Evaluating virulence features of Acinetobacter baumannii resistant to polymyxin B.

The increasing resistance to polymyxins in Acinetobacter baumannii has made it even more urgent to develop new treatments. Anti-virulence compounds have been researched as a new solution. Here, we evaluated the modification of virulence features of A. baumannii after acquiring resistance to polymyxin B. The results showed lineages attaining unstable resistance to polymyxin B, except for Ab7 (A. baumannii polymyxin B resistant lineage), which showed stable resistance without an associated fitness cost. Analysis of virulence by a murine sepsis model indicated diminished virulence in Ab7 (A. baumannii polymyxin B resistant lineage) compared with Ab0 (A. baumannii polymyxin B susceptible lineage). Similarly, downregulation of virulence genes was observed by qPCR at 1 and 3 h of growth. However, an increase in bauE, abaI, and pgAB expression was observed after 6 h of growth. Comparison analysis of Ab0, Ab7, and Pseudomonas aeruginosa suggested no biofilm formation by Ab7. In general, although a decrease in virulence was observed in Ab7 when compared with Ab0, some virulence feature that enables infection could be maintained. In light of this, virulence genes bauE, abaI, and pgAB showed a potential relevance in the maintenance of virulence in polymyxin B-resistant strains, making them promising anti-virulence targets.

Expanding therapeutic potential of Bdellovibrio bacteriovorus against multidrug-resistant pathogens.

Novel treatments toward Gram-negative bacteria are urgently needed to prevent even higher mortality levels associated with resistant bacterial infections. Predatory bacteria have been studied as a new type of treatment against pathogenic bacteria, including resistant species. However, because of limitations related to eradication efficacy, combination therapy using predatory bacteria with other agents has also been tested. Here, we discuss recent advances in the use of predatory bacteria to treat infections and propose novel combinatory strategies with antivirulence compounds.

Recent Advances in Anti-virulence Therapeutic Strategies With a Focus on Dismantling Bacterial Membrane Microdomains, Toxin Neutralization, Quorum-Sensing Interference and Biofilm Inhibition.

Antimicrobial resistance constitutes one of the major challenges facing humanity in the Twenty-First century. The spread of resistant pathogens has been such that the possibility of returning to a pre-antibiotic era is real. In this scenario, innovative therapeutic strategies must be employed to restrict resistance. Among the innovative proposed strategies, anti-virulence therapy has been envisioned as a promising alternative for effective control of the emergence and spread of resistant pathogens. This review presents some of the anti-virulence strategies that are currently being developed, it will cover strategies focused on quench pathogen quorum sensing (QS) systems, disassemble of bacterial functional membrane microdomains (FMMs), disruption of biofilm formation and bacterial toxin neutralization.

Metodología para la obtención y evaluación de un extracto proteínico de una cepa autóctona de Helicobacter pylori / Methodology to obtain and evaluate a protein extract of an autochthonous Helicobacter pylori strain

Helicobacter pylori es una bacteria gram negativa que posee numerosos antígenos que juegan un importante papel en la patogénesis de las enfermedades gastroduodenales. Debido a la necesidad de métodos de diagnóstico estandarizados con antígenos locales u autóctonos nos propusimos el diseño de una estrategia para la obtención de extractos de antígenos con reactividad frente a sueros de pacientes infectados por H. pylori. Dos cepas de H. pylori, una autóctona (IPK196A) y una de referencia ATCC 43504, se cultivaron en un medio líquido modificado. Se sometieron a los protocolos de ruptura por ultrasonido aplicándose tres variantes de precipitación y al fraccionamiento celular mediante ultracentrifugación diferencial. Los extractos proteínicos se visualizaron mediante electroforesis en gel de poliacrilamida y se transfirieron para la detección de antígenos inmunorreactivos a sueros de pacientes con infección por H. pylori e individuos sanos. La variante de ultrasonido y precipitación con Coomasie fue la más efectiva para concentrar las muestras. El método de ultracentrifugación mejoró la resolución de las proteínas reactivas y permitió separarlas según su localización subcelular. El sistema de transferencia húmedo fue ideal para la inmunodetección de los antígenos obtenidos por ultrasonido mientras que el sistema semiseco permitió detectar las proteínas de membrana obtenidas por ultracentrifugación diferencial. La introducción de una metodología en el laboratorio para la obtención y evaluación de extractos proteínicos antigénicos a partir de cepas autóctonas de H. pylori, constituye la antesala para el diseño de futuros diagnosticadores y candidatos vacunales(AU)

Helicobacter pylori is a gram-negative spiral-shaped bacterium, which has many antigens that play an important role in the pathogenesis of gastroduodenal diseases. Due to the lack of standardized methods from native or autochthonous antigens, we proposed in this study, the design of a strategy for extracting and obtaining immunoreactive antigens against H. pylori infected-patient sera. Two H. pylori strains, one autochthonous (IPK196A) and one reference ATCC 43504, were cultured in a modified liquid medium. Both strains were subjected to the ultrasound rupture protocols applying three precipitation variants and cell fractionation by differential ultracentrifugation. Protein extracts were visualized by polyacrylamide gel electrophoresis and transferred for the detection of immunoreactive antigens to sera from patients with H. pylori infection and healthy individuals. The precipitation with Coomasie was the most effective variant. The ultracentrifugation extraction method optimized the resolution of the proteins, which could be separated according to their subcellular location. The wet transfer system was ideal for the immunodetection of the antigens obtained by ultrasound, while the semi-dry system allowed detecting the membrane proteins by differential ultracentrifugation. The introduction of a methodology in the laboratory for obtaining and evaluating antigenic antibodies from autochthonous strains of H. pylori, is the prelude to the design for future diagnostics and vaccine candidates(AU)

Interference With Quorum-Sensing Signal Biosynthesis as a Promising Therapeutic Strategy Against Multidrug-Resistant Pathogens.

Faced with the global health threat of increasing resistance to antibiotics, researchers are exploring interventions that target bacterial virulence factors. Quorum sensing is a particularly attractive target because several bacterial virulence factors are controlled by this mechanism. Furthermore, attacking the quorum-sensing signaling network is less likely to select for resistant strains than using conventional antibiotics. Strategies that focus on the inhibition of quorum-sensing signal production are especially attractive because the enzymes involved are expressed in bacterial cells but are not present in their mammalian counterparts. We review here various approaches that are being taken to interfere with quorum-sensing signal production via the inhibition of autoinducer-2 synthesis, PQS synthesis, peptide autoinducer synthesis, and N-acyl-homoserine lactone synthesis. We expect these approaches will lead to the discovery of new quorum-sensing inhibitors that can help to stem the tide of antibiotic resistance.

Metodología para la obtención y evaluación de un extracto proteínico de una cepa autóctona de Helicobacter pylori / Methodology to obtain and evaluate a protein extract of an autochthonous Helicobacter pylori strain

Helicobacter pylori es una bacteria gram negativa que posee numerosos antígenos que juegan un importante papel en la patogénesis de las enfermedades gastroduodenales. Debido a la necesidad de métodos de diagnóstico estandarizados con antígenos locales u autóctonos nos propusimos el diseño de una estrategia para la obtención de extractos de antígenos con reactividad frente a sueros de pacientes infectados por H. pylori. Dos cepas de H. pylori, una autóctona (IPK196A) y una de referencia ATCC 43504, se cultivaron en un medio líquido modificado. Se sometieron a los protocolos de ruptura por ultrasonido aplicándose tres variantes de precipitación y al fraccionamiento celular mediante ultracentrifugación diferencial. Los extractos proteínicos se visualizaron mediante electroforesis en gel de poliacrilamida y se transfirieron para la detección de antígenos inmunorreactivos a sueros de pacientes con infección por H. pylori e individuos sanos. La variante de ultrasonido y precipitación con Coomasie fue la más efectiva para concentrar las muestras. El método de ultracentrifugación mejoró la resolución de las proteínas reactivas y permitió separarlas según su localización subcelular. El sistema de transferencia húmedo fue ideal para la inmunodetección de los antígenos obtenidos por ultrasonido mientras que el sistema semiseco permitió detectar las proteínas de membrana obtenidas por ultracentrifugación diferencial. La introducción de una metodología en el laboratorio para la obtención y evaluación de extractos proteínicos antigénicos a partir de cepas autóctonas de H. pylori, constituye la antesala para el diseño de futuros diagnosticadores y candidatos vacunales(AU)

Helicobacter pylori is a gram-negative spiral-shaped bacterium, which has many antigens that play an important role in the pathogenesis of gastroduodenal diseases. Due to the lack of standardized methods from native or autochthonous antigens, we proposed in this study, the design of a strategy for extracting and obtaining immunoreactive antigens against H. pylori infected-patient sera. Two H. pylori strains, one autochthonous (IPK196A) and one reference ATCC 43504, were cultured in a modified liquid medium. Both strains were subjected to the ultrasound rupture protocols applying three precipitation variants and cell fractionation by differential ultracentrifugation. Protein extracts were visualized by polyacrylamide gel electrophoresis and transferred for the detection of immunoreactive antigens to sera from patients with H. pylori infection and healthy individuals. The precipitation with Coomasie was the most effective variant. The ultracentrifugation extraction method optimized the resolution of the proteins, which could be separated according to their subcellular location. The wet transfer system was ideal for the immunodetection of the antigens obtained by ultrasound, while the semi-dry system allowed detecting the membrane proteins by differential ultracentrifugation. The introduction of a methodology in the laboratory for obtaining and evaluating antigenic antibodies from autochthonous strains of H. pylori, is the prelude to the design for future diagnostics and vaccine candidates(AU)

Enzymatic analysis of venom from Cuban scorpion Rhopalurus junceus.

Rhopalurus junceus scorpion venom has been identified as a natural extract with anticancer potential. Interestingly, this scorpion venom does not cause adverse symptoms in humans. However, there is scarce information about its composition and enzymatic activity. In this work, we determined the electrophoretic profile of the venom, the gelatinase and caseinolytic activity, and the phospholipase A2 (PLA2) and hemolytic activity. The effect of different venom doses (6.25, 12.5 and 25 mg/kg) on gastrocnemius muscle was also measured as CK and LDH activity in serum. The presence of hyaluronidase was determined by turbidimetric assay. The effect of different fractions obtained by gel filtration chromatography were evaluated at different concentrations (0.05, 0.1, 0.2, 0.4, 0.6mg/ml) against lung cancer cell A549 and lung normal cell MRC-5 using MTT assay. The electrophoretic profile demonstrated the presence of proteins bands around 67kDa, 43kDa, 18.4kDa and a majority band below 14.3kDa. The venom did not showed caseinolytic, gelatinase, PLA2 and hemolytic activity even at highest venom concentration used in the study. Scorpion venom only showed a significant toxic effect on gastrocnemius muscles identified by CK and LDH release after subcutaneous injection of 12.5 and 25mg/kg. Low molecular weight fractions (<4kDa) induced a significant cytotoxicity in A549 cells while high molecular weight proteins (45-60kDa) were responsible for hyaluronidase activity and toxic effect against MRC-5. Experiments indicate that Rhopalurus junceus scorpion venom has low enzymatic activity, which could contribute to the low toxic potential of this scorpion venom.