Occurrence and genetic diversity of prophage sequences identified in the genomes of L. casei group bacteria.

It is widely believed that microorganisms belonging to L. casei group can have positive effects on the human body. Therefore, these bacteria are used in many industrial processes, including the production of dietary supplements and probiotic preparations. When using live microorganisms in technological processes, it is important to use those without phage sequences within their genomes that can ultimately lead to lysis of the bacteria. It has been shown that many prophages have a benign nature, meaning that they don't directly lead to lysis or inhibit microbial growth. Moreover, the presence of phage sequences in the genomes of these bacteria increases their genetic diversity, which may contribute to easier colonization of new ecological niches. In the 439 analyzed genomes of the L. casei group, 1509 sequences of prophage origin were detected. The average length of intact prophage sequences analyzed was just under 36 kb. GC content of tested sequences was similar for all analyzed species (44.6 ± 0.9%). Analyzing the protein coding sequences collectively, it was found that there was an average of 44 putative ORFs per genome, while the ORF density of all phage genomes varied from 0.5 to 2.1. The average nucleotide identity calculated on sequence alignments for analyzed sequences was 32.7%. Of the 56 L. casei strains used in the next part of the study, 32 did not show culture growth above the OD600 value of 0.5, even at a mitomycin C concentration of 0.25 µg/ml. Primers used for this study allowed for the detection of prophage sequences for over 90% of tested bacterial strains. Finally, prophages of selected strains were induced using mitomycin C, phage particles were isolated and then genomes of viruses obtained were sequenced and analyzed.

The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study.

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Detection of immunoreactive proteins of Escherichia coli, Streptococcus uberis, and Streptococcus agalactiae isolated from cows with diagnosed mastitis.

Introduction: Mastitis is a widespread mammary gland disease of dairy cows that causes severe economic losses to dairy farms. Mastitis can be caused by bacteria, fungi, and algae. The most common species isolated from infected milk are, among others, Streptococcus spp., and Escherichia coli. The aim of our study was protein detection based on both in silico and in vitro methods, which allowed the identification of immunoreactive proteins representative of the following species: Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Methods: The study group included 22 milk samples and 13 serum samples obtained from cows with diagnosed mastitis, whereas the control group constituted 12 milk samples and 12 serum samples isolated from healthy animals. Detection of immunoreactive proteins was done by immunoblotting, while amino acid sequences from investigated proteins were determined by MALDI-TOF. Then, bioinformatic analyses were performed on detected species specific proteins in order to investigate their immunoreactivity. Results: As a result, we identified 13 proteins: 3 (molybdenum cofactor biosynthesis protein B, aldehyde reductase YahK, outer membrane protein A) for E. coli, 4 (elongation factor Tu, tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG, GTPase Obg, glyceraldehyde-3-phosphate dehydrogenase) for S. uberis, and 6 (aspartate carbamoyltransferase, elongation factor Tu, 60 kDa chaperonin, elongation factor G, galactose-6-phosphate isomerase subunit LacA, adenosine deaminase) for S. agalactiae, which demonstrated immunoreactivity to antibodies present in serum from cows with diagnosed mastitis. Discussion: Due to the confirmed immunoreactivity, specificity and localization in the bacterial cell, these proteins can be considered considered potential targets in innovative rapid immunodiagnostic assays for bovine mastitis, however due to the limited number of examined samples, further examination is needed.

The Combination of Intestinal Alkaline Phosphatase Treatment with Moderate Physical Activity Alleviates the Severity of Experimental Colitis in Obese Mice via Modulation of Gut Microbiota, Attenuation of Proinflammatory Cytokines, Oxidative Stress Biomarkers and DNA Oxidative Damage in Colonic Mucosa.

Inflammatory bowel diseases (IBD) are commonly considered as Crohn's disease and ulcerative colitis, but the possibility that the alterations in gut microbiota and oxidative stress may affect the course of experimental colitis in obese physically exercising mice treated with the intestinal alkaline phosphatase (IAP) has been little elucidated. Mice fed a high-fat-diet (HFD) or normal diet (ND) for 14 weeks were randomly assigned to exercise on spinning wheels (SW) for 7 weeks and treated with IAP followed by intrarectal administration of TNBS. The disease activity index (DAI), grip muscle strength test, oxidative stress biomarkers (MDA, SOD, GSH), DNA damage (8-OHdG), the plasma levels of cytokines IL-2, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, MCP-1 and leptin were assessed, and the stool composition of the intestinal microbiota was determined by next generation sequencing (NGS). The TNBS-induced colitis was worsened in obese sedentary mice as manifested by severe colonic damage, an increase in DAI, oxidative stress biomarkers, DNA damage and decreased muscle strength. The longer running distance and weight loss was observed in mice given IAP or subjected to IAP + SW compared to sedentary ones. Less heterogeneous microbial composition was noticed in sedentary obese colitis mice and this effect disappeared in IAP + SW mice. Absence of Alistipes, lower proportion of Turicibacter, Proteobacteria and Faecalibacterium, an increase in Firmicutes and Clostridium, a decrease in oxidative stress biomarkers, 8-OHdG content and proinflammatory cytokines were observed in IAP + SW mice. IAP supplementation in combination with moderate physical activity attenuates the severity of murine colitis complicated by obesity through a mechanism involving the downregulation of the intestinal cytokine/chemokine network and oxidative stress, the modulation of the gut microbiota and an improvement of muscle strength.

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

Numerous scientific studies confirm that, apart from environmental and genetic factors, a significant role is played by gastrointestinal microbiota in the aetiology of type 2 diabetes and obesity. Currently, scientists mainly focus on the distal intestinal microbiota, while the equally important proximal parts of the intestine are overlooked. The aim of the study was a qualitative analysis of the structure of the duodenal mucosa microbiota in groups of patients with obesity and with type 2 diabetes and where obesity qualified for bariatric surgery: sleeve gastrectomy. The microbiological results obtained were compared with some clinical parameters. As a result, it was possible to determine the microbiological core that the treatment and control groups had in common, including phyla: Firmicutes, Proteobacteria, and Actinobacteria. The patients with obesity and with type 2 diabetes and obesity presented a significantly lower number of genus Bifidobacterium compared to healthy subjects. Furthermore, the numbers of Bifidobacterium were positively correlated with the high density lipoprotein (HDL) concentration in the groups under study. The obtained results indicate that bacteria of the genus Bifidobacterium should be considered in the future in the context of a potential biomarker in the progress of type 2 diabetes and obesity.

Gut microbiome as a biomarker of cardiometabolic disorders.

INTRODUCTION AND OBJECTIVE: Cardiovascular diseases are the leading cause of death in Europe and worldwide. One of the most important risk factors for atherosclerosis are lipid metabolism disorders, in particular hipercholesterolaemia. The aim of the study was to determine the correlation between gut microbiota composition and atherosclerosis risk factors, so in order that it might be used as a biomarker for coronary artery disease diagnosis. MATERIAL AND METHODS: The study involved middle-aged men in eastern Poland with central obesity (n=20), subjects with atherosclerosis (n=15) and those with no cardiovascular diseases (n=5). The gut microbiota composition was determined using tag-encoded 16S rRNA gene using Illuminal MiSeq. Data were analyzed with the use of t-test. RESULTS: Firmicutes (49.26%) and Bacteroidetes (44.46%) were the dominant Phyla in the middle-aged men in eastern Poland. Subjects with improper levels of total cholesterol were enriched in Prevotella (p=0.03) and decreased level of Clostridium (p=0.02). They also showed a falling tendency in Faecalibacterium (p=0.07). An upward trend was observed in Prevotella (p=0.07) in subjects with improper LDL-C values. CONCLUSIONS: The study showed that intestinal microbiome is likely to play a role in the pathogenesis of atherosclerosis through its role in lipid metabolism. Bacterial genera of particular importance were Prevotella, Bacteroides, Clostridium, Faecalibacterium. However, further studies involving larger groups of subjects are required to confirm these observations.