ß-caryophyllene and docosahexaenoic acid, isolated or associated, have potential antinociceptive and anti-inflammatory effects in vitro and in vivo.

Inflammation is a complex biological response involving the immune, autonomic, vascular, and somatosensory systems that occurs through the synthesis of inflammatory mediators and pain induction by the activation of nociceptors. Staphylococcus aureus, the main cause of bacteremia, is one of the most common and potent causes of inflammation in public health, with worse clinical outcomes in hospitals. Antioxidant substances have been evaluated as alternative therapeutic analgesics, antioxidants, anti-inflammatory agents, antitumor agents, and bactericides. Among these, we highlight the essential oils of aromatic plants, such as ß-caryophyllene (BCP), and polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). The objective of this study was to evaluate the biological activities of BCP-DHA association in in vitro and in vivo experimental models of antinociception and inflammation. To determine the anti-inflammatory effects, monocytes isolated from the peripheral blood of adult male volunteers were infected with methicillin-resistant S. aureus and incubated with treatment for cytokine dosage and gene expression analysis. Antinociceptive effects were observed in the three models when comparing the control (saline) and the BCP-DHA treatment groups. For this purpose, the antinociceptive effects were evaluated in animal models using the following tests: acetic acid-induced abdominal writhing, paw edema induced by formalin intraplantar injection, and von Frey hypernociception. There was a significant reduction in the GM-CSF, TNFα, IL-1, IL-6, and IL-12 levels and an increase in IL-10 levels in the BCP-DHA treatment groups, in addition to negative regulation of the expression of the genes involved in the intracellular inflammatory signaling cascade (IL-2, IL-6, IRF7, NLRP3, and TYK2) in all groups receiving treatment, regardless of the presence of infection. Statistically significant results (p < 0.05) were obtained in the acetic acid-induced abdominal writhing test, evaluation of paw edema, evaluation of paw flinching and licking in the formalin intraplantar injection model, and the von Frey hypernociception test. Therefore, BCP and DHA, either administered individually or combined, demonstrate potent anti-inflammatory and antinociceptive effects.

Heterologous Expression, Purification, and Immunomodulatory Effects of Recombinant Lipoprotein GUDIV-103 Isolated from Ureaplasma diversum.

Ureaplasma diversum is a bacterial pathogen that infects cattle and can cause severe inflammation of the genital and reproductive systems. Lipid-associated membrane proteins (LAMPs), including GUDIV-103, are the main virulence factors in this bacterium. In this study, we heterologously expressed recombinant GUDIV-103 (rGUDIV-103) in Escherichia coli, purified it, and evaluated its immunological reactivity and immunomodulatory effects in bovine peripheral blood mononuclear cells (PBMCs). Samples from rabbits inoculated with purified rGUDIV-103 were analysed using indirect enzyme-linked immunosorbent assay and dot blotting to confirm polyclonal antibody production and assess kinetics, respectively. The expression of this lipoprotein in field isolates was confirmed via Western blotting with anti-rGUDIV-103 serum and hydrophobic or hydrophilic proteins from 42 U. diversum strains. Moreover, the antibodies produced against the U. diversum ATCC 49783 strain recognised rGUDIV-103. The mitogenic potential of rGUDIV-103 was evaluated using a lymphoproliferation assay in 5(6)-carboxyfluorescein diacetate succinimidyl ester−labelled bovine PBMCs, where it induced lymphocyte proliferation. Quantitative polymerase chain reaction analysis revealed that the expression of interleukin-1ß, toll-like receptor (TLR)-α, TLR2, TLR4, inducible nitric oxide synthase, and caspase-3−encoding genes increased more in rGUDIV-103−treated PBMCs than in untreated cells (p < 0.05). Treating PBMCs with rGUDIV-103 increased nitric oxide and hydrogen peroxide levels. The antigenic and immunogenic properties of rGUDIV-103 suggested its suitability for immunobiological application.

Streptococcus agalactiae: Identification methods, antimicrobial susceptibility, and resistance genes in pregnant women.

BACKGROUND: Group B Streptococcus (GBS) is a normal component of the gastrointestinal and genital microbiota in humans and can lead to important infections in newborns. AIM: To compare GBS isolation and identification methods as well as to assess the antibiotic susceptibility and to identify resistance genes in GBS strains from pregnant women attended in healthcare services from the city of Vitória da Conquista, in Bahia State, Brazil. METHODS: From January 2017 to February 2018, vaginorectal swabs were obtained from 186 participants and the samples were seeded onto chromogenic agar for GBS before and after inoculation in selective broth. Confirmatory identification using 3 CAMP and latex tests was performed in samples with GBS-suggestive colonies. Then, disk diffusion antibiograms were performed in GBS-positive samples, and the detection of the resistance genes ermB, ermTR, mefA, and linB in the clindamycin and/or erythromycin-resistant samples was carried out. RESULTS: Thirty-two samples (17.2%) were GBS-positive. The culture in chromogenic agar after sample incubation in selective broth was the most sensitive method (96.9%) for GBS detection. All isolates were susceptible to penicillin, ampicillin, cefotaxime, and vancomycin. Clindamycin resistance was observed in 6 samples (18.8%), while 8 samples (25%) were erythromycin-resistant. All erythromycin and/or clindamycin-resistant GBS strains had negative D-tests. Two strains (25%) presented an M phenotype and 6 isolates (75%) presented a cMLSB phenotype. The ermB gene was identified in 4 samples (44.4%), the mefA gene was also found in 4 samples (44.4%), the ermTR gene was identified in 1 isolate (11.1%), and the linB gene was not found in any isolate. CONCLUSION: This study evidenced that the screening for SGB can be performed by means of various methods, including chromogenic media, and that the chemoprophylaxis for pregnant women who cannot use penicillin must be susceptibility-guided.

Immunoinformatics and analysis of antigen distribution of Ureaplasma diversum strains isolated from different Brazilian states.

BACKGROUND: Ureaplasma diversum has numerous virulence factors that contribute to pathogenesis in cattle, including Lipid-associated membrane proteins (LAMPs). Therefore, the objectives of this study were to evaluate in silico important characteristics for immunobiological applications and for heterologous expression of 36 LAMPs of U. diversum (UdLAMPs) and, also, to verify by conventional PCR the distribution of these antigens in strains of Brazilian states (Bahia, Minas Gerais, São Paulo, and Mato Grosso do Sul). The Manatee database was used to obtain the gene and peptide sequences of the antigens. Similarity and identity studies were performed using BLASTp and direct antigenicity was evaluated by the VaxiJen v2.0 server. Epitope prediction for B lymphocytes was performed on the BepiPred v2.0 and CBTOPE v1.0 servers. NetBoLApan v1.0 was used to predict CD8+ T lymphocyte epitopes. Subcellular location and presence of transmembrane regions were verified by the software PSORTb v3.0.2 and TMHMM v2.2 respectively. SignalP v5.0, SecretomeP v2.0, and DOLOP servers were used to predict the extracellular excretion signal. Physico-chemical properties were evaluated by the web-software ProtParam, Solpro, and Protein-sol. RESULTS: In silico analysis revealed that many UdLAMPs have desirable properties for immunobiological applications and heterologous expression. The proteins gudiv_61, gudiv_103, gudiv_517, and gudiv_681 were most promising. Strains from the 4 states were PCR positive for antigens predicted with immunogenic and/or with good characteristics for expression in a heterologous system. CONCLUSION: These works contribute to a better understanding of the immunobiological properties of the UdLAMPs and provide a profile of the distribution of these antigens in different Brazilian states.

Identification and characterization of resistance and pathogenicity of Enterococcus spp. in samples of donor breast milk.

BACKGROUND: Breast milk is the primary source of nutrition for newborns. Hospitalized babies frequently need nutritional support from Human Milk Banks. As bacterial species of the genus Enterococcus are part of the microbiota of healthy donors, they may contaminate samples of pumped breast milk. AIM: To identify and characterize the bacterial virulence and resistance in samples isolated from the nipple-areolar region, hands, and breast milk aliquots from donors at the Human Milk Bank of Municipal Hospital Esaú Matos in the city of Vitória da Conquista, Bahia State, Brazil. METHODS: The personal hygiene and sanitation of donors were analyzed with the aim of identifying possible reasons for contamination of pumped milk. Cutaneous samples as well as aliquots of unpasteurized and pasteurized milk from 30 participants were obtained. Each Enterococcus spp. isolate underwent a disk diffusion susceptibility test and molecular biology techniques to determine resistance and virulence genes. RESULTS: Enterococcus spp. were identified in 30% of donors (n = 9), and 11 specimens were isolated. Resistance to tetracycline was highly prevalent, being detectable in 63% of the isolates (n = 7) and followed by intermediate sensitivity to ciprofloxacin, observed in 27% of the specimens (n = 3). The efaA gene was found in 63% (n = 7) of the isolates, while the ace gene was detected in 27% (n = 3). CONCLUSION: This study illustrates the importance of microbiological monitoring by Human Milk Banks and the need for alternatives to prevent the presence of Enterococcus spp. in hospital settings.

Anti-inflammatory activity of ß-caryophyllene combined with docosahexaenoic acid in a model of sepsis induced by Staphylococcus aureus in mice.

BACKGROUND: Sepsis is a set of serious organic manifestations caused by an infection, whose progression culminates in exacerbated inflammation and oxidative stress, poor prognosis, and high hospital costs. Antioxidants used against sepsis have been evaluated, including essential oils such as ß-caryophyllene (BCP), and polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). The aim of this study was to evaluate the anti-inflammatory activity of the association of these two compounds. RESULTS: Treatment with BCP-DHA, at a dose of 200 µL/animal, significantly inhibited the migration of neutrophils in a Cg-induced peritonitis model. After Staphylococcus aureus infection, in the groups treated with BCP-DHA there was a significant decrease in the total and differential count of leukocytes, increased expression of cytokines TNF-α and IFN-γ in treated groups, an increase of IL-4 and IL-5 in B/D and B/D + SA groups, and an augmentation of IL-6 and IL-12 groups in B/D + SA groups. Histological and bacterial analysis revealed lower neutrophil migration and lower bacterial load in the infected and treated groups. CONCLUSION: In general, the BCP-DHA association presented anti-inflammatory activity against two different models of acute inflammation and infection, showing promising potential as a therapeutic adjuvant in sepsis. © 2019 Society of Chemical Industry.