Łukasz Grabowski

Neurodegeneration with brain iron accumulation (NBIA) is a group of rare neurodegenerative disorders characterized by excessive iron deposition in the central nervous system. The disease typically manifests in early childhood with progressive dystonia, muscle rigidity, spasticity, ataxia, as well as personality changes (such as nervousness, aggression, and learning difficulties) and psychiatric symptoms (including disorientation, confusion, seizures, and dementia). The most common forms – BPAN, PKAN, MPAN, and PLAN—are associated with mutations in the WDR45, PANK2, C19orf12, and PLA2G6 genes, respectively, leading to disruptions in mitochondrial functions, autophagy, and coenzyme A metabolism. These abnormalities result in oxidative stress and neurodegeneration; however, the connection between the observed mutations and iron accumulation in the central nervous system remains unclear. There is ongoing debate as to whether iron accumulation is a primary pathological factor or a secondary consequence of cellular dysfunction. Unfortunately, despite numerous therapeutic attempts, including the use of iron chelators, treatment efficacy remains very limited. Therefore, the aim of this review was to present the latest findings on the relationship between WDR45, PANK2, C19orf12, and PLA2G6 mutations and iron metabolism disorders. The data presented may help explain the previously undescribed roles of these mutations in the excessive iron accumulation observed in the nervous system of patients with NBIA, while also highlighting the complexity of NBIA pathogenesis. A better understanding of these mechanisms may contribute to the development of new, more effective therapeutic strategies, which are greatly needed by patients.

Fatty acids play important, yet different roles in bacterial physiology, specifically their growth, either stimulating or inhibiting this parameter which are of biotechnological importance. Here, we present results showing to what degree short- and medium-chain fatty acids (butyric acid (butanoic acid, C4:0); caproic acid (hexanoic acid, C6:0); caprylic acid (octanoic acid, C8:0)), used at relatively low concentrations (in a range of μg/mL, contrary to previously reported mg/mL which revealed inhibitory effects on bacterial growth) affect growth of Escherichia coli K-12 (MG1655 laboratory strain) depending on various conditions. In rich medium (LB) positive effects of all tested fatty acids on E. coli growth were observed, while temperature of incubation (growth at 25 °C and 37 °C was assessed) modulated these effects. In contrast, a slight but significant growth inhibition by fatty acids was observed in a minimal medium (M9) supplemented with glucose. Nonetheless, in minimal medium containing acetate, the effects of these compounds varied, being either positive or negative depending on their concentrations. No measurable bacterial growth was observed in the case of the presence of any tested fatty acids when primary carbon source (glucose or acetate) was removed from a minimal medium before addition of butyric acid, caproic acid or caprylic acid. Our results indicated that effects of low concentrations of fatty acids on E. coli cells depend on growth conditions of bacterial cultures. This may be of biotechnological importance, especially for modulating E. coli growth by using different compositions of media and incubation temperatures.

Fatty acids play important, yet different roles in bacterial physiology, specifically their growth. On the one hand, these compounds may be used by bacteria to produce storage materials or serve as a carbon source. On the other hand, due to their influence on biolog-ical membranes, fatty acids have been reported to exhibit antimicrobial effects. It was in-dicated that the length of the fatty acid chain is crucial for specific effects on bacterial cells. However, the literature still contains reports of contradictory findings in studies on these molecules. Here, we present results showing to what degree short- and medium-chain fatty acids (butyric acid (butanoic acid, C4:0); caproic acid (hexanoic acid, C6:0); caprylic acid (octanoic acid, C8:0)), used at relatively low concentrations (in a range of g/mL, spe-cifically 0.25-16 g/mL, contrary to previously reported mg/mL which revealed inhibitory effects on bacterial growth) affect growth of Escherichia coli K-12 (MG1655 laboratory strain) depending on various conditions. In rich medium (LB) positive effects of all tested fatty acids on E. coli growth were observed, while temperature of incubation (growth at 25oC and 37oC was assessed) modulated these effects. In contrast, a slight but significant growth inhibition by fatty acids was observed in a minimal medium (M9) supplemented with glucose. Nonetheless, in minimal medium containing acetate, the effects of these compounds varied, being either positive or negative depending on their concentrations. No measurable bacterial growth was observed in the case of the presence of any tested fatty acids when primary carbon source (glucose or acetate) was removed from a minimal medium before addition of butyric acid, caproic acid or caprylic acid. These results indi-cated that effects of fatty acids on E. coli cells depend on growth conditions of bacterial cultures, and may suggest that previously reported discrepancies between results ob-tained by various groups might arise, at least partially, from using various cultivation procedures used for different experiments.

The overuse of antibiotics in both humans and livestock has led to the antibiotic resistance phenomenon which is now considered one of the biggest problems in the modern world. Some antibiotics used to control or prevent infections in livestock poultry were registered a long time ago, and as a result, data on the possible side effects of their use, both for birds and humans, are incomplete and should be updated. An example of such an antibiotic is enrofloxacin which has been widely used in poultry since 1989. Data in recent years have begun to indicate that this antibiotic induces the process of apoptosis in diverse types of eukaryotic cells. Unfortunately, such studies have never been conducted on chicken models even though it is in poultry that this antibiotic is most commonly used. Therefore, the purpose of this work was to investigate whether enrofloxacin induces apoptosis in chicken cells of the UMNSAH/DF-1 line and to study the molecular mechanism of its action. The results of these experiments indicated that enrofloxacin induces apoptosis in chicken cells but not in human HEK-293 and PC3 cells. This induction was accompanied by changes in the morphology and size of mitochondria, the process of apoptosome formation and activation of executive caspases, which clearly indicates the role of the mitochondrial pathway in the induction of apoptosis by enrofloxacin. This study is the first to show the toxicity of enrofloxacin against chicken cells and to demonstrate the exact mechanism of its action. The results presented in this work show the need to monitor the concentration of antibiotic residues in poultry foods as well as to study their impact on public health to guarantee consumer safety and prevent the phenomenon of antibiotic resistance in bacteria.

Previous studies indicated that the use of a phage cocktail, composed of bacteriophages vB_SenM-2 and vB_Sen-TO17, is effective in killing cells of Salmonella enterica serovars Typhimurium and Enteritidis in vitro and in the Galleria mellonella animal model as efficiently as antibiotics (enrofloxacin or colistin) and induced fewer deleterious changes in immune responses. Here, we investigated the effects of this phage cocktail on the hematological parameters and selected biochemical markers in chickens infected with S. enterica serovar Typhimurium, in comparison to those caused by enrofloxacin or colistin. We found that treatment with antibiotics (especially with enrofloxacin) caused nonbeneficial effects on red blood cell parameters, including hematocrit, MCV, MCH, and MCHC. However, Salmonella-induced changes in the aforementioned parameters were normalized by the use of the phage cocktail. Importantly, hepatotoxicity was suggested to be induced by both antibiotics on the basis of increased alanine transaminase (ALT) and aspartate aminotransferase (AST) activities, in contrast to the phage cocktail, which did not influence these enzymes. We conclude that phage therapy with the cocktail of vB_SenM-2 and vB_Sen-TO17 in Salmonella-infected chickens is not only as effective as antibiotics but also significantly safer for the birds than enrofloxacin and colistin.

Bacterial functional amyloids, apart from their many other functions, can influence the resistance of bacteria to antibiotics and other antibacterial agents. Mechanisms of modulation of susceptibility of bacterial cells to antimicrobials can be either indirect or direct. The former mechanisms are exemplified by the contribution of functional amyloids to biofilm formation, which may effectively prevent the penetration of various compounds into bacterial cells. The direct mechanisms include the effects of bacterial proteins revealing amyloid-like structures, like the C-terminal region of the Escherichia coli Hfq protein, on the expression of genes involved in antibiotic resistance. Therefore, in this paper, we describe methods by which effects and mechanisms of action of bacterial amyloids on antibiotic resistance can be studied. Assessment of formation of biofilms, determination of the efficiency of antibiotic resistance in solid and liquid media, and determination of the effects on gene expression at levels of mRNA abundance and stability and protein abundance are described. Key words Amyloids in antibiotic resistance Biofilm Minimal inhibitory concentration (MIC) mRNA abundance and stability Protein level determination WES—automatic Western blotting system

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed.

The “antibiotic crisis”, defined as the appearance of microbial strains resistant to most, if not all, already known antibiotics, indicates that searching for previously unknown antimicrobial agents is crucial for further development of novel drugs that can be used to combat infections caused by bacteria and fungi. Bacteria living in untypical and extreme habitats appear to be a potentially reached source of such compounds. We recently reported an isolation of newly identified strains of Actinobacteria from the Szczelina Chochołowska cave (Tatra Mountains, Poland). Some of them produced molecules revealing antibacterial, antifungal and anticancer properties. Here, we describe further characterization of the selected strains. Their microbiological properties, ability to form biofilms and antimicrobial activities against various strains of bacteria and fungi are reported. The selected strains of newly isolated Actinobacteria belonging to the genus Streptomyces appear a promising source of previously unknown antimicrobial agents.