Extracellular Protease Production Research Articles

The Ecological Strategy Determines the Response of Fungi to Stress: A Study of the 2,4-diacetylphloroglucinol Activity Against Aspergillus and Fusarium Species.

Aspergillus and Fusarium are two economically important genera of fungi. They cause significant yield losses and contamination of crops with mycotoxins. In this study we aimed to evaluate the impact of 2,4-diacetylphloroglucinol (2,4-DAPG) on Aspergillus and Fusarium fungi. It is hypothesized that two fungal genera, which have different ecological strategies, react differently to stress caused by a secondary metabolite produced by rhizosphere Pseudomonas species. We found that 2,4-DAPG was able to reduce biofilm formation of Aspergillus and Fusarium, as reflected in biomass and its chemical composition. Furthermore, subinhibitory concentrations of 2,4-DAPG increased the levels of ergosterol and polysaccharides (α- and β-glucans, chitin) in the cell membrane and cell wall of Aspergillus, while decreasing them in Fusarium. 2,4-DAPG altered the production of secondary metabolites, especially mycotoxins and extracellular proteases. The production of ochratoxin A was decreased in A. ochraceus, and T-2 toxin and zearalenone, on the contrary, were increased in F. culmorum and F. sporotrichioides, respectively. Thus, using 2,4-DAPG we demonstrated that the ecological role of fungi determines their reaction to antibiotic substances produced by the plant microbiome. Our data contributes to understanding the molecular mechanisms behind symbiotic relationships in natural communities, which are mediated by the biosynthesis of antibiotics.

Isolation of Pathogenic Cronobacter Species as Bacteriological Risks Indicator in Powdered Infant Formula Available to Deprived Infants in Lagos Metropolis, Nigeria.

Powdered infant formula (PIF) is rich in nutrients that support the survival and growth of bacteria that trigger food safety disorders in deprived infants through life-threatening illnesses. The study aims to examine and identify the incidence of pathogenic bacteria of concern in PIF upon reconstitution in lukewarm water. A total of 172 samples consisting of 38 brands of PIF available in the Lagos metropolis were sampled, suspended in water (10g in 100mL), and bacteria strains were isolated using combinational enrichment and selective culture techniques. Pure bacterial strains were characterized and identified based on their physiology and 16S rRNA gene sequence homology. While 85 bacterial strains were isolated from the enriched culture system, 20 strains were selectively isolated based on tolerance to sodium deoxycholate. Approximately 13% of the selected bacteria were identified as Cronobacter spp., exhibiting virulence traits including extracellular protease production, coagulation and proteolysis of casein, haem-agglutination, and β-haemolysis of human blood. Approximately 82% of the Cronobacter strains tolerated NaCl (10%) and bile salt; and exhibited resistance to cefotaxime, ceftriaxone, gentamicin, and Amoxicillin-clavulanic acid antibiotics. The presence of Cronobacter spp. in 13% of the PIF brands available to infants calls for concern about the safety of deprived infants that might be fed with such PIF. Consequently, PIF safety alerts need to be activate while further studies on critical points at which the pathogens get introduced to the PIFs need to be identified.

Evaluation of the potential probiotic Bacillus subtilis isolated from darkbarbel catfish (Pelteobagrus fulvidraco) on growth performance, serum immunity, and disease resistance of Aeromonas hydrophila.

This study aimed to identify potential probiotic strains of Bacillus subtilis from healthy fish gut microbiota for application in aquaculture. The effects of dietary B. subtilis administration on growth performance, serum enzyme activity, immune gene expression, and disease resistance in darkbarbel catfish (Pelteobagrus fulvidraco) were investigated. The isolate, identified through gene sequencing and biochemical tests, demonstrated resilience to pH 3.0% and 6.0% bile, and exhibited extracellular protease, cellulose, lipase, and amylase production. Darkbarbel catfish were fed diets with varying B. subtilis concentrations (0 CFU/kg [T0], 107 CFU/kg [T1], 108 CFU/kg [T2], and 109 CFU/kg [T3]). After 8 weeks, significant increases (p < 0.05) were observed in final body weight, weight gain rate, specific growth rate, serum lysozyme, serum superoxide dismutase, alkaline phosphatase, and total antioxidant capacity, whereas malondialdehyde levels significantly decreased. Feeding darkbarbel catfish with B. subtilis diets increased immunoglobulin M (IgM) and C3 gene expression (p < 0.05), indicating a positive impact on the fish's immune system. The strain upregulated interleukin 10 (IL-10) and transforming growth factor-β (TGF-β) expression and downregulated TNF-α and IL-1β, suggesting potential anti-inflammatory effects. Following a 7-day challenge with Aeromonas hydrophila, fish fed with B. subtilis exhibited lower mortality, with higher survival rates in the T2 and T3 groups. In conclusion, supplementing darkbarbel catfish diets with B. subtilis effectively enhances growth performance, immune response, and disease resistance.

Bacillus spp. Isolated from Miang as Potential Probiotics in Nile Tilapia Culture-In Vitro Research.

Among 79 Bacillus spp. isolated from Miang, a fermented tea in north Thailand, 17 Bacillus strains were selected with probiotic potential in Nile tilapia culture based on the capabilities of bacteriocin production and associated antimicrobial activities against fish pathogens, Aeromonas hydrophila and Streptococcus agalactiae. However, only six isolates were selected for further extensive studies based on the strength of their antimicrobial activities and their tolerance against simulated gastrointestinal conditions. The molecular identification by 16S rRNA gene sequence analysis revealed that five isolates, K2.1, K6.1, K7.1, K15.4, and K22.6, were Bacillus tequilensis, and the isolate K29.2 was Bacillus siamensis. B. siamensis K29.2 showed complete susceptibility to antibiotics tested in this study, while B. tequilensis K 15.4 showed moderate resistance to some antibiotics; therefore, both strains were selected as potential probiotic bacteria. B. tequilensis K15.4 and B. siamensis K29.2 were capable of the production and secretion of extracellular protease and polysaccharide degrading enzymes, including cellulase, xylanase, and β-mannanase. The tannin tolerant test also demonstrated their ability to grow on selective agar plates and secrete cellulase and β-mannanase in the presence of hydrolyzable tannin. In addition, in vitro digestion of commercial fish substrate revealed that the extracellular enzymes produced by both strains efficiently reacted with feed protein and polysaccharides. Based on the results from this study, B. siamensis K29.2 was deemed to have the highest potential multifunctional probiotic qualities for application in Nile tilapia culture, while the antibiotic-resistant gene in B. tequilensis K15.4 must be clarified before field application.

Insights into the efficient degradation mechanism of extracellular proteases mediated by Purpureocillium lilacinum.

Protease secretion is crucial for degrading nematode cuticles using nematophagous fungus Purpureocillium lilacinum, but the secretion pattern of protease remains poorly understood. This study aimed to explore the degradation mechanism of proteases by investigating the characteristics of protease secretion under various carbon and nitrogen sources, and different carbon to nitrogen (C:N) ratios in P. lilacinum. The results showed that corn flour as a carbon source and yeast extract as a nitrogen source specifically induced protease secretion in P. lilacinum. P. lilacinum produced significant amounts of gelatinase and casein enzyme at C:N ratios of 10:1, 20:1, and 40:1, indicating that higher C:N ratios were more beneficial for secreting extracellular proteases. Proteomic analysis revealed 14 proteases, including 4 S8 serine endopeptidases and one M28 aminopeptidase. Among four S8 serine peptidases, Alp1 exhibited a high secretion level at C:N ratio less than 5:1, whereas PR1C, PR1D, and P32 displayed higher secretion levels at higher C:N ratios. In addition, the transcription levels of GATA transcription factors were investigated, revealing that Asd-4, A0A179G170, and A0A179HGL4 were more prevalent at a C:N ratio of 40:1. In contrast, the transcription levels of SREP, AreA, and NsdD were higher at lower C:N ratios. The putative regulatory profile of extracellular protease production in P. lilacinum, induced by different C:N ratios, was analyzed. The findings offered insights into the complexity of protease production and aided in the hydrolytic degradation of nematode cuticles.

Increased production of aureolysin and staphopain A is a primary determinant of the reduced virulence of Staphylococcus aureus sarA mutants in osteomyelitis.

We previously demonstrated that mutation of sarA in Staphylococcus aureus limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic sarA mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 sarA mutants by subsequent mutation of aur and scpA, while mutation of aur had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of sspAB was also required to mimic the phenotype of sarA mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of aur and scpA had the greatest impact on restoring the virulence of LAC and UAMS-1 sarA mutants, with concurrent mutation of sspAB and the spl operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of S. aureus sarA mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of S. aureus virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of sarA mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that sarA-mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of S. aureus virulence factors in diverse clinical isolates and diverse forms of S. aureus infection.

Discovery of psoralen as a quorum sensing inhibitor suppresses Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines.Key points• Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa.• Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes.• Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Assessment of Proteinase and Phospholipases Enzymes Isolated from Pathogenic Candida species from Women Attending Antenatal Clinic at Mbeya Zonal Referral Hospital, Southern Highland Regions of Tanzania

Fungi are a global cause of vaginal infections, with vaginal candidiasis largely afflicting tropical regions including most parts of sub-Saharan Africa. In Tanzania, baseline studies have established the prevalence of Candida species among symptomatic and asymptomatic pregnant women in Dar es Salaam (Namkinga et al., 2005; Namkinga 2012) and in Mwanza (Mushi et al., 2019). However, no study that has been done to determine the above-mentioned enzymes form Candida species in Southern Highland Regions of Tanzania. Several factors have been reported to contribute to the virulence and pathogenicity of Candida, among others the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. Therefore, this study was designed to investigate the in vitro production of phospholipases and proteinases enzymes from isolated pathogenic Candida spp from pregnant women.Of the 280 samples collected from symptomatic and asymptomatic pregnant women attending antenatal clinics at Mbeya Zonal Referral Hospital were tested for several microbiological methods such as the wet mount microscopic preparations (hanging drop) and the Gram stained smears for microscopy, cultures on Sabouraud’s dextrose agar and on Conidia enhancing medium (Corn meal agar), germ tube test, biochemical tests, sugar fermentation-assimilation tests and molecular test to characterize Candida species.Phospholipase production was performed in egg yolk medium while the production of proteinase was done in a medium containing bovine serum albumin. All analyses were performed in triplicates. The results showed that; out of the 280 tested, 155 (74.3%) isolates were phospholipase positive while 201 (96.6%) were positive for proteinase activity. C. albicans was the species with the highest number of positive isolates for proteinase and phospholipase 110 (95.6%), and most strains of C. albicans produce both enzymes (phospholipases and proteinases). The non-albicans Candida isolates were also producers of hydrolytic enzymes that, consequently, might be able to cause infections as favorable conditions arises.

Phenotypic heterogeneity unveils a negative correlation between antibiotic resistance and quorum sensing in Pseudomonas aeruginosa clinical isolates.

Colonization of Pseudomonas aeruginosa in the lung environments frequently leads to the enrichment of strains displaying enhanced antibiotic resistance and reduced production of quorum-sensing (QS) controlled products. However, the relationship between the emergence of QS deficient variants and antibiotic resistance remains less understood. In this study, 67 P. aeruginosa strains were isolated from the lungs of 14 patients with chronic obstructive pulmonary disease, followed by determining their genetic relationship, QS-related phenotypes and resistance to commonly used antibiotics. The integrity of P. aeruginosa QS system was checked by DNA sequencing. The relationship between the QS system and antibiotic resistance was then assessed by correlation analyses. The function of the LasR protein and bacterial virulence were evaluated through homology modeling and nematode-infection assay. The influence of antibiotic on the development of extracellular protease production ability of P. aeruginosa was tested by an evolutionary experiment. The results showed that P. aeruginosa clinical strains displayed abundant diversity in phenotype and genotype. The production of extracellular proteases was significantly negatively correlated with antibiotic resistance. The strains with enhanced antibiotic resistance also showed a notable overlap with the mutation of lasR gene, which is the core regulatory gene of P. aeruginosa QS system. Molecular docking and Caenorhabditis elegans infection assays further suggested that P. aeruginosa with impaired LasR protein could also have varying pathogenicity. Moreover, in vitro evolution experiments demonstrated that antibiotic-mediated selective pressure, particularly from Levofloxacin contributed to the emergence of extracellular protease-negative strains. Therefore, this study provides evidence for the connection of P. aeruginosa QS system and antibiotic resistance, and holds significance for developing targeted strategies to address antibiotic resistance and improving the management of antibiotic-resistant infections in chronic respiratory diseases.

Approach towards Submerge Production, Characterization and Application of Extracellular Alkaline Protease from Pseudomonas aeruginosa YPVC

Approach towards Submerge Production, Characterization and Application of Extracellular Alkaline Protease from Pseudomonas aeruginosa YPVC

Investigation of optimal parameters for aspartic protease production from Aspergillus terreus using solid-state fermentation

The current study presents extracellular Aspartic protease production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus terreus, and parameters that largely affect enzyme production, namely fermentation time, pH, moistening solution, temperature, inoculum volume, and different solutions, were utilized for enzyme extraction. The isolated fungus was identified as A. terreus (registered as A. terreus SFH5 in the gene bank) by phenotypic and molecular characterization, showed the highest aspartic protease activity of 258.6 units/mL and 272.2 units/mg using a solid-state fermentation technique during screening, and was further used for media component screening and optimization studies. The study employed wheat bran as the preferred production medium and casein as the nitrogen source. The fermentation process was conducted at a temperature of 30°C and a pH of 5. The moistening solution to fermentation medium ratio was set at 3:1, while an inoculum volume of 2 mL (containing 1 × 106 spores/10 g solid) was used. Distilled water was selected as the most effective solution for extracting the enzyme. It has been discovered that the locally isolated novel strain of A. terreus, SFH5, can efficiently produce a substantial amount of aspartic protease by utilizing agro-industrial waste. This makes it a viable candidate for large-scale optimized production of the enzyme to satisfy industrial requirements.

Bioconversão de resíduos de aves para produção de proteases por Aspergillus sp. isolado do solo da floresta Amazônica

ABSTRACT: Feathers are by-products that are generated in significant quantities by the poultry industry. Microbial bioconversion has been investigated as a promising strategy for the processing of feathers, since, along with the degradation of these keratinous materials, bioprocessing can result in value-added products. Thus, from the perspective of industrial microbiology, chicken feathers can be considered a raw material for obtaining microbial proteases. Within this context, this research investigated and characterized the production of extracellular proteases by Aspergillus sp., isolated from soil of the Amazon Rainforest. The enzymatic production was evaluated using several growth substrates (whole feathers, feather meal, human hair, casein, gelatin, peptone and chicken beaks). With highest enzyme production was obtained the feather meal (FM) and peptone. After 48 h of fermentation, FM degradation was 15.82%. The crude protease showed optimal activity at pH 5.0 and 37 °C and enzymatic activity was enhanced with the addition of 1 and 5 mM of CaCl2, MnSO4, KCl, MgSO4 and CuSO4. The detergents Tween 20 and Triton x-100, at concentrations 0.5 and 1% (v/v), tended to stimulate activity. The presence of 0.5 and 1% (v/v) of organic solvents (methanol, acetone, butanol, acetonitrile, isopropanol and DMSO), maintained the enzymatic activity. β-mercaptoethanolstimulated proteolytic activity in the enzymatic assays. This study suggested new direction for waste management with industrial applications giving rise to green technology for sustainable development.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Evaluation of Anti-Foodborne Bacterial Activity, Digestive Enzyme Secretion, and Antimicrobial Resistant Genes as Probiotic Strains Selection for Industrial Interest

Beneficial microbes, such as probiotic bacteria, are increasingly in demand in the food and feed industry. Lactic acid bacteria and bifidobacteria are commonly used as commercial probiotics, only a few species have been isolated from Southeast Asia areas. This study employed criteria including antimicrobial activity, the release of digestive enzymes, and the absence of antibiotic-resistant (AMR) genes to screen potential local isolates. The results revealed that 4 out of 16 isolates met these criteria, displaying anti-foodborne bacterial activities and a lack of fifty-one tested AMR genes. Furthermore, the four selected isolates demonstrated the production of extracellular digestive enzymes, including amylase, lipase, protease, β-glucanase, and cellulase, with enzyme indices ranging from 1.09–1.31. Among these isolates, two potential probiotics were identified as Bifidobacterium animalis subsp. lactis (strain H9-01) and Lactobacillus reuteri (strain P4-S03). Importantly, both species are approved for use as food and feed supplements in accordance with Thai regulations. This research outlines an approach for screening potential probiotics for industrial-scale applications.

Extracellular activity of proteases from Yarrowia lipolytica IPS21 as a function of the carbon and nitrogen source

Abstract The yeast strain Yarrowia lipolytica IPS 21 was tested for its ability to produce the protease enzyme on analytically pure carbon sources as well as on waste carbon sources. It was confirmed that the yeast Y. lipolytica IPS21 can have a higher proteolytic activity in the presence of waste carbon sources in chrome-tanned leather shavings (CTLS) than on yeast extract alone. This is confirmed by the high concentration of amino acids in samples with CTLS, suggesting increased degradation of CTLS by Y. lipolytica or secretion of proteases into the medium. It was also confirmed that metals accumulate mainly in the biomass and not in the supernatant. The biomass was also found to contain high levels of Ca, K and P, which are essential for plant growth. These results show that Y. lipolytica strain IPS21 can be used for the production of extracellular alkaline proteases and for the degradation of protein waste.

DegQ is an important policing link between quorum sensing and regulated adaptative traits in Bacillus subtilis.

Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study, Bacillus subtilis was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (comQ::kan) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P srfAA hypersensitivity of the comQ::kan mutant is linked to a 168 strain-specific mutation in the P degQ region. Hence, the markerless ΔcomQ mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P srfAA and not detected in another ComX-regulated promoter, the P aprE , which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P srfAA and P aprE acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production. IMPORTANCE DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.

Isolation and Identification Antagonistic Bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli.

In this study, we aimed to screen antagonistic microorganisms against Acidovorax citrulli, the causal agent of bacterial fruit blotch, which is known to induce sever diseases in cucurbit crops. From 240 bacterial strains isolated, only one unknown bacterial isolate, named YM002, showed significant antagonistic activity against A. citrulli KACC17909. Further experiments revealed that YM002 shows antagonistic activity against all tested A. citrulli strains, including KACC17000, KACC17001 and KACC17005, to different degrees. The phylogenetic analysis of 16S rRNA sequences identified YM002 as Paenibacillus tianmuensis. Importantly, pretreatment of cucumber (Cucumis sativus) leaves with YM002 enhanced disease resistance as observed by significantly reduced necrotic symptom development and bacterial growth. YM002-induced resistance accompanied by enhanced expression of defense-related genes, such as PAL1, PR1-1a and CTR1. Importantly, culture filtrate of YM002 significantly suppressed biofilm formation and swimming motility of A. citrulli, which is indispensable for its full virulence. In addition to its antagonistic activity, YM002 showed a various plant growth promotion (PGP)-related traits, such as production of ammonia production, amylase production, ACC deaminase production, inodole-3-acetic acid production, extracellular protease production, siderophore production, and zinc solubilization activities. Indeed, treatment of cucumber roots with YM002 significantly enhanced plant growth parameters, such as fresh and dry weight of leaves or roots. This study suggests the potential of YM002 as an effective PGPR with biological control activity against Acidovorax citrulli in cucumber plants.

Antibacterial and Antibiofilm Efficacy and Mechanism of Ginger (Zingiber officinale) Essential Oil against Shewanella putrefaciens.

Ginger (Zingiber officinale) has unique medicinal value and can be used to treat colds and cold-related diseases. The chemical composition and antibacterial activity of ginger essential oil (GEO) against Shewanella putrefaciens were determined in the present study. Zingiberene, α-curcumene, and zingerone were the main active compounds of GEO. GEO displayed significant antibacterial activity against S. putrefaciens, with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 2.0 and 4.0 μL/mL, respectively. Changes in intracellular ATP content, nucleic acid and protein structure, exopolysaccharides (EPS) content, and extracellular protease production indicated that GEO disrupted the membrane integrity of S. putrescens. At the same time, changes in biofilm metabolic activity content and the growth curve of biofilm showed that GEO could destroy the biofilm. Both scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) observations confirmed that GEO destroyed the cell membrane and lead to the leakage of the constituents. The above results indicate that GEO entered the cells via contact with bacterial membranes, and then inhibited the growth of S. putrefaciens and its biofilms by increasing membrane permeability and inhibiting various virulence factors such as EPS. The findings showed that GEO could destroy the structure of cell membrane and biofilm of tested S. putrefaciens, indicating its potential as a natural food preservative.

Salmon oyster mushroom: growth characteristics and protease production on a laboratory scale

Pleurotus have an easy cultivation and are excellent sources of proteolytic enzymes. The aimed was evaluate the mycelial growth and extracellular protease activity of Pleurotus ostreatoroseus. The mushroom was grown on potato dextrose agar (BDA) with yeast extract (YE) 0.5% (w/v). The radial mycelial growth velocity (RCV) and mycelial density occurred on different media containing 0.5% (w/v) YE: Potato Dextrose agar (BDA), GYP agar (glucose, yeast extract and peptone), malt extract agar (MEA), purple yam extract agar (EIR), torn yam extract agar (CEIN), aria extract agar (EA). Proteolytic activity was determined by the gelose block method. In mycelial growth (VCM) is used: cupuaçu exocarp (CC), açaí (Sac), pineapple (CsAb) and sawdust (SER), supplemented with rice bran (FA) or wheat bran (FT) were used. Proteases were extracted in sterile distilled water under 180 rpm agitation at 30 °C. Significant RCV was observed in MEA+YE (12.32 ± 0.10 mm/day). However, GYP agar showed strong mycelial density. Significant substrate mixture with strong mycelial density occurred on Sac+FA (0.61 ± 0.09 cm/day). The tested media were efficient in the production of extracellular proteases by this mushroom.

The major role of sarA in limiting Staphylococcus aureus extracellular protease production in vitro is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

ABSTRACT We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus.