Influence of Oat β-Glucan on the Survival and Proteolytic Activity of Lactobacillus rhamnosus GG in Milk Fermentation: Optimization by Response Surface

β-glucans come from cereals that have been located within compounds with prebiotic activity. They have presented several bioactivities that have determined their high functional value. The aim of this study was to identify the influence of oat β-glucan on the survival and proteolytic activity of Lactobacillus rhamnosus GG in a milk fermentation through an experimental design to optimize the process. For β-glucan extraction after dry milling of oats, two methods were applied: with and without enzymatic inactivation of the semolina. The highest extraction yield (45.25 g/L) was obtained with enzymatic inactivation. For the optimization of survival and proteolytic activity, a central design composed of axial points with two factors on three levels was used. Control factors were β-glucan and inoculum concentrations. According to response surface, the best survival growth rate of probiotic was observed with 4.38% of inoculum and 22.46 g/L of β-glucan, and the highest production of free amino groups was observed with 4.18% of inoculum and 22.71 g/L of β-glucan. Thus, β-glucan promotes the proteolytic activity of Lb. rhamnosus GG in milk fermentation.

C25-modified rifamycin derivatives with improved activity against Mycobacterium abscessus

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

ANTIBIOTIC RESISTANCE OF BACTERIA OF THE FAMILY PASTEURELLACEAE, PATHOGENS OF RESPIRATORY INFECTIONS OF CATTLE AND PIGS

In the article a literature review of scientific papers on the topic of antimicrobial resistance of bacteria of the family Pasteurellaceae, pathogens of respiratory diseases in pigs and cattle, is presented. The main mechanisms of the development of Pasteurellaceae resistance to β-lactam antibiotics are the synthesis of β-lactamases by bacteria, what are able to break the beta-lactam ring, thereby inactivating β-lactams, or alteration of the penicillin-binding proteins structure. Other mechanisms, such as reduced permeability of the outer membrane or the process of active removal of antibiotics from the bacterial cell (efflux), are very rare. Resistance among Pasteurellaceae to β-lactams is often associated with plasmids. Eflux and ribosomal protection are the main mechanisms for the development of resistance of Pasteurellaceae to tetracyclines. At least nine tetracycline resistance genes (tet genes) have been identified in bacteria of the genus Pasteurella, Mannheimia, Actinobacillus and Haemophilus, what encode these processes. Resistance to aminoglycosides and aminocyclitols is mainly caused by enzymatic inactivation of antibiotics, as well as through mutations in chromosomal genes. Many plasmids carry genes of resistance to aminoglycosides, causing resistance to antibiotics of other groups. Chemical modification of a ribosomal target by rRNA methylases and mutations in ribosomal proteins are the main resistance mechanisms of bacteria of the family Pasteurellaceae to macrolides. Many gram-negative bacteria have a natural resistance to macrolide antibiotics. The development of lincosamide resistance is influenced by methyltransferase 23S rRNA, active efflux proteins, enzymatic inactivation and chromosomal mutations. Resistance of bacteria of the family Pasteurellaceae to chloramphenicol is caused mainly by enzymatic inactivation, while the emergence of resistance to fluorophenicol is associated with the efflux of an antibiotic from a bacterial cell. Plasmids carrying phenicol resistance genes were detected in isolates of P. multocida, M. haemolytica, A. pleuropneumoniae and H. parasuis. Usually the level of bacteria sensitivity of the genus Pasteurella, Mannheimia, Actinobacillus and Haemophilus to quinolones is quite high. Resistance to quinolones mainly occurs due to mutational alterations in chromosomal genes, and may also be in consequence of the export antibiotics from the cell by membrane proteins or thanks to qnr genes of plasmids. The main mechanism of resistance to sulfonamides and trimethoprim is both plasmid-mediated and mutation-induced production of altered dihydropteroate synthetase and dihydrofolate reductase with reduced affinity with these antimicrobials. Monitoring of antibiotic resistance with the determination of its mechanism phenomenon will facilitate the choice of an effective agent of etiotropic therapy of respiratory diseases of cattle and pigs caused by bacteria of the family Pasteurellaceae.

Clinically relevant mutations in core metabolic genes confer antibiotic resistance

Although metabolism plays an active role in antibiotic lethality, antibiotic resistance is generally associated with drug target modification, enzymatic inactivation, and/or transport rather than metabolic processes. Evolution experiments of Escherichia coli rely on growth-dependent selection, which may provide a limited view of the antibiotic resistance landscape. We sequenced and analyzed E. coli adapted to representative antibiotics at increasingly heightened metabolic states. This revealed various underappreciated noncanonical genes, such as those related to central carbon and energy metabolism, which are implicated in antibiotic resistance. These metabolic alterations lead to lower basal respiration, which prevents antibiotic-mediated induction of tricarboxylic acid cycle activity, thus avoiding metabolic toxicity and minimizing drug lethality. Several of the identified metabolism-specific mutations are overrepresented in the genomes of >3500 clinical E. coli pathogens, indicating clinical relevance.

Effect of High Pressure Processing on enzymatic activity for strawberries, sour cherries and red grapes

Color degradation is an important factor that affect the quality and acceptability of fruit juices and purees; several enzymes, as well as the microbial endogenous population are not only responsible for this phenomenon but for changes in flavor and texture. Traditional stabilization methods have been used to preserve these kind of products; however, there is a negative impact on vitamins and bioactive compounds composition. High Pressure Processing (HPP) is a non-thermal alternative that has been applied for the extension of shelf life of fresh products, reducing the adverse effects of classical treatments. The aim of this review is to provide a scientific base on the effect of HPP technology in terms of enzymatic inactivation (peroxidase, polyphenol oxidase, ascorbate oxidase and β-glucosidase) in comparison with a conventional pasteurization process in strawberries, sour cherries and red grapes, and to propose an optimization strategy for the operational parameters to achieve the greatest inactivation

Permeability evaluation of gemcitabine-CPP6 conjugates in Caco-2 cells

Cancer is one of the most alarming diseases due to its high mortality and still increasing incidence rate. Currently available treatments for this condition present several shortcomings and new options are continuously being developed and evaluated, aiming at increasing the overall treatment efficiency and reducing associated adverse side effects. Gemcitabine has proven activity and is used in chemotherapy. However, its therapeutic efficiency is limited by its low bioavailability as a result of rapid enzymatic inactivation. Additionally, tumor cells often develop drug resistance after initial tumor regression related to transporter deficiency. We have previously developed three gemcitabine conjugates with cell-penetrating hexapeptides (CPP6) to facilitate intracellular delivery of this drug while also preventing enzymatic deamination. The bioactivity of these new prodrugs was evaluated in different cell lines and showed promising results. Here, we assessed the absorption and permeability across Caco-2 monolayers of these conjugates in comparison with gemcitabine and the respective isolated cell-penetrating peptides (CPPs). CPP6-2 (KLPVMW) and respective Gem-CPP6-2 conjugate showed the highest permeability in Caco-2 cells.