Gut microbiota in a healthy population is shaped by various geographic, demographic and lifestyle factors. Although the majority of research remains focused on the bacterial community, recent efforts to include the remaining microbial members like viruses, archaea and especially fungi revealed various functions they perform in the gut. Using the amplicon sequencing approach we analysed bacterial and fungal gut communities in a Slovenian cohort of 186 healthy volunteers. In the bacterial microbiome we detected 253 different genera. A core microbiome analysis revealed high consistency with previous studies, most prominently showing that genera Faecalibacterium, Bacteroides and Roseburia regularly comprise the core community. We detected a total of 195 fungal genera, but the majority of these showed low prevalence and are likely transient foodborne contaminations. The fungal community showed a low diversity per sample and a large interindividual variability. The most abundant fungi were Saccharomyces cerevisiae and Candida albicans. These, along with representatives from genera Penicillium and Debaryomyces, cover 82% of obtained reads. We report three significant questionnaire-based host covariates associated with microbiota composition. Bacterial community was associated with age and gender. More specifically, bacterial diversity was increased with age and was higher in the female population compared to male. The analysis of fungal community showed that more time dedicated to physical activity resulted in a higher fungal diversity and lower abundance of S. cerevisiae. This is likely dependent on different diets, which were reported by participants according to the respective rates of physical activity.

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The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDI-TOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches, Staphylococcus and Streptococcus were found as predominant genera in HMM and the abundance of Staphylococcus was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, lower microbial load, and higher abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events.

Typical disease-associated microbiota changes are widely studied as potential diagnostic or therapeutic targets. Our aim was to analyze a hospitalized cohort including various gastroenterological pathologies in order to fine-map the gut microbiota dysbiosis. Bacterial (V3 V4) and fungal (ITS2) communities were determined in 121 hospitalized gastrointestinal patients from a single ward and compared to 162 healthy controls. Random Forest models implemented in this study indicated that the gut community structure is in most cases not sufficient to differentiate the subjects based on their underlying disease. Instead, hospitalized patients in our study formed three distinct disease non-related clusters (C1, C2, and C3), partially explained by antibiotic use. Majority of the subjects (cluster C1) closely resembled healthy controls, showing only mild signs of community disruption; most significantly decreased in this cluster were Faecalibacterium and Roseburia. The remaining two clusters (C2 and C3) were characterized by severe signs of dysbiosis; cluster C2 was associated with an increase in Enterobacteriaceae and cluster C3 by an increase in Enterococcus. According to the cluster affiliation, subjects also showed different degrees of inflammation, most prominent was the positive correlation between levels of C-reactive protein and the abundance of Enterococcus.

Clostridium difficile infection (CDI) is typically associated with disturbed gut microbiota and changes related to decreased colonization resistance against C. difficile are well described. However, nothing is known about possible effects of C. difficile on gut microbiota restoration during or after CDI. In this study, we have mimicked such a situation by using C. difficile conditioned medium of six different C. difficile strains belonging to PCR ribotypes 027 and 014/020 for cultivation of fecal microbiota. A marked decrease of microbial diversity was observed in conditioned medium of both tested ribotypes. The majority of differences occurred within the phylum Firmicutes, with a general decrease of gut commensals with putative protective functions (i.e. Lactobacillus, Clostridium_XIVa) and an increase in opportunistic pathogens (i.e. Enterococcus). Bacterial populations in conditioned medium differed between the two C. difficile ribotypes, 027 and 014/020 and are likely associated with nutrient availability. Fecal microbiota cultivated in medium conditioned by E. coli, Salmonella Enteritidis or Staphylococcus epidermidis grouped together and was clearly different from microbiota cultivated in C. difficile conditioned medium suggesting that C. difficile effects are specific. Our results show that the changes observed in microbiota of CDI patients are partially directly influenced by C. difficile.