Antonio Starcevic

Research background. Numerous biosynthetic gene clusters (BGCs) encoding unknown structures have been discovered in the genomes of diverse microorganisms, representing a potentially rich source of novel natural products. However, most of the identified BGCs do not seem to be active, since we cannot detect any corresponding metabolites. Therefore, a better understanding of the regulation and biosynthesis of secondary metabolites encoded by these so-called ‘silent’ BGCs is of great importance. Experimental approach. We conducted a bioinformatic analysis of the Streptomyces rimosus ATCC 10970 strain, a producer of the antibiotic oxytetracycline, focusing on the expression of identified BGCs. We then reviewed experimentally identified compounds and putative structures predicted from genome data and similarity to known metabolites. We analysed available data on the regulation of two major metabolites – oxytetracycline and rimocidin, and experimentally evaluated the effect of the deletion of two oxytetracycline-competing pathways. Finally, we evaluated the effect of overexpressing BGC encoding the biosynthesis of the carotenoid isorenieratene, which cannot be detected in the culture of the native strain. Results and conclusions. We identified 48 BGCs in the genome of Streptomyces rimosus ATCC 10970. However, only about 15 structures were predicted or identified in the culture of this strain. Transcriptional analysis of identified BGCs demonstrated a very high variability in expression strength. Interestingly, around 30 % of BGCs were ‘silent’. In trans overexpression of one such silent BGC, encoding the biosynthesis of the carotenoid isorenieratene resulted in strong production of this metabolite, suggesting that silent BGCs are likely still functional. We also demonstrated that BGCs encoding two major metabolites, oxytetracycline and rimocidin, both derived from malonyl-coenzyme A (malonyl-CoA), are not competitive pathways. Surprisingly, deletion of one silent BGC, also derived from malonyl-CoA, has a very strong effect on the biosynthesis of oxytetracycline. Novelty and scientific contribution. We observed that the expression strength of genes from BGCs identified in Streptomyces rimosus does not correspond to the experimental data obtained from the engineered strains, suggesting much more complex regulatory mechanisms than previously thought. Engineered Streptomyces rimosus host strains thus represent a very good model system to study the expression of ‘silent’ BGCs.

Objectives: The application of whole fermented foods in sports nutrition for gut microbiota personalization is a promising area of investigation. Sauerkraut, a traditional fermented food, has not been extensively studied in this context. This study aimed to replicate earlier findings of a proof-of-concept study on the potential of sauerkraut for gut microbiota personalization in active athletes. Methods: A pilot follow-up study was conducted on active recreational athletes who consumed 250 g of organic pasteurized sauerkraut daily for 10 days. Changes in gut microbiota composition and functionality were assessed using 16S rRNA sequencing and metabolic pathway analysis across three time points: pre-intervention, postintervention, and one-month postintervention. Laboratory parameters, bowel function, and side effects were monitored throughout the study. Results: In total, 11 male participants with an average age of 30 years completed the study. The pilot follow-up study did not replicate the original study’s findings on sauerkraut’s short-term effects on β-diversity and taxonomic and functional groups. However, long-term effects of sauerkraut supplementation were demonstrated, including a significant reduction in α-diversity variance and increased gut microbiota composition similarity (β-diversity) as well as several significant changes in bacterial taxa and metabolic pathways after a washout period. The intervention also induced a transient decrease in B12 vitamin levels and a short- and long-term increase in leukocyte concentrations. The probability for physiological types of stools increased after one week of sauerkraut supplementation as well as the incidence of gastrointestinal side effects, such as bloating, diarrhea, pain, nausea, and constipation. Conclusions: This study suggests that the short-term effects on the gut microbiota of sauerkraut supplementation depend on its baseline status, but it can induce long-term effects. Sauerkraut supplementation requires a seven-day adaptation period. Further research is needed to explore the mechanisms behind the short- and long-term effects of sauerkraut supplementation.

Objectives: The application of whole fermented foods in sports nutrition for gut microbiota personalization is a promising area of investigation. Sauerkraut, a traditional fermented food, has not been extensively studied in this context. This study aimed to replicate earlier findings of a proof-of-concept study on the potential of sauerkraut for gut microbiota personalization in active athletes. Methods: A follow-up study was conducted on active recreational athletes who consumed 250 g of organic pasteurized sauerkraut daily for 10 days. Changes in gut microbiota composition and functionality were assessed using 16S rRNA sequencing and metabolic pathway analysis across three time points: pre-intervention, post-intervention, and one-month post-intervention. Laboratory parameters, bowel function, and side effects were monitored throughout the study. Results: The follow-up study did not replicate the original study's findings on sauerkraut's short-term effects on β-diversity and taxonomic and functional groups. However, long-term effects of sauerkraut supplementation were demonstrated, including a significant reduction in α-diversity variance and increased gut microbiota composition similarity (β-diversity), as well as several significant changes in bacterial taxa and metabolic pathways after a wash-out period. The intervention also induced a transient decrease in B12 vitamin levels and a short- and long-term increase in leukocyte concentrations. The probability for physiological types of stools increased after one week of sauerkraut supplementation, as well as the incidence of gastrointestinal side effects, such as bloating, diarrhea, pain, nausea and constipation. Conclusions: This study suggests that the short-term effects on the gut microbiota of sauerkraut supplementation depend on its baseline status, but that it can induce long-term effects. Sauerkraut supplementation requires a seven-day adaptation period. Further research is needed to explore the mechanisms behind the short- and long-term effects of sauerkraut supplementation.

Oxford-Nanopore PromethION sequencing is a PCR-free method that retains epigenetic markers and provides direct quantitative information about DNA methylation. Using this long-read sequencing technology, we successfully assembled 5 myxozoan genomes free from discernible host DNA contamination, surpassing previous studies in both quality and completeness. Genome assembly revealed DNA methylation patterns within myxozoan genomes, particularly in GC-rich regions within gene bodies. The findings not only refute the notion of myxozoans lacking DNA methylation capability but also offer a new perspective on gene regulation in these parasites. The high-quality genome assemblies lay a solid foundation for future research on myxozoans, including new strategies to control these commercially significant fish pathogens.

Background: Since the gut microbiota is important for athlete health and performance, its optimization is increasingly gaining attention in sports nutrition, for example, with whole fermented foods. Sauerkraut is a traditional fermented food rich in pro-, pre-, and postbiotics, which has not yet been investigated in the field of sports nutrition. Methods: To determine whether sauerkraut could be used for gut microbiota optimization in sports nutrition, a proof-of-concept study was conducted. The microbiota composition of organic pasteurized sauerkraut was analyzed, and then healthy active athletes were provided with the same sauerkraut for 10 days as an intervention. The effects of sauerkraut on the athlete’s gut microbiota, laboratory parameters, and bowel function were assessed. Results: Significant changes in the gut microbiota composition were seen on taxonomic and functional levels, independent of baseline microbiota composition, even after short-term supplementation. Most notably, there was an increase in several health-promoting genera of the family Lachnospiraceae, as well as significant alterations in metabolic pathways regarding cell wall synthesis and the metabolism of nucleotide bases. An increase in the proportion of lymphocytes and a decrease in B12 vitamin levels was observed, as well as a risk of indigestion in certain athletes, which significantly resolved after seven days of supplementation in all athletes. It is unclear whether the observed effects are attributable to the sauerkraut’s own microbiome or its pre- and postbiotics since it is a whole food. Conclusions: Our study has demonstrated that the concept of whole fermented foods, such as sauerkraut, could potentially be feasible and effective in sports nutrition for gut microbiota optimization.

Background: Since the gut microbiota is important for athlete health and performance, its optimization is increasingly gaining attention in sports nutrition, for example with whole fermented foods. Sauerkraut is a traditional fermented food rich in pro-, pre- and postbiotics, which has not yet been investigated in the field of sports nutrition. Methods: To determine whether sauerkraut could be used for gut microbiota optimization in sports nutrition a proof-of-concept study was conducted. The microbiota composition of organic pasteurized sauerkraut was analyzed, and then healthy active athletes were provided with the same sauerkraut for 10 days as an intervention. The effects of sauerkraut on the athlete’s gut microbiota, laboratory parameters and bowel function were assessed. Objectives: Significant changes in the gut microbiota composition were seen on taxonomic and functional level, independent of baseline microbiota composition, even after short-term supplementation. Most notably an increase of several health-promoting genera of family Lachnospiraceae, as well as significant alterations in metabolic pathways regarding cell wall synthesis and the metabolism of nucleotide bases. An increase in the proportion of lymphocytes and decrease in B12 vitamin levels was observed as well as a risk of indigestion in certain athletes, which significantly resolved after seven days of supplementation in all athletes. It is unclear whether the observed effects are attributable to the sauerkraut's own microbiome, or its pre- and postbiotics since it is a whole food. Our study has demonstrated that the concept of whole fermented foods, such as sauerkraut, could potentially be feasible and effective in sports nutrition for gut microbiota optimization.

To enhance recovery, athletes often consume ready-to-eat foods, specialized sports foods and/or supplements that belong to the group of ultra-processed foods (UPFs), which may affect their health. The aim of this study was to estimate consumption of UPFs in basketball players and to determine whether there is a difference in anthropometric characteristics, neuromuscular and cardiovascular fitness and microbiota composition between players in relation to the proportion of daily energy from processed foods. The study involved 17 elite male basketball players (>18 years) from Croatia during the competitive season. After cluster analysis of daily energy contribution from NOVA food groups, 35% of players were clustered into higher UPFs consumption group. No differences in anthropometric characteristics, neuromuscular and cardiovascular fitness were observed between players who consumed more UPFs and those who consumed less UPFs. Players with higher consumption of UPFs had lower abundance of the order Veillonellales-Selenomonadales in their respective microbiotas, more precisely, of family Veillonellaceae (p = 0.040) and the genus Agathobacter (p = 0.025). These results suggest that the consumption of UPFs did not affect athletes’ performance but does impact their gut microbiota. Further studies on this issue are warranted to establish nutritional guidelines for athletes regarding the consumption of UPFs.

Research background. In this study, we investigated the effects of soluble dietary fibre on improving neuromuscular and cardiovascular endurance and perception of fatigue in a closely monitored group of basketball players. Prebiotics have been sidelined in sports nutrition and their effect on performance remains poorly investigated and understood. Experimental approach. Eighteen healthy male basketball players were divided into two groups; one received 17 g/day of soluble dietary fibre (Nutriose®) for four weeks and the other group received placebo. Their morphological characteristics, neuromuscular and cardiovascular endurance, and rating of perceived exertion according to the rating of perceived exertion (RPE) scale were assessed. Measurements were taken before supplementation and after four weeks of supplementation. Faecal samples were collected from all participants immediately before and after the supplementation period, their total DNA extracted and sent for amplicon sequencing. Results and conclusions. In this study, fibre had no statistically significant effect on the vertical-type explosive power, no statistically significant effect on sprint-type explosive power, nor on aerobic and anaerobic endurance in the experimental group. Soluble fibre had a statistically significant effect on reducing the rating of perceived exertion of basketball players during the competitive part of the season (RPE 7.27±0.04 versus 8.82±0.81). This was confirmed by two-way ANOVA with replication, which showed that within-group interaction (p=0.0193), before and after dietary intake (p=0.0049), and between-group interaction before and after dietary intake (p=0.0313) had a significant effect on the result. The overall conclusion of the study is that soluble dietary fibre supplementation does not improve neuromuscular and cardiovascular endurance over a 4-week period. However, fibre supplementation could have a significant effect on reducing the rating of perceived exertion, as shown by the statistics. Both amplicon sequencing and subsequent bioinformatics results suggest that this could be the result of the beneficial effect on the intestinal microbiota and its metabolites. Novelty and scientific contribution. This work highlights the importance of prebiotics in sports nutrition. Dietary fibre has been a neglected component of sports nutrition. This study demonstrated a statistically significant positive effect on the perception of fatigue, highlighting the need for further studies in this direction.

An increasing amount of evidence implies that native microbiota is a constituent part of a healthy urinary tract (UT), making it an ecosystem on its own. What is still not clear is whether the origin of the urinary microbial community is the indirect consequence of the more abundant gut microbiota or a more distinct separation exists between these two systems. Another area of uncertainty is the existence of a link between the shifts in UT microbial composition and both the onset and persistence of cystitis symptoms. Cystitis is one of the most common reasons for antimicrobial drugs prescriptions in primary and secondary care and an important contributor to the problem of antimicrobial resistance. Despite this fact, we still have trouble distinguishing whether the primary cause of the majority of cystitis cases is a single pathogen overgrowth or a systemic disorder affecting the entire urinary microbiota. There is an increasing trend in studies monitoring changes and dynamics of UT microbiota, but this field of research is still in its infancy. Using NGS and bioinformatics, it is possible to obtain microbiota taxonomic profiles directly from urine samples, which can provide a window into microbial diversity (or the lack of) underlying each patient’s cystitis symptoms. However, while microbiota refers to the living collection of microorganisms, an interchangeably used term microbiome referring to the genetic material of the microbiota is more often used in conjunction with sequencing data. It is this vast amount of sequences, which are truly “Big Data”, that allow us to create models that describe interactions between different species contributing to an UT ecosystem, when coupled with machine-learning techniques. Although in a simplified predator—prey form these multi-species interaction models have the potential to further validate or disprove current beliefs; whether it is the presence or the absence of particular key players in a UT microbial ecosystem, the exact cause or consequence of the otherwise unknown etiology in the majority of cystitis cases. These insights might prove to be vital in our ongoing struggle against pathogen resistance and offer us new and promising clinical markers.

Seawater is an environment in which numerous microorganisms have evolved, some with a great potential for biotechnology. In recent years, many scientists have moved away from the assumption that the origin of life was in pools of water, and instead propose that life on Earth probably originated in accumulations of warm, nutrient-rich mud. This mud, also called peloid is a rich source of organisms that, due to their adaptation to this unique environment, produce a wide variety of primary and secondary metabolites with numerous and diverse activities, including anti-cancer, anti-inflammatory, antiviral, and immunomodulatory ones. In this research, two questions were addressed using collected samples of a peloid with demonstrated healing properties. Firstly biodiversity in this ecological niche was explored in order to assess microbial communities present and secondly natural products were screened for in order to assess whether predicted activities could be linked to healing properties of the peloid. The use of peloids in medical therapy dates back to ancient times. Abiotic components such as clay and mineral water are believed to be the main contributors of the healing properties of natural peloids. The places where peloids are usually found are characteristically shallow and enclosed lagoons. The constant UV exposure and increased salt concentration classify peloid as an extreme environment. The spectrum of relief’s peloid therapy is claimed to provide ranges from purely cosmetic and skin-related to musculoskeletal and immunological problems. These claims can hardly be supported by mineral content and heat-retaining properties alone. However, organic compounds from present microorganisms as well as secondary metabolites could help explain the observed range of health benefits. The fact that the relationship between the therapeutic activity of peloids and their composition besides mineral and physicochemical properties has not been extensively studied indicates untapped biotechnological potential.