The Brucella abortus two-component system response regulator BvrR binds to three DNA regulatory boxes in the upstream region of omp25.

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and ß-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales.

Increased ompW and ompA expression and higher virulence of Acinetobacter baumannii persister cells.

BACKGROUND: Acinetobacter baumannii is one of the main causes of healthcare-associated infections that threaten public health, and carbapenems, such as meropenem, have been a therapeutic option for these infections. Therapeutic failure is mainly due to the antimicrobial resistance of A. baumannii, as well as the presence of persister cells. Persisters constitute a fraction of the bacterial population that present a transient phenotype capable of tolerating supra-lethal concentrations of antibiotics. Some proteins have been suggested to be involved in the onset and/or maintenance of this phenotype. Thus, we investigated the mRNA levels of the adeB (AdeABC efflux pump component), ompA, and ompW (outer membrane proteins) in A. baumannii cells before and after exposure to meropenem. RESULTS: We found a significant increase (p-value < 0.05) in the expression of ompA (> 5.5-fold) and ompW (> 10.5-fold) in persisters. However, adeB did not show significantly different expression levels when comparing treated and untreated cells. Therefore, we suggest that these outer membrane proteins, especially OmpW, could be part of the mechanism of A. baumannii persisters to deal with the presence of high doses of meropenem. We also observed in the Galleria mellonella larvae model that persister cells are more virulent than regular ones, as evidenced by their LD50 values. CONCLUSIONS: Taken together, these data contribute to the understanding of the phenotypic features of A. baumannii persisters and their relation to virulence, as well as highlight OmpW and OmpA as potential targets for drug development against A. baumannii persisters.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Immunopotential of Pasteurella multocida bivalent outer membrane protein-based vaccine entrapped in aluminum hydroxide nanoparticles.

Swine pasteurellosis is one of the most economically important diseases of pig caused by Pasteurella multocida (P. multocida) capsular types A and D. These organisms are commensals and opportunistic pathogens in the upper respiratory tract in pig. In the present study, we extracted whole outer membrane proteins (OMP) from P. multocida capsular types A and D and were mixed together in the ratio of 1:1 forming bivalent outer-membrane proteins. The bivalent OMP was adsorbed onto aluminum hydroxide nanoparticles. The size of aluminum hydroxide nanoparticles adsorbed outer membrane protein was found to be in the range of 125 to 130 nm. We observed that aluminum hydroxide nanoparticles adjuvanted bivalent OMP-based vaccine elicited quicker immune kinetics in terms of IgG response as compared to aluminum hydroxide microparticles adjuvanted bivalent bacterin vaccine against P. multocida capsular type A and D.

Aggregatibacter actinomycetemcomitans Outer Membrane Proteins 29 and 29 Paralogue Induce Evasion of Immune Response.

Aggregatibacter actinomycetemcomitans (Aa) is abundant within the microbial dysbiotic community of some patients with periodontitis. Aa outer membrane protein 29 (OMP29), a member of the OMPA family, mediates the invasion of Aa to gingival epithelial cells (GECs). This study evaluated the effect of OMP29 and its paralogue OMP29par on the response of GECs to Aa. The omp29 or/and omp29 par deletion mutants AaΔ29, AaΔ29P, and AaΔ29Δ29P were constructed, and recombinant Aa OMP29His was obtained. Microarray analysis and the evaluation of cxcl-8 gene expression were performed to examine the response of GECs line OBA-09 to Aa and its mutants. The expression of cxcl-8 and its product CXCL-8 was examined in LPS-stimulated OBA-09 cells with Aa OMP29His. Proteomics analysis showed that the deletion of omp29 led to overexpression of both OMP29par and another membrane protein OMP39, the expression of which was further increased in AaΔ29Δ29P. OBA-09 cells challenged with AaΔ29Δ29P exhibited a higher expression of cxcl-8 in comparison to wildtype Aa strain AaD7S or single-deletion mutants AaΔ29 or AaΔ29P. LPS-stimulated OBA-09 cells challenged with Aa OMP29His showed reduced expressions of cxcl-8 and its product CXCL-8. OBA-09 cells challenged with AaΔ29Δ29P in comparison to Aa strain AaD7S resulted in higher expressions of genes involved in apoptosis and inflammatory response such as bcl2, birc3, casp3, c3, ep300, fas, fosb, grb2, il-1α, il-1ß, il-6, cxcl-8, nr3c1, prkcq, socs3, and tnfrsf1ß and reduced expressions of cd74, crp, faslg, tlr1, and vcam1. The results suggested a novel strategy of Aa, mediated by OMP29 and OMP29par, to evade host immune response by inhibiting CXCL-8 expression and modulating the genes involved in apoptosis and inflammatory response in GECs. Pending further confirmation, the strategy might interfere with the recruitment of neutrophils and dampen the host inflammatory response, leading to a more permissive subgingival niche for bacterial growth.

Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl ß-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Genetic and molecular Omp25 analyses from worldwide Brucella canis strains: Possible mutational influences in protein function.

Brucella canis is responsible for canine brucellosis, a neglected zoonotic disease. The omp25 gene has been described as an important marker for Brucella intra-species differentiation, in addition to the ability to interact with the host immune system. Therefore, this study investigated the omp25 sequence from B. canis strains associated to a phylogenetic characterization and the unveiling of the molecular structure. In vitro analyses comprised DNA extraction, PCR, and sequencing of omp25 from 19 B. canis strains. Moreover, in silico analyses were performed at nucleotide level for phylogenetic characterization and evolutionary history of B. canis omp25 gene; and in amino acid level including modeling, dynamics, and epitope prediction of B. canis Omp25 protein. Here, we identified a new mutation, L109P, which diverges the worldwide omp25 sequences in two large branches. Interestingly, this mutation appears to have epidemiology importance, based on a geographical distribution of B. canis strains. Structural and molecular dynamics analyses of Omp25 revealed that Omp25L109P does not sustain its native ß-barrel. Likewise, the conformation of B-cell epitope on the mutated region was changed in Omp25L109P protein. Even without an evolutive marker, the new identified mutation appears to affect the basic function of B. canis Omp25 protein, which could indicate virulence adaptation for some B. canis strains in a context of geographical disposition.

Adhesive Functions or Pseudogenization of Type Va Autotransporters in Brucella Species.

Adhesion to host cells is a key step for successful infection of many bacterial pathogens and may define tropism to different host tissues. To do so, bacteria display adhesins on their surfaces. Brucella is an intracellular pathogen capable of proliferating in a wide variety of cell types. It has been described that BmaC, a large protein that belongs to the classical (type Va) autotransporter family, is required for efficient adhesion of Brucella suis strain 1330 to epithelial cells and fibronectin. Here we show that B. suis 1330 harbors two other type Va autotransporters (BmaA and BmaB), which, although much smaller, share significant sequence similarities with BmaC and contain the essential domains to mediate proper protein translocation to the bacterial surface. Gain and loss of function studies indicated that BmaA, BmaB, and BmaC contribute, to a greater or lesser degree, to adhesion of B. suis 1330 to different cells such as synovial fibroblasts, osteoblasts, trophoblasts, and polarized epithelial cells as well as to extracellular matrix components. It was previously shown that BmaC localizes to a single bacterial pole. Interestingly, we observed here that, similar to BmaC, the BmaB adhesin is localized mostly at a single cell pole, reinforcing the hypothesis that Brucella displays an adhesive pole. Although Brucella species have strikingly similar genomes, they clearly differ in their host preferences. Mainly, the differences identified between species appear to be at loci encoding surface proteins. A careful in silico analysis of the putative type Va autotransporter orthologues from several Brucella strains showed that the bmaB locus from Brucella abortus and both, the bmaA and bmaC loci from Brucella melitensis are pseudogenes in all strains analyzed. Results reported here evidence that all three autotransporters play a role in the adhesion properties of B. suis 1330. However, Brucella spp. exhibit extensive variations in the repertoire of functional adhesins of the classical autotransporter family that can be displayed on the bacterial surface, making them an interesting target for future studies on host preference and tropism.

Microevolution in the major outer membrane protein OmpA of Acinetobacter baumannii.

Acinetobacter baumannii is nowadays a relevant nosocomial pathogen characterized by multidrug resistance (MDR) and concomitant difficulties to treat infections. OmpA is the most abundant A. baumannii outer membrane (OM) protein, and is involved in virulence, host-cell recognition, biofilm formation, regulation of OM stability, permeability and antibiotic resistance. OmpA members are two-domain proteins with an N-terminal eight-stranded ß-barrel domain with four external loops (ELs) interacting with the environment, and a C-terminal periplasmic domain binding non-covalently to the peptidoglycan. Here, we combined data from genome sequencing, phylogenetic and multilocus sequence analyses from 975 strains/isolates of the Acinetobacter calcoaceticus/Acinetobacter baumannii complex (ACB), 946 from A. baumannii, to explore ompA microevolutionary divergence. Five major ompA variant groups were identified (V1 to V5) in A. baumannii, encompassing 52 different alleles coding for 23 different proteins. Polymorphisms were concentrated in five regions corresponding to the four ELs and the C-terminal end, and provided evidence for intra-genic recombination. ompA variants were not randomly distributed across the A. baumannii phylogeny, with the most frequent V1(lct)a1 allele found in most clonal complex 2 (CC2) strains and the second most frequent V2(lct)a1 allele in the majority of CC1 strains. Evidence was found for assortative exchanges of ompA alleles not only between separate A. baumannii lineages, but also different ACB species. The overall results have implications for A. baumannii evolution, epidemiology, virulence and vaccine design.

Temporal evolution of Acinetobacter baumannii ST107 clone: conversion of blaOXA-143 into blaOXA-231 coupled with mobilization of ISAba1 upstream occAB1.

Nine carbapenem-resistant Acinetobacter baumannii isolates carrying blaOXA-231 and an ISAba1 upstream occAB1 were evaluated. They were clonally related and belonged to ST107. An OXA-143-producing A. baumannii ST107 strain (Ac-148) that did not possess ISAba1 upstream occAB1 was included in the analysis. Reduction in the expression of occAB1 and a 4-fold increase of carbapenem MICs were observed for all isolates, except for the Ac-148 strain, probably due to the presence of ISAba1 upstream occAB1 but in the same transcriptional orientation. We reported an A. baumannii ST107 clone carrying blaOXA-143 that acquired a mutation resulting into blaOXA-231 and mobilized ISAba1 upstream occAB1.

Solubilization, Folding, and Purification of a Recombinant Peptidoglycan-Associated Lipoprotein (PAL) Expressed in Escherichia coli.

Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL-enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.

Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology.

Leptospira spp. are diderm (two membranes) bacteria that infect mammals causing leptospirosis, a public health problem with global implications. Thousands of people die every year due to leptospirosis, especially in developing countries with tropical climates. Prophylaxis is difficult due to multiple factors, including the large number of asymptomatic hosts that transmit the bacteria, poor sanitation, increasing numbers of slum dwellers, and the lack of an effective vaccine. Several leptospiral recombinant antigens were evaluated as a replacement for the inactivated (bacterin) vaccine; however, success has been limited. A prospective vaccine candidate is likely to be a surface-related protein that can stimulate the host immune response to clear leptospires from blood and organs. In this study, a comprehensive bioinformatics approach based on reverse and structural vaccinology was applied toward the discovery of novel leptospiral vaccine candidates. The Leptospira interrogans serovar Copenhageni strain L1-130 genome was mined in silico for the enhanced identification of conserved ß-barrel (ßb) transmembrane proteins and outer membrane (OM) lipoproteins. Orthologs of the prospective vaccine candidates were screened in the genomes of 20 additional Leptospira spp. Three-dimensional structural models, with a high degree of confidence, were created for each of the surface-exposed proteins. Major histocompatibility complex II (MHC-II) epitopes were identified, and their locations were mapped on the structural models. A total of 18 ßb transmembrane proteins and 8 OM lipoproteins were identified. These proteins were conserved among the pathogenic Leptospira spp. and were predicted to have epitopes for several variants of MHC-II receptors. A structural and functional analysis of the sequence of these surface proteins demonstrated that most ßb transmembrane proteins seem to be TonB-dependent receptors associated with transportation. Other proteins identified included, e.g., TolC efflux pump proteins, a BamA-like OM component of the ßb transmembrane protein assembly machinery, and the LptD-like LPS assembly protein. The structural mapping of the immunodominant epitopes identified the location of conserved, surface-exposed, immunogenic regions for each vaccine candidate. The proteins identified in this study are currently being evaluated for experimental evidence for their involvement in virulence, disease pathogenesis, and physiology, in addition to vaccine development.

Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

Detección de Chlamydia trachomatis en muestras de exudado endocervical mediante una prueba de diagnóstico rápido y dos técnicas de reacción en cadena de la polimerasa / Detection of Chlamydia trachomatis in endocervical swab using rapid test and two reaction techniques polymerase chain

Introducción: Chlamydia trachomatis es el principal agente bacteriano que produce infecciones de transmisión sexual.Objetivo: detectar la presencia de C. trachomatis utilizando una prueba de diagnóstico rápido y compararla con la reacción en cadena de la polimerasa (RCP).Métodos: se procesaron 50 muestras de exudado endocervical, de mujeres sintomáticas del municipio 10 de Octubre. A las muestras se les aplicó la prueba Chlamy-check-1, un ensayo de RCP del gen del plásmido críptico y una RCP en tiempo real (RCP-TR) de la proteína mayor de la membrana externa (MOMP) de C. trachomatis, que fue utilizada como referencia. Se calculó, sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN).Resultados: de las muestras estudiadas, 44 resultaron positivas por la prueba rápida, mientras que por la RCP del plásmido críptico solo 3 muestras (6 porciento) amplificaron. Al aplicar la RCP-TR, 4 muestras (8 porciento) se confirmaron como positivas, coincidiendo 3 por los tres métodos de diagnóstico. Al evaluar la prueba Chlamy-check-1 frente a la prueba de referencia se observó una sensibilidad de 100 porciento, mientras que la especificidad fue de 13 porciento, así como un VPP de 9,1 porciento y VPN de 100 porciento. Por el contrario, la RCP del plásmido críptico mostró una sensibilidad y especificidad de 75 y 100 porciento, respectivamente; un VPP de 100 porciento y VPN de 97,9 porciento.Conclusiones: se obtuvo diferencia entre los porcentajes de positividad detectados con la prueba rápida, y las técnicas de RCP. La baja especificidad de la prueba rápida indica la necesidad de realizar estudios de evaluación de este estuche diagnóstico.

Introduction: Chlamydia trachomatis is the leading bacterial agent that causes sexually transmitted infections.Objective: to detect the presence of C. trachomatis using a rapid test and compare it with the chain reaction (PCR).Methods: 50 endocervical exudates taken from symptomatic women were processed in Diez de October municipality. The samples were applied the Chlamy-check-1 test, a PCR assay of the cryptic plasmid gene and a real-time PCR (RT-PCR) of major outer membrane protein (MOMP) of C. trachomatis which was used as reference. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive value were calculated.Results: 44 samples were positive by the rapid test, whereas only three samples (6 percent) amplified by cryptic plasmid PCR. Applying RT-PCR, 4 samples (8 percent) were confirmed as positive, 3 samples matched with three diagnostic methods. In assessing the Chlamy-check-1 versus the reference test, 100 percent of sensitivity was observed, while the specificity was 13 percent> Also PPV was 9.1percent and NPV was 100 percent. On the contrary, the cryptic plasmid PCR had 75 and 100 percent of sensitivity and specificity respectively, 100 percent PPV and 97.9 percent NPV.Conclusions: the difference was obtained between the percentages of positivity detected with both the rapid test, and CPR techniques. The low specificity of the rapid test indicates the need for further studies to evaluate this diagnostic kit.

Computational modeling and preliminary iroN, fepA, and cirA gene expression in Salmonella Enteritidis under iron-deficiency-induced conditions.

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (µx = 0.36 h(-1)) and biomass productivity (Px = 0.9 g•h(-1)•L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.

Expression of the Escherichia coli ompW colicin S4 receptor gene is regulated by temperature and modulated by the H-NS and StpA nucleoid-associated proteins.

The OmpW family consists of a ubiquitous group of small outer membrane (OM) ß-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts.

Comparison of microscopic agglutination test and indirect elisa in the diagnosis of bovine leptospirosis / Comparação da soroaglutinação microscópica e elisa indireto para o diagnóstico da leptospirose bovina

The objective of this research was to compare the results obtained by the Microscopic Agglutination Test (MAT) and indirect ELISA using outer membrane proteins (OMP) of the serovar Hardjo as antigen (ELISA/OMP-Hardjo). Ninety-three samples of blood serum from 3 to 6-year-old cattle of both sexes with no history of vaccination for leptospirosis were used. To perform the MAT, live cultures of 14 Leptospira serovars representing 11 serogroups of L. interrogans were used as antigen. Outer membrane proteins (OMP) of the serovar Hardjo (ELISAOMP/ Hardjo) were used as the ELISA antigen. Of the 93 samples tested by MAT, 37 (40%) were positive. None of the samples testing positive in the MAT were negative in the ELISA. Sensitivity was 100% and specificity 64%. A comparison of the MAT and ELISA-OMP/Hardjo tests showed 78% agreement and the Kappa index was 0.58 (p<0.0001). The ELISA-OMP/Hardjo proved to be a sensitive test for the diagnosis of bovine leptospirosis, indicating its potential use as a screening test and for epidemiological studies. The titration results obtained by MAT and by optical density (OD) in the ELISA-OMP/Hardjo test showed a positive correlation, and as the antibody titers in the MAT increased, so did the OD in ELISA, demonstrating the correspondence between the tests evaluated here.

O objetivo desta pesquisa foi comparar os resultados obtidos pela Soroaglutinação Microscópica (SAM) e pelo ELISA indireto empregando proteínas de membrana externa (PME) do sorovar Hardjo como antígeno (ELISA/PME-Hardjo). Utilizou-se 93 amostras de soro sanguíneo de bovinos de ambos os sexos com idade entre três a seis anos e sem histórico de vacinação para leptospirose. Para a realização da SAM utilizou-se como antígeno culturas vivas de 14 sorovares de Leptospira, representando 11 sorogrupos de L. interrogans. Empregou-se como antígeno do ELISA, proteínas de membrana externa (PME) do sorovar Hardjo (ELISA-PME/Hardjo). Das 93 amostras testadas na SAM, 37 (40%) foram positivas. Já o ELISA-PME/Hardjo identificou 57 (62%) positivas e 36 (38%) negativas. Nenhuma amostra positiva na SAM foi negativa no ELISA. A sensibilidade foi de 100% e especificidade 64%. A comparação dos testes de SAM e ELISA-PME/Hardjo demonstrou concordância de 78% e índice Kappa de 0,58 (p<0,0001). O ELISAPME/ Hardjo revelou-se como um teste sensível para o diagnóstico da leptospirose bovina, indicando seu uso potencial como exame de triagem e para estudos epidemiológicos. A correlação dos resultados obtidos pela titulação na SAM e DO no ELISA-PME/Hardjo foi positiva, sendo que a medida que os títulos de anticorpos na SAM aumentaram, a DO no ELISA também aumentou, demonstrando haver correspondência entre os exames avaliados.

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica

OBJECTIVES: To study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. RESULTS: Five Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2-4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gramnegative clinical isolates.

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica.

OBJECTIVES: To study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. RESULTS: Five Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2-4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gram-negative clinical isolates.

Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens / Eficácia de bactéria inativada (bacterina), proteína da membrana externa e extrato de fimbrias no controle de infecção experimental por Salmonella Enteritidis (SE) em galinhas

The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE) phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP) and fimbriae crude extract (FE). The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9) CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were performed for the recovery of shedding SE, and SE was recovered from the liver and spleen. Additionally, antibody titers were measured in the serum by micro-agglutination test. The results indicated that the vaccine produced with bacterin yielded better results and resulted in reduction of fecal shedding and organ invasion by SE after oral challenge, although no vaccine was 100% effective for the control of SE experimental infection.

A eficácia de três vacinas de Salmonella Enteritidis fagotipo 4, produzidas na forma de bacterina, proteínas de membrana externa (OMP) e extrato bruto de fímbrias (FE) foi avaliada para proteção de aves infectadas experimentalmente. As aves foram vacinadas por via intramuscular com duas doses de cada vacina as 12 e 15 semanas de idade e desafiadas com 10(9) UFCs de Salmonella Enteritidis fagotipo 4 às 18 semanas de idade, por via oral. A eficácia foi determinada através do reisolamento da bactéria nas fezes e no fígado e baço, e os anticorpos foram mensurados no soro. Os resultados demonstraram que a vacina produzida com a bacterina foi mais eficaz em comparação às outras vacinas examinadas, para reduzir a excreção fecal e a invasão de órgãos após o desafio por SE.