Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

AIMS: Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. METHODS AND RESULTS: In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35 kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P < 0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P < 0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. CONCLUSIONS: These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

A hypothetical adhesin protein induces anti-biofilm antibodies against multi-drug resistant Acinetobacter baumannii.

The increase in multidrug-resistant (MDR) Acinetobacter baumannii strains in hospital environments has generated great concern around the world. Biofilm is one of the forms of bacterial adaptation that is increasingly leading to antimicrobial resistance and therapeutic failure. The search for alternative therapeutic strategies, especially non-antibiotic-based, is urgently needed. In this study, we produce polyclonal antibodies (pAbs) in murine models against recombinant CAM87009.1 antigen, an A. baumannii fimbriae protein. The pAbs produced were isotyped and anti-biofilm activity evaluated in the A. baumannii ATCC® 19606 standard strain and nine MDR clinical isolates. All clinical isolates were analyzed for the presence of the cam87009.1 gene using the PCR technique, and one of the isolates did not have the gene in its genome. After four intraperitoneal immunizations (days 0, 14, 21, and 28) of mice with rCAM87009.1 and Freund's adjuvant, a significant antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA) since the first immunization (1:6400), and the level increased until the 4th immunization (1:819,200). IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 isotypes were identified in the serum of immunized mice (P < 0.001). The anti-rCAM87009.1 pAb was able to inhibit biofilm formation in 80 % of the strains evaluated in this study, including the ATCC® 19606 strain. The rCAM87009.1 proves to be a promising target in the development of alternative strategies to control biofilm-forming in A. baumannii MDR strains.

Antibodies anti-rFilF protein has anti-biofilm activity against carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic bacterium that causes infection in several sites. Carbapenem-resistant A. baumannii strains (CRAb) lead the World Health Organization's list of 12 pathogens considered a priority for developing new antimicrobials. The pathogenicity of A. baumannii is related to the different virulence factors employed in the colonization of biotic and abiotic surfaces, biofilm formation and multidrug resistance. We analyze the outer membrane protein FilF from A. baumannii in silico and produce it in recombinant form (rFilF). rFilF protein was successfully expressed in Escherichia coli BL21 Star in an insoluble form. Immunization with rFilF induced significant anti-rFilF IgG antibody production in mice, detected by indirect enzyme-linked immunosorbent assay, since the first evaluation until 49th. On the last experimentation day, the predominant immunoglobulin found was IgG1 followed by IgG2a, IgG2b, IgM, IgG3, and IgA. We observe that interleukins 4 and 10 show significant production after the 28th day of experimentation in mice immunized with rFilF. Anti-rFilF pAbs were able to inhibit biofilm formation in nine CRAb strains evaluated, and in the standard strain ATCC® 19606. These results demonstrate the anti-biofilm activity of anti-rFilF antibodies, promising in the development of a non-antibiotic approach based on the control of CRAb strains.

Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil.

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult.Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil.Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method.Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers.Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.