Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms.

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1-130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SIGNIFICANCE: The comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

Symbiotic microparticles produced through spray-drying-induced in situ alginate crosslinking for the preservation of Pediococcus pentosaceus viability.

Probiotic microorganisms are a promising alternative to antibiotics in preventing and treating bacterial infections. Within the probiotic group, the lactic acid bacteria (LAB)stand out for their health benefits and for being recognized as safe by regulatory agencies. However, these microorganisms are sensitive to various environmental conditions, including the acidic environment of the stomach. Faced with these obstacles, this work aimed to promote the symbiotic microencapsulation of LAB in a composite matrix of alginate and prebiotics to enhance their survival and improve their probiotic activity during gastrointestinal transit. We evaluated the effect of inulin, fructo-oligosaccharides (FOS) and mannan-oligosaccharides (MOS) as prebiotic sources on the growth of Pediococcus pentosaceus LBM34 strain, finding that MOS favored LAB growth and maintenance of microencapsulated cell viability. The symbiotic microparticles were produced using the spray-drying technique with an average size of 10 µm, a smooth surface, and a composition that favored the stabilization of live cells according to the FTIR and the thermal analysis of the material. The best formulation was composed of 1 % of alginate, 10 % MOS and 1 % M10 (% w/v), which presented notable increases in the survival rates of the probiotic strain in both alkaline and acidic conditions. Therefore, this industrially scalable approach to symbiotic LAB microencapsulation can facilitate their growth and colonization within the host. This effort aims to contribute to reducing antibiotic reliance and mitigating the emergence of new zoonotic diseases, which pose significant challenges to public health.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

Dual antibacterial mechanism of [K4K15]CZS-1 against Salmonella Typhimurium: a membrane active and intracellular-targeting antimicrobial peptide.

Salmonella genus is a leading cause of food-borne infections with strong public health impact and economic ramifications. The development of antimicrobial resistance added complexity to this scenario and turned the antibiotic drug discovery into a highly important challenge. The screening of peptides has served as a successful discovery platform to design new antibiotic candidates. Motivated by this, the antimicrobial and cytotoxic properties of three cruzioseptins against Salmonella Typhimurium and RAW 264.7 murine macrophage cells, respectively, were investigated. [K4K15]CZS-1 was the most potent antimicrobial peptide identified in the screening step with a minimum inhibitory concentration (MIC) of 16 µg/mL (7.26 µM) and moderate cytotoxicity. From a structural point of view, in vitro and in silico techniques evidenced that [K4K15]CZS-1 is a α-helical cationic antimicrobial peptide. In order to capture mechanistic details and fully decipher their antibacterial action, we adopted a multidimensional approach, including spectroscopy, electron microscopy and omics analysis. In general lines, [K4K15]CZS-1 caused membrane damage, intracellular alterations in Salmonella and modulated metabolic pathways, such as the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and lipid metabolism. Overall, these findings provide deeper insights into the antibacterial properties and multidimensional mode of action of [K4K15]CZS-1 against Salmonella Typhimurium. In summary, this study represents a first step toward the screening of membrane-acting and intracellular-targeting peptides as potential bio-preservatives to prevent foodborne outbreaks caused by Salmonella.

Antimicrobial Activity of Peptides Produced by Lactococcus lactis subsp. lactis on Swine Pathogens.

Swine production is of great importance worldwide and has huge economic and commercial impact. Due to problems with bacterial infection, the use of antimicrobials has increased in the last decades, particularly in Latin America and Asia. This has led to concerns about antimicrobial resistance, which poses risks to human health and the environment. The use of probiotic organisms has been proposed as an alternative to this use, as these beneficial bacteria can produce antimicrobial peptides, such as bacteriocins, which allow the induction of inhibitory effects against pathogenic microorganisms. Among probiotics, some bacteria stand out with the inhibition of animal pathogens. The bacteriocin-like inhibitory substances (BLISs) of Lactococcus lactis subsp. lactis strain L2, present in its cell-free supernatant, were tested against pathogenic strains isolated from pig samples, such as Escherichia coli, Salmonella enterica, Streptococcus suis, Streptococcus dysgalactiae, Staphylococcus hyicus, and Enterococcus faecalis. Compounds secreted by L. lactis L2 have been shown to inhibit the growth of some pathogenic species, particularly Gram-positive bacteria, with S. suis being the most prominent. Antimicrobial peptides with a molecular size of 500-1160 Daltons were isolated from BLISs. The results highlight the potential of L. lactis BLISs and its peptides as natural antimicrobials for use in the food industry and to reduce the use of growth promoters in animal production.

Salmonella and other Enterobacteriaceae in conventional and organic vegetables grown in Brazilian farms.

This study aimed to assess the microbiological profile of conventional and organic vegetables grown in Brazilian farms through the detection of Salmonella and other Enterobacteriaceae. A total of 200 samples (100 conventional and 100 organic), including leafy greens, spices/herbs, and other unusual vegetables, were submitted to the enumeration of Enterobacteriaceae by plating on VRBG agar. Moreover, colonies of Enterobacteriaceae were randomly selected and submitted to identification by MALDI-TOF MS. Samples were also tested for Salmonella, using culture-based and PCR-based enrichment methods. The mean counts of Enterobacteriaceae in conventional and organic vegetables were 5.1 ± 1.5 and 5.4 ± 1.4 log CFU/g, respectively (P > 0.05). A total of 18 genera (including 38 species) of Enterobacteriaceae were identified, and the most frequent ones found in samples from both farming systems were Enterobacter (76%) and Pantoea (68%). Salmonella was identified in 17 samples (8.5%): nine (4.5%) in conventional and eight (4.0%) in organic vegetables. These results indicate that the farming system had no impact on the Enterobacteriaceae populations and rates of Salmonella and revealed unsatisfactory microbiological safety of some samples, mainly due to the presence of Salmonella. These findings highlight the need for control measures during vegetable production, regardless of the farming system, to reduce microbial contamination and the risks of foodborne illnesses.

Agricultural practices in Brazilian organic farms and microbiological characteristics of samples collected along the production chain.

AIMS: To gather data on agricultural practices in organic farms in Sao Paulo, Brazil, and evaluate their relationship with the microbiological characteristics of samples collected along the production chain. METHODS AND RESULTS: Practices data were based on field observations and interviews with farmers in 10 selected organic lettuce producing farms. Counts of Enterobacteriaceae and surveys for Salmonella were performed in samples of lettuce (before and after washing), fertilizers, irrigation and washing water, all collected in the same farm. Water samples were also tested for total coliforms and generic Escherichia coli. Isolated Enterobacteriaceae were identified by MALDI-TOF MS. Contamination of lettuce was influenced by some agricultural practices: chicken manure-based fertilization resulted in higher Enterobacteriaceae counts in lettuce when compared to other types of manure, whereas pre-washed lettuces presented lower microbial counts than non-pre-washed samples. Salmonella was detected in one lettuce sample by qPCR. Escherichia coli was detected in all irrigation water samples. All sample types contained Enterobacteriaceae species commonly reported as opportunistic human pathogens. CONCLUSIONS: The data highlight the need for improvement in the good agricultural practices in the studied farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on agricultural practices and microbiological characteristics of organic lettuce, contributing to the development of more accurate risk assessments.

Identification of Possible Salivary Metabolic Biomarkers and Altered Metabolic Pathways in South American Patients Diagnosed with Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) represents 90% of oral malignant neoplasms. The search for specific biomarkers for OSCC is a very active field of research contributing to establishing early diagnostic methods and unraveling underlying pathogenic mechanisms. In this work we investigated the salivary metabolites and the metabolic pathways of OSCC aiming find possible biomarkers. Salivary metabolites samples from 27 OSCC patients and 41 control individuals were compared through a gas chromatography coupled to a mass spectrometer (GC-MS) technique. Our results allowed identification of pathways of the malate-aspartate shuttle, the beta-alanine metabolism, and the Warburg effect. The possible salivary biomarkers were identified using the area under receiver-operating curve (AUC) criterion. Twenty-four metabolites were identified with AUC > 0.8. Using the threshold of AUC = 0.9 we find malic acid, maltose, protocatechuic acid, lactose, 2-ketoadipic, and catechol metabolites expressed. We notice that this is the first report of salivary metabolome in South American oral cancer patients, to the best of our knowledge. Our findings regarding these metabolic changes are important in discovering salivary biomarkers of OSCC patients. However, additional work needs to be performed considering larger populations to validate our results.

Chlorella vulgaris phycoremediation at low Cu+2 contents: Proteomic profiling of microalgal metabolism related to fatty acids and CO2 fixation.

The aim of this work was to correlate metabolic changes with copper ions (Cu+2) bioremediation by microalgae C. vulgaris 097 CCMA-UFSCar at low Cu+2 content. The metabolic effects include proteome changes related to fatty acid biosynthesis (value-added product) and carbon fixation (climate change mitigation). Cu+2, even at low concentration, showed a significant negative impact on C. vulgaris growth. The microalgal bioremediation reached 100, 74, 38 and 26% for Cu+2 content at 0.1; 0.3; 0.6 and 0.9 mg L-1, respectively. Regarding proteomics, the numbers of proteins reduced (≈37%) from 581 proteins (control) to 369 proteins (0.9 mg of Cu+2 L-1) compared to control. The microalgal CO2 fixation is strictly related to the Calvin cycle, particularly phase 1, in which ribulose bisphosphate carboxylase large chain (RuBisCO) produces two phosphoglycerate molecules from CO2 and ribulose 1,5-bisphosphate. Then, phosphoglycerate can be metabolically reduced into glucose. When compared to control, the RuBisCO was underexpressed (≈50%). Similar changes in proteomic profiling of metabolism-related to fatty acid biosynthesis was observed. Nevertheless, no protein was found for the cultivation at 0.9 mg of Cu+2 L-1. Thus, the analysis of C. vulgaris proteomic data indicated that even at low concentration, Cu+2 lead to drastic metabolic changes.

Identification of bioactive peptides released from in vitro gastrointestinal digestion of yam proteins (Dioscorea cayennensis).

Bioactive peptides have been broadly studied for their contribution to human health. This study aimed to identify bioactive peptides generated by in vitro gastrointestinal digestion of yam proteins. Yam protein concentrate (YPC) was submitted to simulated digestion. Gastric phase hydrolysate (GPH) and total gastrointestinal phase hydrolysate (GIPH) had their peptides identified by nanoLC-ESI-MS/MS. Peptide sequences were subjected to a database-driven (BIOPEP) bioactivity search. In vitro tests included: Antioxidant activity, DNA damage protection, ACE-inhibitory activity and antibacterial activity against the bacteria Escherichia coli, Salmonella sp. and Lysteria monocytogenes. Simulated digestion generated small peptides (mostly MW < 3500 Da), several of them with potential bioactive sequences predicted in silico. In both GPH and GIPH biological activities were detected, although GIPH displayed stronger DNA damage protection and antibacterial activity against Escherichia coli. The digestion of yam proteins releases promising biologically active peptides which can contribute to the prevention of bacterial infection and chronic degenerative diseases, with beneficial effects to human health.

Metabolomic and secretomic approach to the resistance features of the fungus Aspergillus niger IOC 4687 to copper stress.

Metabolomic and secretomic analyses of Aspergillus niger IOC 4687 indicated the features of resistance of this strain to copper stress. To investigate the metabolites produced under oxidative stress conditions, gas chromatography-mass spectrometry analysis was performed. The secretome principal component analysis results showed that mannitol could be the main metabolite responsible for conferring resistance to the fungus, and gluconic acid is the possible cause of copper desorption because of its chelating ability. The meta-analysis of the metabolome of A. niger IOC 4687 indicated that a low concentration of sorbitol and ribonolactone during growth may be an indicator of oxidative stress.

Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment.

The production of bioactive peptides from organic by-waste materials is in line with current trends devoted to guaranteeing environmental protection and a circular economy. The objectives of this study were i) to optimize the conditions for obtaining bioactive hydrolysates from chicken combs and wattles using Alcalase, ii) to identify the resulting peptides using LC-ESI-MS2 and iii) to evaluate their chelating and antioxidant activities. The hydrolysate obtained using a ratio of enzyme to substrate of 5% (w/w) and 240 min of hydrolysis showed excellent Fe2+ chelating and antioxidant capacities, reducing Fe3+ and inhibiting 2, 2'-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The mapping of ion distribution showed that a high degree of hydrolysis led to the production of peptides with m/z ≤ 400, suggesting low mass peptides or peptides with multiple charge precursor ions. The peptides derived from the proteins of cartilage like Collagen alpha-2(I), Collagen alpha-1(I), Collagen alpha-1(III) and elastin contributed to generation of bioactive compounds. Hydrolysates from chicken waste materials could be regarded as candidates to be used as ingredients to design processed foods with functional properties.

Label-free peptide quantification coupled with in silico mapping of proteases for identification of potential serum biomarkers in gastric adenocarcinoma patients.

OBJECTIVES: We aimed to identify serum level variations in protein-derived peptides between patients diagnosed with gastric adenocarcinoma (GAC) and non-cancer persons (control) to detect the activity changes of proteases and explore the auxiliary diagnostic value in the context of GAC physiopathology. METHODS: The label-free quantitative peptidome approach was applied to identify variants in serum levels of peptides that can differentiate GAC patients from the control group. Peptide sequences were submitted against Proteasix tool predicting proteases potentially involved in their generation. The activity change of proteases was subsequently estimated based on the peptides with significantly altered relative abundance. In turn, activity change prediction of proteases was correlated with relevant protease expression data from the literature. RESULTS: A total of 191 peptide sequences generated by the cleavage of 36 precursor proteins were identified. Using the label-free quantification approach, 33 peptides were differentially quantified (adjusted fold change ≥ 1.5 and p-value < 0.05) in which 19 were up-regulated and 14 were down-regulated in GAC samples. Of these peptides, fibrinopeptide A was significantly decreased and its phosphorylated form ADpSGEGDFLAEGGGVR was upregulated in GAC samples. Activity change prediction yielded 10 proteases including 6 Matrix Metalloproteinases (MMPs), Thrombin, Plasmin, and kallikreins 4 and 14. Among predicted proteases in our analysis, MMP-7 was presented as a more promising biomarker associated with useful assays of clinical practice for GAC diagnosis. CONCLUSION: Our experimental results demonstrate that the serum levels of peptides were significantly differentiated in GAC physiopathology. The hypotheses built on protease regulation could be used for further investigations to measure proteases and their activity levels that have been poorly studied for GAC diagnosis.

Response mechanism of mine-isolated fungus Aspergillus niger IOC 4687 to copper stress determined by proteomics.

Proteomic analysis of the fungus Aspergillus niger showed that its capacity to absorb metals was boosted by physiological modification under metal stress conditions. To investigate the proteome elicited by copper stress, the mine-isolated strain A. niger IOC 4687 was cultured in the absence (control) or presence of copper ions (50 mg L-1) for 72 h. Protein extract from each treatment was analyzed by nano-liquid chromatography-mass spectrometry and proteins were identified using PEAKS Studio 8.5 software. Grouping proteins by functional category showed that antioxidant enzymes, such as catalase, superoxide dismutase, and cytochrome c peroxidase, were present in both treatments. However, heat shock proteins (Hsp60 and Hsp70) and some metalloproteins (LMBR1 domain protein and A. niger contig An09c0040) were only observed after copper treatment. These proteins were the cellular response to the stress conditions. In conclusion, significant changes in the proteome of A. niger were observed due to the presence of copper.

Identification of microorganisms in biofluids of individuals with periodontitis and chronic kidney disease using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE: Chronic kidney disease (CKD) and periodontitis (PD) are important health issues. There is a large variety of microorganisms related to the pathogenesis of periodontitis, and optimising the time and the cost of laboratory assays to detect these organisms is highly valuable in the medical field. METHODS: Bacteria were isolated from saliva and oral biofilm of 30 adolescents and young adults with definite medical and dental diagnosis of CKD and PD, respectively, and proteins were extracted for microorganism identification by means of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) technique. RESULTS: The results showed that the most incident microorganisms were Actinomyces dentalis (43%), Acinetobacter ursingi (60%), Aggregatibacter actinomycetencomitans (60%), Corynebacterium argentoctens (63%), Staphylococcus aureus (93%), Streptococcus salivarius (97%) and Tannerella forsythensis (43%). The analysis of oral biofilm showed higher incidences for Actinomyces dentalis (33%), Acinetobacter ursingi (50%), Aggregatibacter actinomycetencomitans (50%), Corynebacterium argentoctens (70%), Pseudomonas aeruginosa (40%), Staphylococcus aureus (73%) and Streptococcus salivarius (87%). CONCLUSIONS: Based on these results, we concluded that the MALDI Biotyper protocol proves useful as a rapid and reliable assay for distinguishing different microorganisms possibly related to CKD and PD. Copyright © 2016 John Wiley & Sons, Ltd.

Environmental contamination by fluoroquinolones

Over the past few decades, a high number of pharmaceuticals have been detected in surface, ground and drinking waters. This contamination comes from domestic sewage, livestock, hospitals and chemical-pharmaceutical industries. Typical examples of these pollutants are the fluoroquinolones - powerful antibiotics used in human and veterinary medicine. The presence of fluoroquinolones in the environment can pose a serious threat to the ecosystem and to human health due to their high consumption globally: in 1998, around 120 tons were produced. Even at low environmental concentrations, antibiotics stimulate bacterial resistance. The consequences of the presence of fluoroquinolones in the environment are not fully understood, but are known to be toxic to plants and aquatic organisms. Approximately 85% of the fluoroquinolones present in influents can be removed by conventional wastewater treatment plants, but the removed fraction is frequently accumulated in the sludge, which is sometimes used as fertilizer, representing an additional input route into the environment. The removal of fluoroquinolones by biological treatment is ineffective, and it is believed that only advanced oxidation technologies are able to destroy these emerging pollutants.

Nas últimas décadas, um grande número de fármacos tem sido identificado em águas superficiais, subterrâneas e potáveis. Tal contaminação advém do esgoto doméstico, hospitais, criação de animais e das indústrias químico-farmacêuticas. Exemplos típicos desses poluentes são as fluoroquinolonas – potentes antibióticos empregados na medicina humana e veterinária. A presença de fluoroquinolonas no meio ambiente pode representar uma séria ameaça para o ecossistema e para a saúde humana devido ao alto consumo mundial: em 1998 foram produzidas, aproximadamente, 120 toneladas. Mesmo em baixas concentrações, antibióticos podem estimular a resistência bacteriana. As consequências da presença de fluoroquinolonas no ambiente não são completamente compreendidas, mas sabe-se que são tóxicas para plantas e organismos aquáticos. Aproximadamente 85% das fluoroquinolonas presentes em efluentes podem ser removidos em estações de tratamento de efluentes convencionais, porém a fração removida é frequentemente acumulada no lodo, muitas vezes usado como fertilizante, o que representa uma rota adicional de entrada desses compostos no ambiente. A remoção de fluoroquinolonas por meio de tratamento biológico não é eficiente, e acredita-se que somente as tecnologias de oxidação avançada sejam capazes de degradar esses poluentes emergentes.