2021 Jan 22.

2nd Department of General Surgery, Jagiellonian University Medical College, Krakow, Poland; Centre for Research, Training and Innovation in Surgery, Krakow, Poland. Electronic address:

Background: The composition of the gastrointestinal microbiota is associated with obesity. We hypothesized that the gut microbiota influences the outcomes of bariatric surgery.

Objectives: We aimed to analyze using oral swabs and stool samples the microbiota of patients with morbid obesity who were undergoing laparoscopic sleeve gastrectomy (SG).

Setting: A university hospital in Poland.

Methods: This prospective cohort study was conducted between November 2018 and June 2019. Participants underwent SG or no surgery (controls). Results were then analyzed as a group 1 (surgical participants who achieved a percentage of excess weight loss [%EWL] >50%), group 2 (surgical participants who achieved a %EWL <50%), and group 3 (nonsurgical controls). %EWL was measured 6 months following surgery. Before surgery, oral swabs were obtained and stool samples were provided. The endpoint was the composition of the gut microbiota.

Results: Group 1 comprised 19 participants, group 2 comprised 11 participants, and group 3 comprised 16 participants. No participants were lost to follow-up during the study. Participants in group 1 had an oral microbiota that was enriched in the phyla Proteobacteria, and Bacteroidetes. Their intestinal microbiota was enriched in the Proteobacteria. In contrast, the oral microbiota of group 2 was enriched in the Actinobacteria and the intestinal microbiota was enriched in the phyla Bacteroidetes and Firmicutes.

Conclusions: The compositions of the microbiota of the oral cavity and large intestine are related to the weight loss achieved following SG.
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http://dx.doi.org/10.1016/j.soard.2021.01.011DOI Listing
May 2021

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

Microorganisms 2021 Jan 12;9(1). Epub 2021 Jan 12.

Department of Metabolic Diseases, Jagiellonian University Medical College, 2 Jakubowskiego Street, 30-688 Krakow, Poland.

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 ( = 0.02), and Shannon's ( = 0.04) and Simpson's diversity indexes ( = 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 () was enriched in patients with HbA1c < 53 mmol/mol, whereas one family () and four species (, unclassified species of , , and ) 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.
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http://dx.doi.org/10.3390/microorganisms9010155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826603PMC
January 2021

Does Postoperative Oral and Intestinal Microbiota Correlate with the Weight-Loss Following Bariatric Surgery?-A Cohort Study.

J Clin Med 2020 Nov 27;9(12). Epub 2020 Nov 27.

2nd Department of General Surgery, Faculty of Medicine, Jagiellonian University Medical College, 30-688 Cracow, Poland.

The composition of the gastrointestinal microbiota is associated with obesity. The aim of this study was to verify if, six months after bariatric surgery, patients who achieve satisfying weight-loss after sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) have a different composition of oral and intestinal microbiota in comparison with those who do not. This prospective cohort study was conducted between November 2018 and November 2020. Participants underwent either SG or RYGB and were allocated into: Group 1-participants who achieved a percentage of excess weight loss (%EWL) of ≥ 50%, and Group 2-patients with %EWL of < 50%. The %EWL was measured 6 months following surgery. At this time, oral swabs were obtained and stool samples were provided. The endpoint was the composition of the gut microbiota. Group 1 comprised 20 participants and Group 2 comprised 11 participants. Group 1 had oral microbiota more abundant in phylum Fusobacteria and intestinal microbiota more abundant in phylum Firmicutes. Group 2 had oral microbiota was more enriched in phylum Actinobacteria and intestinal microbiota was more enriched in phylum Bacteroidetes. The compositions of the microbiota of the oral cavity and large intestine 6 months after bariatric surgery are related to the weight-loss.
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http://dx.doi.org/10.3390/jcm9123863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761068PMC
November 2020

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

Microorganisms 2020 Nov 18;8(11). Epub 2020 Nov 18.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland.

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 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 and . Statistically significant differences in the quantitative composition between trimesters were observed in relation to , , spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: spp., , and 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.
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http://dx.doi.org/10.3390/microorganisms8111813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698737PMC
November 2020

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

Adv Med Sci 2020 Sep 23;65(2):348-353. Epub 2020 Jun 23.

Jagiellonian University Medical College, Faculty of Medicine, Department of Pediatrics, Gastroenterology and Nutrition, Krakow, Poland. Electronic address:

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.
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http://dx.doi.org/10.1016/j.advms.2020.06.003DOI Listing
September 2020

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

J Clin Med 2020 Mar 4;9(3). Epub 2020 Mar 4.

Jagiellonian University Medical College, Faculty of Medicine, Department of Pediatrics, Gastroenterology and Nutrition, 30-633 Kraków, Poland.

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 ( = 0.003, adj. = 0.010) and observed operational taxonomic units (OTUs) ( = 0.007, adj. = 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.
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http://dx.doi.org/10.3390/jcm9030687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141282PMC
March 2020

Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the "Gold Standard"?

Microorganisms 2020 Feb 29;8(3). Epub 2020 Feb 29.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland.

Standard blood cultures require at least 24-120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets taken from post-operative septic patients: 82 negative aerobic blood cultures, 82 anaerobic blood cultures, and 82 blood samples, as well as 57 blood samples taken from healthy volunteers. From the eighty-two blood sets analyzed from the septic patients, Gram stain visualized bacteria in 62.2% of blood samples, 35.4% of the negative aerobic bottles, and in 31.7% of the negative anaerobic bottles. Utilizing FISH, we detected bacteria in 75.6%, 56.1%, and 64.6% respectively. Among the blood samples from healthy volunteers, FISH detected bacteria in 64.9%, while Gram stain detected bacteria in only 38.6%. The time needed to obtain the study results using Gram stain was 1 h, for FISH 4 h, and for the culture method, considering the duration of growth, 5 days. Gram stain and FISH allow quick detection of bacteria in the blood taken directly from a patient. Finding phagocytosed bacteria, which were also detected among healthy individuals, confirms the hypothesis that blood microbiome exists.
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http://dx.doi.org/10.3390/microorganisms8030346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143506PMC
February 2020

Metagenomic Analysis of Duodenal Microbiota Reveals a Potential Biomarker of Dysbiosis in the Course of Obesity and Type 2 Diabetes: A Pilot Study.

J Clin Med 2020 Jan 29;9(2). Epub 2020 Jan 29.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland.

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 compared to healthy subjects. Furthermore, the numbers of were positively correlated with the high density lipoprotein (HDL) concentration in the groups under study. The obtained results indicate that bacteria of the genus should be considered in the future in the context of a potential biomarker in the progress of type 2 diabetes and obesity.
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http://dx.doi.org/10.3390/jcm9020369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074165PMC
January 2020

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

Sci Rep 2019 12 11;9(1):18880. Epub 2019 Dec 11.

Department of Pediatrics, Gastroenterology and Nutrition, Faculty of Medicine, Jagiellonian University Medical College, Wielicka 265, Kraków, 30-663, Poland.

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.
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http://dx.doi.org/10.1038/s41598-019-55290-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906406PMC
December 2019

Exacerbations of Chronic Rhinosinusitis-Microbiology and Perspectives of Phage Therapy.

Antibiotics (Basel) 2019 Oct 5;8(4). Epub 2019 Oct 5.

Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland.

The chronically inflamed mucosa in patients with chronic rhinosinusitis (CRS) can additionally be infected by bacteria, which results in an acute exacerbation of the disease (AECRS). Currently, AECRS is universally treated with antibiotics following the guidelines for acute bacterial rhinosinusitis (ABRS), as our understanding of its microbiology is insufficient to establish specific treatment recommendations. Unfortunately, antibiotics frequently fail to control the symptoms of AECRS due to biofilm formation, disruption of the natural microbiota, and arising antibiotic resistance. These issues can potentially be addressed by phage therapy. In this study, the endoscopically-guided cultures were postoperatively obtained from 50 patients in order to explore the microbiology of AECRS, evaluate options for antibiotic treatment, and, most importantly, assess a possibility of efficient phage therapy. and coagulase-negative staphylococci were the most frequently isolated bacteria, followed by and . Alarmingly, mechanisms of antibiotic resistance were detected in the isolates from 46% of the patients. Bacteria not sensitive to amoxicillin were carried by 28% of the patients. The lowest rates of resistance were noted for fluoroquinolones and aminoglycosides. Fortunately, 60% of the patients carried bacterial strains that were sensitive to bacteriophages from the Biophage Pharma collection and 81% of the antibiotic-resistant strains turned out to be sensitive to bacteriophages. The results showed that microbiology of AECRS is distinct from ABRS and amoxicillin should not be the antibiotic of first choice. Currently available bacteriophages could be used instead of antibiotics or as an adjunct to antibiotics in the majority of patients with AECRS.
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http://dx.doi.org/10.3390/antibiotics8040175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963383PMC
October 2019

"Command" surfaces with thermo-switchable antibacterial activity.

Mater Sci Eng C Mater Biol Appl 2019 Oct 27;103:109806. Epub 2019 May 27.

Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland.

In the presented work "smart" antibacterial surfaces based on silver nanoparticles (AgNPs) embedded in temperature-responsive poly(di(ethylene glycol)methyl ether methacrylate) - (POEGMA188) as well as poly(4-vinylpyridine) - (P4VP) coatings attached to a glass surface were successfully prepared. The composition, thickness, morphology and wettability of the resulting coatings were analyzed using ToF-SIMS, XPS, EDX, ellipsometry, AFM, SEM and CA measurements, respectively. Temperature-switched killing of the bacteria was tested against Escherichia coli ATCC 25922 (representative of Gram-negative bacteria) and Staphylococcus aureus ATCC 25923 (representative of Gram-positive bacteria) at 4 and 37 °C. In general at 4 °C no significant difference was observed between the amounts of bacteria accounted on the grafted brush coatings and within the control sample. In contrast, at 37 °C almost no bacteria were visible for temperature-responsive coating with AgNPs, whereas the growth of bacteria remains not disturbed for "pure" coating, indicating strong temperature-dependent antibacterial properties of AgNPs integrated into brushes.
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http://dx.doi.org/10.1016/j.msec.2019.109806DOI Listing
October 2019

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

Pol J Microbiol 2019 ;68(1):121-126

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College , Krakow , Poland.

The aim of this study was to determine if there are quantitative differences in 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 decreased during both therapeutic intervention but the difference was statistically significant for the IFX intervention only ( = 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 ( = 0.013). The total distribution of with both IFX and EEN as well as in the control group differed significantly ( = 0.01) before treatment only. No correlation between the numbers of 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 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 decreased during both therapeutic intervention but the difference was statistically significant for the IFX intervention only ( = 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 ( = 0.013). The total distribution of with both IFX and EEN as well as in the control group differed significantly ( = 0.01) before treatment only. No correlation between the numbers of 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.
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http://dx.doi.org/10.21307/pjm-2019-014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256697PMC
May 2019

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

Pol J Microbiol 2018;67(4):479-486

Chair of Microbiology, Department of Molecular Medical Microbiology, Faculty of Medicine, Jagiellonian University Medical College , Cracow , Poland.

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 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.
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http://dx.doi.org/10.21307/pjm-2018-056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256870PMC
June 2019

The dynamics of vaginal and rectal Lactobacillus spp. flora in subsequent trimesters of pregnancy in healthy Polish women, assessed using the Sanger sequencing method.

BMC Pregnancy Childbirth 2018 Aug 29;18(1):350. Epub 2018 Aug 29.

Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, Czysta 18, 31-121, Krakow, Poland.

Background: Lactobacilli play an important role in maintaining vaginal health and protection against bacterial infections in the genital tract. The aim of this study is to show the dynamics of changes of the vaginal and rectal Lactobacillus flora during pregnancy by using the Sanger sequencing method.

Method: The study included 31 healthy pregnant women without clinical signs of genitourinary infections. The material was taken in the three trimesters of pregnancy by vaginal and rectal swabs and grown on the MRS agar quantitatively to estimate the number of Lactobacillus spp. [CFU/ml]. Afterwards, 3 to 8 morphologically different lactobacilli colonies were taken for identification. Bacterial species identification was performed by 16 s rDNA sequence fragment analyses using the Sanger method.

Results: Among the patients tested, the most common species colonizing the vagina in the first trimester were: L. crispatus 29%, L. gasseri 19.4% and L. rhamnosus 16.1%, in the second trimester: L. crispatus 51.6%, L. gasseri 25.8%, L. rhamnosus 19.4% and L. amylovorus 16.1%, and in the third trimester the most common Lactobacillus species were: L. crispatus 25.8%, L. gasseri 25.8% and L. johnsonii 19.4%. In rectal species, the number decreased in the second and third trimesters in comparison to the first trimester (p = 0.003). An analysis of rectal dynamics showed that in the first trimester, the most common species were: L. johnsonii 19.4%, and L. plantarum 9.7%, in the second trimester: L. crispatus 9.7% and L. mucosae 6.5%, and in the third trimester: L. casei 9.7% and L. rhamnosus 9.7%. Individual dynamics of the Lactobacillus species composition showed variability, characterized by continuous, intermittent, or periodic colonization. The patients examined were mostly colonized by three Lactobacillus species in vagina (32.3%), whereas for the rectum, one Lactobacillus species during the whole pregnancy duration was common (32.3%).

Conclusion: This study showed that in the examined group of healthy, pregnant Polish women, the vaginal Lactobacillus flora, both qualitative and quantitative, was stable during the three subsequent trimesters. In contrast, the number of rectal Lactobacillus species dramatically decreased after the first trimester.
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http://dx.doi.org/10.1186/s12884-018-1987-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114255PMC
August 2018

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

Pol Arch Intern Med 2018 06 15;128(6):336-343. Epub 2018 Apr 15.

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). Next‑generation 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 high‑density 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. Next‑generation sequencing allows a rapid identification of the DNA of all bacteria present in the sample and their taxonomic classification.
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http://dx.doi.org/10.20452/pamw.4246DOI Listing
June 2018

Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood.

Med Dosw Mikrobiol 2016;68(1):47-56

Introduction: Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to consider- able defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient's blood.

Methods: The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time am- plification and its equivalent, in nested version. The remaining part earmarked for serologi- cal testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version.

Results: The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the single- variety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples.

Conclusions: These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells. By contrast, the results of serological and molecular tests should always be carried out tak- ing into account the patient's clinical status.
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March 2017

Using of the 16S rDNA sequencing for identification of Lactobacillus species.

Med Dosw Mikrobiol 2016;68(1):5-11

Introduction: Lactobacilli play an important role in maintaining vaginal health and pro- tecting the genital tract from bacterial infections, so very often they are used as probiotics. Despite the scientific consensus on the significance of the genus Lactobacillus, its species identification still poses several difficulties. The aim of this study was to find out if the 16S rDNA sequencing method allows exact genotyping of Lactobacillus species.

Methods: 78 isolates from healthy pregnant women were tested: 57 from the vagina and 21 from the rectum. We also examined seven reference strains: L. acidophilus ATCC 4356, L.fermentum ATCC 20052, L. plantarum ATCC 20174, L. plantarum ATCC 14431, L. delbrueckii ssp. bulgaricus ATCC 20074, L. crispatus ATCC 20225 and L. gasseri ATCC 20243 to confirm the effectiveness sequencing method. A fragment of the 16S rDNA was amplified. After amplification, the amplicons were separated by gel electrophoresis and then sequenced. Furthermore, the received consensus sequences were checked for species specificity in the National Center for Biotechnology Information (NCBI) database with BLAST software. Sequences with a ;> 98% match to a database sequence were considered to be the same species.

Results: We have confirmed the genus of seven tested reference strains of lactobacilli with 100% probability. Of the analyzed isolates, all were identified to the species level. 14 spe- cies were identified in the 78 respondents, 9 of which colonized the vagina and 11 appeared in the rectum. The species colonizing the vagina were: L. gasseri 31.6%, followed by L. crispatus 28.2%, L. rhamnosus 14%, L. amylovorus 14%, L. helveticus 3.5%, L. reuteri 3.5%, L. casei 1.7%, L. salivarius 1.7% and L. delbrueckii 1.7%. The species colonizing the anus were: L. caseil L. paracasei 28.6%, L. plantarum 14.3%, L. crispatus 14.3%, L. gasseri 9.5%, L. reuteri 9.5%, L. salivarius 4.8%, L. rhamnosus 4.8%, L. acidophilus 4.8%, L. ruminis 4.8% and L. sakei 4.8%.

Conclusions: Using the 16S rDNA sequencing method made it possible to genotype 100% of the tested isolates of Lactobacillus.
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March 2017

Qualitative Parameters of the Colonic Flora in Patients with HNF1A-MODY Are Different from Those Observed in Type 2 Diabetes Mellitus.

J Diabetes Res 2016 11;2016:3876764. Epub 2016 Oct 11.

Department of Metabolic Diseases, Jagiellonian University Medical College, 15 Kopernika Street, 31-501 Kraków, Poland; University Hospital, 36 Kopernika Street, 31-501 Kraków, Poland.

. Type 2 diabetes mellitus (T2DM) is determined by genetic and environmental factors. There have been many studies on the relationship between the composition of the gastrointestinal bacterial flora, T2DM, and obesity. There are no data, however, on the gut microbiome structure in monogenic forms of the disease including Maturity Onset Diabetes of the Young (MODY). . The aim of the investigation was to compare the qualitative parameters of the colonic flora in patients with HNF1AMODY and T2DM and healthy individuals. 16S sequencing of bacterial DNA isolated from the collected fecal samples using the MiSeq platform was performed. . There were significant between-group differences in the bacterial profile. At the phylum level, the amount of Proteobacteria was higher ( = 0.0006) and the amount of Bacteroidetes was lower ( = 0.0005) in T2DM group in comparison to the control group. In HNF1A-MODY group, the frequency of Bacteroidetes was lower than in the control group ( = 0.0143). At the order level, Turicibacterales was more abundant in HNF1A-MODY group than in T2DM group. . It appears that there are differences in the gut microbiome composition between patients with HNF1A-MODY and type 2 diabetes. Further investigation on this matter should be conducted.
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http://dx.doi.org/10.1155/2016/3876764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078663PMC
June 2017

Synergic Interaction of Rifaximin and Mutaflor (Escherichia coli Nissle 1917) in the Treatment of Acetic Acid-Induced Colitis in Rats.

Gastroenterol Res Pract 2016 28;2016:3126280. Epub 2016 Jun 28.

Department of Internal Medicine II, Thuringia Clinic, Teaching Hospital of the University of Jena, Rainweg 68, 07318 Saalfeld, Germany.

Background. Inflammatory bowel disease results from the dysregulation of immune response to environmental and microbial agents in genetically susceptible individuals. The aim of the present study was to examine the effect of rifaximin and/or Mutaflor (Escherichia coli Nissle 1917, EcN) administration on the healing of acetic acid-induced colitis. Methods. Colitis was induced in male Wistar rats by rectal enema with 3.5% acetic acid solution. Rifaximin (50 mg/kg/dose) and/or Mutaflor (10(9) CFU/dose) were given intragastrically once a day. The severity of colitis was assessed at the 8th day after induction of inflammation. Results. Treatment with rifaximin significantly accelerated the healing of colonic damage. This effect was associated with significant reversion of the acetic acid-evoked decrease in mucosal blood flow and DNA synthesis. Moreover, administration of rifaximin significantly reduced concentration of proinflammatory TNF-α and activity of myeloperoxidase in colonic mucosa. Mutaflor given alone was without significant effect on activity of colitis. In contrast, Mutaflor given in combination with rifaximin significantly enhanced therapeutic effect of rifaximin. Moreover, Mutaflor led to settle of the colon by EcN and this effect was augmented by pretreatment with rifaximin. Conclusion. Rifaximin and Mutaflor exhibit synergic anti-inflammatory and therapeutic effect in acetic acid-induced colitis in rats.
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http://dx.doi.org/10.1155/2016/3126280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940557PMC
July 2016

A study of the effects of therapeutic doses of ionizing radiation in vitro on Lactobacillus isolates originating from the vagina - a pilot study.

BMC Microbiol 2016 May 31;16:99. Epub 2016 May 31.

Department of Bacteriology, Microbial Ecology and Parasitology, Chair of Microbiology, Jagiellonian University Medical College, 18 Czysta Street, 31-121, Krakow, Poland.

Background: Ionizing radiation is used as a therapeutic option in the treatment of certain neoplastic lesions located, among others, in the pelvic region. The therapeutic doses of radiation employed often result in adverse effects manifesting themselves primarily in the form of genital tract infections in patients or diarrhea. The data available in the literature indicate disorders in the microbial ecosystem caused by ionizing radiation, which leads to the problems mentioned above. In the present study, we examined the influence of ionizing radiation on 52 selected strains of bacteria: Lactobacillus crispatus, L. fermentum, L. plantarum, L. reuteri, L. acidophilus L. amylovorus, L. casei, L. helveticus, L. paracasei, L. rhamnosus, L. salivarius and L. gasseri. This collection of Lactobacillus bacteria isolates of various species, obtained from the genital tract and gastrointestinal tract of healthy women, was tested for resistance to therapeutic doses of ionizing radiation.

Results: The species studied, were isolated from the genital tract (n = 30) and from the anus (n = 22) of healthy pregnant women. Three doses of 3 Gy (fractionated dose) and 50 Gy (total dose of the whole radiotherapy cycle) were applied. The greatest differences in survival of the tested strains in comparison to the control group (not subjected to radiation) were observed at the dose of 50 Gy. However, the results were not statistically significant. Survival decrease to zero was not demonstrated for any of the tested strains.

Conclusions: Therapeutic doses of radiation do not affect the Lactobacillus bacteria significantly.
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http://dx.doi.org/10.1186/s12866-016-0716-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886408PMC
May 2016

The use of PFGE method in genotyping of selected bacteria species of the Lactobacillus genus.

Methods Mol Biol 2015 ;1301:225-40

Jagiellonian University Medical College, 18 Czysta St., 31-121, Krakow, Poland,

Bacteria belonging to the genus Lactobacillus are a group of microorganisms in which there are numerous strains with high probiotic potential. In recent years, interest in probiotics as nutritional supplements and as drugs has significantly increased. In order to carry out research on probiotic bacterial strains, their beneficial effects on the human body should be confirmed and molecular typing should be carried out with the aim of marking a specific strain. Pulse-field gel electrophoresis (PFGE) is the reference method employed for bacterial genotyping, but it is commonly used for typing mainly pathogenic bacteria. The following study describes a way of performing PFGE analysis in genotyping of bacteria species belonging to the genus Lactobacillus: L. fermentum, L. gasseri, L. plantarum and L. rhamnosus.
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http://dx.doi.org/10.1007/978-1-4939-2599-5_18DOI Listing
December 2015

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.

BMC Microbiol 2014 Dec 11;14:313. Epub 2014 Dec 11.

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.
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http://dx.doi.org/10.1186/s12866-014-0313-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302608PMC
December 2014

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

Gut Pathog 2014 15;6(1):43. Epub 2014 Oct 15.

Department of Microbiology, Jagiellonian University Medical College, 18 Czysta St, 31-121 Krakow, Poland.

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.
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http://dx.doi.org/10.1186/s13099-014-0043-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201707PMC
October 2014

Dynamics of colonization with group B streptococci in relation to normal flora in women during subsequent trimesters of pregnancy.

New Microbiol 2014 Jul 1;37(3):307-19. Epub 2014 Jul 1.

Department of Bacteriology, Microbial Ecology and Parasitology, Chair of Microbiology, Jagiellonian University Medical College, Krakow, Poland;

The main objective of the study was to compare the qualitative and quantitative composition of vaginal and rectal flora in GBS-positive (n=15) and GBS-negative (n=27) pregnant women examined in three subsequent trimesters of their pregnancy. Study samples consisted of vaginal and rectal smears and urine samples. GBS numbers were determined by the quantitatively cultured method [cfu/ml] and with the use of qPCR. Five GBS colonies were isolated per each positive sample and genotyped by PFGE and serotyping. The normal flora components: Lactobacillus, Bifidobacterium and Candida were quantitatively cultured. Carriage of GBS in subsequent trimesters in vagina/anus was variable and fluctuated between 17% and 28%. Quantitative GBS analyses showed that the vaginal population was at a constant level with the mean value equal to 3.94×104 cfu/ml, in contrast to the rectal population where the highest values appeared in the third trimester 4.37×105. The use of qPCR gave 7% more positive results for vaginal/rectal swabs. Genetic similarity analysis showed that one GBS clone was present in 73% of carriers during pregnancy, while in 27% of patients, 2 clones were found. H2O2-positive vaginal lactobacilli were detected in all women, while H2O2-negative lactobacilli and Bifidobacterium occurred more frequently in the anus in about 50% of women. Candida was present in the vagina in 30% of women. The analysis of women in three consecutive trimesters of pregnancy on the basis of a study group and control group showed no statistically significant differences in either the species (qualitative) or quantitative composition in vaginal and rectal flora in both of the groups. Therefore, GBS should be considered as a component of the microbiota and an opportunistic microorganism rather than a typical pathogen, because it does not distort the composition of women's normal genital tract flora.
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July 2014