COVID-19 vaccine confidence in the post-vaccination era: Perceptions among adults with immune-mediated inflammatory diseases.

Objective: Assess the perceived protection afforded by a range of COVID-19 vaccines in immune-mediated inflammatory diseases patients previously vaccinated against SARS-CoV-2. Study design: Survey. Methods: On-line cross-sectional survey aimed at evaluating the perceived protection (and its determinants) afforded by a range of COVID-19 vaccines among immune-mediated inflammatory diseases previously vaccinated for COVID-19. Results: Out of 493 eligible respondents who lived in Brazil, 397 (80.5%) were confident that their primary vaccination series would protect them against severe COVID-19. In multivariate analysis, only overlapping immune-mediated inflammatory diseases remained (negatively) associated with the perception of protection. Conclusions: No influence was found between COVID-19 vaccine types and the perception of protection after initial vaccinations.

Lactobacillus plantarum Lp62 exerts probiotic effects against Gardnerella vaginalis ATCC 49154 in bacterial vaginosis.

The severe side-effects elicited by conventional antibiotic therapy and the recurrence of Bacterial vaginosis-associated bacteria and bacterial resistance have led to the development of novel alternative therapies, among which genital probiotics are widely used. In this study, we aimed to evaluate the antimicrobial activities of Lactobacillus plantarum Lp62 and its supernatant against Gardnerella vaginalis, using both in vitro and in vivo approaches. In vitro assays were used to evaluate the viability of the strain and the antimicrobial activities of the supernatant in different pH ranges. An in vivo assay was performed on female BALB/c mice, wherein the animals were divided into eight groups: four control groups and four treated groups (for curative and preventive therapies). After infecting and treating the mice, the animals were killed to quantify the bacterial load using qPCR, evaluate leucocyte cellular response, determine vaginal cytokine levels and perform cytokine tissue gene expression. Our analyses revealed significant activity of the strain and its supernatant against G. vaginalis. Preliminary in vitro tests showed that the strain grew with equal efficiency in different pH ranges. Meanwhile, the presence of halo and inhibition of pathogen growth established the significant activity of the supernatant against G. vaginalis. We observed that both micro-organisms are resident bacteria of mouse microbiota and that the lactobacilli population growth was affected by G. vaginalis and vice versa. We also observed that the treated groups, with their low bacterial load, absence of leucocyte recruitment, reduced cytokine levels in the vaginal lavage and normalized cytokine gene expression, successfully controlled the infection.

Functional screening for cellulolytic activity in a metagenomic fosmid library of microorganisms associated with coral.

Cellulases are enzymes that degrade cellulosic materials. Cellulose is the most abundant renewable carbon resource on Earth, and cellulases are used in various industrial sectors. Although cellulases are obtained from a variety of sources, this is the first description of cellulolytic activity isolated from a coral metagenomic library. A metagenomic fosmid library of microorganisms associated with the coral Siderastrea stellata, comprising 3552 clones, was screened for cellulolytic activity; this allows access to non-cultivable microorganisms by exploiting the full biotechnological potential. Clones were grown on LB agar plates supplemented with 0.5% carboxymethylcellulose and cellulase positive clones revealed by staining with Congo red. Using this approach, six positive clones with cellulolytic activity were identified. The enzymatic index (EI) of the positive clones was calculated by the ratio between the hydrolysis zone diameter and colony diameter. All positive clones had an EI greater than 1.5. Digestion of the DNA isolated from the six positive clones, using the HindIII restriction endonuclease, revealed different restriction patterns in each clone, indicating that the DNA of each clone is different. There is a growing interest for new cellulolytic enzymes in various industry sectors. Here, we present the initial selection of potential clones for cellulose degradation that could be targets for future studies of enzymatic characterization.

Effect of organic matter enrichment on the fungal community in limestone cave sediments.

Caves are considered major touristic attractions. The management plans of many such caves include limiting the number of visitors; however, the human impact on microbial communities within caves is rarely considered. Therefore, the aim of this study was to evaluate the impact of human-transferred organic matter on the fungal microcosms growing on cave sediments. Samples were collected from a Brazilian limestone cave and cultured with 0.25 or 0.5% 1:1 (w/w) beef and yeast extract (simulating organic matter) under laboratory conditions. The contaminated fungal community was then evaluated at days 0, 30, 180, and 365 after inoculation by polymerase chain reaction denaturing gradient gel electrophoresis. We observed changes in the fungal communities with time, as well as the concentration of added organic matter, compared to the control fungal communities. Additionally, the contaminated microcosms showed a greater number of operational taxonomic units compared to the controls. These findings suggest that tourist activity could cause fungal outbreaks of possible human pathogens, demonstrating the importance of fungal monitoring in these caves.

Efficacy of oral administration of lactic acid bacteria isolated from cocoa in a fermented milk preparation: reduction of colitis in an experimental rat model.

We investigated the probiotic potential of lactic acid bacteria (LAB) obtained from cocoa fermentation using an experimental rat model of colitis. Cocoa beans were collected from fermentation boxes every 12 h for 5 days to isolate the microorganisms. Strains were isolated by serial dilution and plating on MRS agar. Gram-positive and catalase-negative rods were subjected to DNA extraction, polymerase chain reaction, and sequencing. Ten strains were randomly pooled and used to prepare a fermented milk drink that was used to treat the experimental colitis. A parallel group was treated with a single strain drink. Serum concentrations of cytokines and IgA, total and differential counts of blood leukocytes, and histological appearance were compared with the untreated control colitis group. Eighty strains of LAB were identified as Lactobacillus fermentum (68) and Lactobacillus plantarum (12). The multi-strain LAB pool significantly reduced the total number of leukocytes. There was a significant reduction in the percentage of neutrophils and monocytes compared with the control colitis group. IFN-γ concentration was downregulated in animals treated with the LAB pool. IL-10 and IgA increased significantly in the group treated with the strains. Histological analysis showed that the LAB pool reduced the inflammatory infiltrate and restored tissue architecture. The group treated with the single strain LAB drink (L. fermentum) showed no signs of inflammation remission. The results confirm the probiotic action of cocoa-derived LAB in the treatment of experimental colitis. Studies using isogenic models and humans will clarify the mechanisms of immune response modulation in inflammatory bowel disease.

Immunogenicity of pneumococcal polysaccharide vaccine in adult systemic lupus erythematosus patients undergoing immunosuppressive treatment.

OBJECTIVE: To evaluate the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in adult systemic lupus erythematosus patients undergoing (IS group) and not undergoing (non-IS group) immunosuppressive treatment. METHODS: In this prospective open-label study from February 2013 to April 2014, 54 patients had blood samples collected immediately before PPSV23 immunization and 4-6 weeks thereafter for the ELISA measurement of IgG antibody levels against seven pneumococcal serotypes. Positive vaccine response for each serotype was defined as a four-fold or greater antibody response over baseline levels or as a post-vaccine anti-pneumococcal IgG level ≥1.3 µg/ml when baseline values were <1.3 µg/ml. Patients should have responded appropriately to ≥70% of the tested serotypes. We also calculated the mean ratio of post- to pre-vaccination anti-pneumococcal IgG levels. RESULTS: Twenty-eight patients were classified into the IS group and 26 into non-IS group. The median dose of prednisone at baseline was ≤5 mg/day in both groups. Serotype-specific vaccine response rates were not significantly different between the groups. Less than 40% of patients responded adequately by both vaccine response criteria, being numerically lower among IS patients. The mean ratio of increase in anti-pneumococcal levels was 6.4 versus 4.7 (p = 0.001) in non-IS and IS groups, respectively. CONCLUSION: The vaccine was poorly immunogenic, especially among adult systemic lupus erythematosus patients under immunosuppressive therapy.

Microbiological quality and bacterial diversity of the tropical oyster Crassostrea rhizophorae in a monitored farming system and from natural stocks.

Microbiological evaluation is one of the most important parameters for analyzing the viability of an oyster farming system, which addresses public health and ecological concerns. Here, the microbiological quality of the oyster Crassostrea rhizophorae cultivated in a monitored environment and from natural beds in Bahia, northeastern Brazil, was determined. Bacterial diversity in oysters was measured by polymerase chain reaction-denaturing gradient gel electrophoresis. Sequence analysis revealed that most bacterial species showed similarity with uncultured or unidentified bacteria from environmental samples, and were clustered into the phylum Proteobacteria. Diverse bacteria from cultivated (monitored) oyster samples were grouped in the same cluster with a high similarity index (above 79%). Microbiological analyses revealed that these oysters did not contain pathogens. These results reflect the natural balance of the microbial communities essential to the maintenance of health and in inhibiting pathogen colonization in the oyster. On the other hand, bacterial diversity of samples from native stocks in extractive areas displayed a similarity index varying between 55 and 77%, and all samples were clustered separately from each other and from the cluster of samples derived from the cultivation area. Microbiological analyses showed that oysters from the extractive area were not fit for human consumption. This reflected a different composition of the microbial community in this area, probably resulting from anthropic impact. Our study also demonstrated that low temperatures and high rainfall limits the bacterial concentration in tropical oysters. This is the first study analyzing the total bacterial community profiles of the oyster C. rhizophorae.

Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects.

Leishmaniasis is an endemic disease present in 98 countries. In Brazil, the northeast region accounts for approximately half of the cases in humans, and has experienced an increased number of positive cases in dogs. In this study, we investigated the epidemiology of canine leishmaniasis in the city of Ilhéus, Bahia, using serological and molecular techniques and evaluated the possible environmental risk factors and associated clinical signs. Blood samples were collected from 560 dogs in urban and peri-urban areas in Ilhéus, northeastern Brazil. Genomic DNA was extracted from the selected animals and subjected to molecular analysis using Leishmania species-specific primers and diagnosis of Trypanosoma cruzi. A total of 54.72% of dogs were positive for Leishmania braziliensis, and animals positive for both Leishmania infantum and T. cruzi were not identified. Hematologic variables were not statistically associated with cases of L. braziliensis. However, the positive animal group showed lower red blood cell and platelet counts and higher levels of urea and serum creatinine. Few dogs presented clinical signs compatible with the presence of Leishmania. Age of more than 2 years and specific hair colors were associated with positive results for L. braziliensis. The geoclimatic characteristics of the region may improve parasite survival, reproduction, and vectors. This may explain the higher rate of dogs identified as positive in this study.

Short Communication: Elucidation of bacterial community structure on thin-spined porcupine (Chaetomys subspinosus) spines by denaturing.

Thin-spined porcupines (Chaetomys subspinosus) are threatened with extinction and are categorized as vulnerable. This is because of alteration to and loss of their habitat and possible hunting activities in their distribution area. Their spines constitute one of their defense mechanisms, which can be fomites for pathogens to humans. However, little is known about such pathogens. The present study aimed to detect bacteria on spines of C. subspinosus, from the Una Biological Reserve, South of Bahia, northeastern Brazil, by analyzing metagenomic DNA, isolating bacterial culture, using the denaturing gradient gel electrophoresis (DGGE) technique, and sequencing. Six anatomical points were selected for withdrawing spine samples from an individual C. subspinosus. At all sample points, bacteria were detected by bacteriological culture and/or DGGE and sequencing of excised bands. When all samples were combined, standard PCR-DGGE analysis of bacteria present in the spines identified 15 distinct bands, thereby revealing a distinct bacterial community. The main pathogens identified through sequencing were Bacillus cereus, B. thuringiensis, B. anthracis, and B. pumilus. The present study demonstrated the isolation and identification of non-pathogenic and pathogenic bacteria on the spines of C. subspinosus.

High yield of functional metagenomic library from mangroves constructed in fosmid vector.

In the present study, metagenomic technique and fosmid vectors were used to construct a library of clones for exploring the biotechnological potential of mangrove soils by isolation of functional genes encoding hydrolytic enzymes. The library was built with genomic DNA from the soil samples of mangrove sediments and the functional screening of 1824 clones (~64 Mbp) was performed to detect the hydrolytic activity specific for cellulases, amylases (at acidic, neutral and basic pH), lipases/esterases, proteases, and nitrilases. Significant numbers of clones, positive for the tested enzyme activities were obtained. Our results indicate the importance and biotechnological potential of mangrove soils especially when compared to those obtained using other soil metagenomic libraries.

DGGE and multivariate analysis of a yeast community in spontaneous cocoa fermentation process.

Cocoa bean is the main raw material used in the production of chocolate. In southern Bahia, Brazil, cocoa farming and processing is an important economic activity. The fermentation of cocoa is the processing stage that yields important chocolate flavor precursors and complex microbial involvement is essential for this process. In this study, PCR-denaturing gradient gel electrophoreses (DGGE) was used to investigate the diversity of yeasts present during the spontaneous fermentation of cocoa in southern Bahia. The DGGE analysis revealed a richness of 8 to 13 distinct bands of varied intensities among the samples; and samples taken at 24, 36, and 48 h into the fermentation process were found to group with 70% similarity and showed the greatest diversity of bands. Hierarchical clustering showed that all samples had common operational taxonomic units (OTUs) and the highest number of OTUs was found in the 48 h sample. Variations in pH and temperature observed within the fermenting mass over time possibly had direct effects on the composition of the existing microbial community. The findings reported here indicate that a heterogeneous yeast community is involved in the complex cocoa fermentation process, which is known to involve a succession of specialized microorganisms.

Evaluation of the biodegradability of petroleum in microcosm systems by using mangrove sediments from Camamu Bay, Bahia, Brazil.

We investigated the biodegradability of oil in mangrove sediment from Camamu Bay and measured its effect on the bacterial community. Microcosms of mangrove sediment were contaminated with 0.1, 0.5, 1, 2, and 5% (w/v) oil, and the microbial activity was compared to that in uncontaminated sediment. The evolution of CO2 and gas chromatography showed the mineralization of oil compounds, which could reach 100%. Bacterial diversity was determined by polymerase chain reaction using a set of primers for the V3 and V6-V8 regions of 16S rDNA. The band profile obtained by denaturing gradient gel electrophoresis of the amplicons that were obtained for the V3 region showed a negative correlation between band number and oil concentration, whereas that of the V6-V8 region showed a positive correlation between band numbers and oil concentration. The latter also gave similar results for microcosms that were contaminated with 2 and 5% oil. These results demonstrate the mangrove sediment's capacity to recover from oil contamination (in vitro) and suggest that native mangrove microorganisms contain enzymes necessary for the catabolism of oil.

Construction and validation of metagenomic DNA libraries from landfarm soil microorganisms.

Landfarming biodegradation is a strategy used by the petrochemical industry to reduce pollutants in petroleum-contaminated soil. We constructed 2 metagenomic libraries from landfarming soil in order to determine the pathway used for mineralization of benzene and to examine protein expression of the bacteria in these soils. The DNA of landfarm soil, collected from Ilhéus, BA, Brazil, was extracted and a metagenomic library was constructed with the Copy Control(TM) Fosmid Library Production Kit, which clones 25-45-kb DNA fragments. The clones were selected for their ability to express enzymes capable of cleaving aromatic compounds. These clones were grown in Luria-Bertani broth plus L-arabinose, benzene, and chloramphenicol as induction substances; they were tested for activity in the catechol cleavage pathway, an intermediate step in benzene degradation. Nine clones were positive for ortho-cleavage and one was positive for meta-cleavage. Protein band patterns determined by SDS-polyacrylamide gel electrophoresis differed in bacteria grown on induced versus non-induced media (Luria-Bertani broth). We concluded that the DNA of landfarm soil is an important source of genes involved in mineralization of xenobiotic compounds, which are common in gasoline and oil spills. Metagenomic library allows identification of non-culturable microorganisms that have potential in the bioremediation of contaminated sites.

Application of denaturing gradient gel electrophoresis for detection of bacterial and yeast communities along a salinity gradient in the estuary of the Cachoeira River in Brazil.

An estuary is a transition zone between freshwater and marine ecosystems, resulting in dilution of seawater. Estuaries are also considered environments of intense biological activity related to the processes of nutrient cycling. The aim of this study was to evaluate the microbial community composition along a salinity gradient in the estuary of the Cachoeira River, located in southern Bahia, Brazil. The analysis of bacterial and yeast communities was performed by determining the denaturing gradient gel electrophoresis band richness. Formation of zones with similar profiles of bands was observed, and the increasing richness at the intermediate zone demonstrated a clear spatial distinction of communities depending on salinity. In addition, the dissolved oxygen content, temperature, pH, salinity, and dissolved inorganic nutrient contents (NH3(+), NO2(-), NO3(-), PO4(-)) were determined. Nutrients were distributed in similar patterns, with decreasing concentrations as the salinity increases.

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA to monitor changes in mouse gut bacterial communities during Salmonella enterica serovar Enteritidis latent infection.

Changes in intestinal microbial flora during a 4-week period of Salmonella enterica serovar Enteritidis colonization in resistant mice (latent carrier animals) were evaluated using a culture independent method involving denaturing gradient gel electrophoresis. The contents of the ileocecal portion of the intestines produced 26 bands. Fifty-seven percent of the bands were expressed in more than 80% of the samples. Forty percent of the bands present in the negative control were common to all samples, and 60% differed from those obtained 12 h and 1, 5, 10, and 28 days post-inoculation (PI). A dendrogram distinguished the negative control as the external group, and 2 clusters were formed with 76% similarity, separating the 12-h PI and 3-day PI time points from the others. These groupings were also revealed through multivariate analysis in a principal component analysis and the Venn diagram. The production of interferon γ 12 h and 3 days PI may explain this brief imbalance in microbiota that was quickly reversed in the subsequent days. These findings demonstrate that S. enterica serovar Enteritidis can colonize the gut and persist in balance with the microbiota of resistant hosts.

Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Comparison of different protocols for the extraction of microbial DNA from reef corals

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content.

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.