Bacterial Protease Activity Research Articles

Identification of High-Risk Lower Extremity Wounds Using Point-of-Care Test for Bacterial Protease Activity; A Single-Centre, Single-Blinded, Prospective Study.

Clinician observation is the mainstay to determine if wound infection is present, and focuses on presence of erythema, purulence, and odour. However, non-visible bacterial protease activity can delay wound healing and lead to complications. In this study, a point-of-care test to detect the presence of bacterial protease activity (BPA, tested with Woundchek Bacterial Status test) was appraised. A total of 130 patients with lower extremity wounds were recruited in vascular and podiatry clinics, and across two time-points 182 BPA tests were conducted subsequent to initial (blinded) clinician's wound appraisal. Clinical opinion ('no infection', 'possible' or 'definite' infection) and BPA result (negative or positive test) had a moderate Kendall's tau-c rank correlation coefficient of 0.32 (P < 0.001). Binary logistic regression analysis and principal component analysis showed that infection determined by clinical opinion was significantly associated with abovementioned clinical signs and a positive BPA test. However, a positive BPA result was also significantly linked with wound severity, such as number of lesions, chronicity and size. Throughout a 12-week follow-up period, median ulcer size was larger for wounds positive for BPA test at baseline (P 0.001) and week-12 (P 0.036; both Mann-Whitney U-test) respectively. As a pilot initiative, clinical staff were allowed to act on the BPA result if they wished; in 11 out of 71 test-positive cases (15%) this happened and antimicrobial dressing was applied instead of planned standard dressing. These results show that protease-releasing bacteria may be active in ulcers that do not (yet) exhibit hallmark signs of infection, and are associated with delayed healing. Targeted point-of-care testing for bacterial protease activity may have the potential to identify and enable pro-active (antimicrobial) management of these high-risk wounds.

Compound K14 inhibits bacterial killing and protease activity in Dictyostelium discoideum phagosomes.

Phagocytic cells of the mammalian innate immune system play a critical role in protecting the body from bacterial infections. The multiple facets of this encounter (chemotaxis, phagocytosis, destruction, evasion and pathogenicity) are largely recapitulated in the phagocytic amoeba Dictyostelium discoideum. Here we identified a new chemical compound (K14; ZINC19168591) which inhibited intracellular destruction of ingested K. pneumoniae in D. discoideum cells. Concomitantly, K14 reduced proteolytic activity in D. discoideum phagosomes. In kil1 KO cells, K14 lost its ability to inhibit phagosomal proteolysis and to inhibit intra-phagosomal bacterial destruction, suggesting that K14 inhibits a Kil1-dependent protease involved in bacterial destruction. These observations stress the key role that proteases play in bacterial destruction. They also reveal an unsuspected link between Kil1 and phagosomal proteases. K14 can be used in the future as a tool to probe the role of different proteases in phagosomal physiology and in the destruction of ingested bacteria.

Identifying infection in chronic wounds in a community setting: A systematic review of diagnostic test accuracy studies.

To determine the diagnostic accuracy of different methods currently available to identify infection in chronic wounds applicable to adult patients in a community setting. Systematic review of diagnostic test accuracy studies. Two authors independently completed screening, data extraction and quality and bias assessments (QUADAS2). Eligible studies compared a method (index test) for detecting infection (diagnosis of interest) with microscopy and culture of either deep tissue biopsy or wound swab (reference test) in adult patients with wounds of >4 weeks duration (participants). The results were synthesized narratively. We systematically searched CINAHL, Embase and Medline from 2011 to April 2022. Four studies were included, all recruiting from secondary care wound clinics. Two studies assessed the diagnostic accuracy of Moleculight i:X, a bacterial fluorescence imaging device against deep tissue biopsy culture. One study assessed the diagnostic accuracy of the elevation of various enzymes detected in wound fluid against wound swab microscopy of culture. One study assessed the diagnostic accuracy of bacterial protease activity against wound swab microscopy and culture. Sensitivities of these methods ranged from 50 to 75% and specificities from 47 to 100%. Only a small number of studies were included in this systematic review due to our strict inclusion criteria. We have not identified any methods for diagnosing infection in chronic wounds with either a sufficient quality of evidence to recommend their use in community settings at present. Further research is needed to develop and evaluate appropriate diagnostics for this purpose. This study highlights the paucity of research into wound diagnostics in a community setting and should prompt further research in this area. Accurate diagnostic tests have the potential to improve community-based wound care by optimizing antibiotic use and potentially improving healing time. PRISMA-DTA checklist. The PPI group for the NIHR Community Healthcare MIC were supportive of this topic of work.

Bioluminescence resonance energy transfer biosensor for measuring activity of a protease secreted by Pseudomonas fluorescens growing in milk

Bacterial proteases are sporadic contributors to milk spoilage, reducing the quality of ultra-heat treated (UHT) milk and other dairy products. Current methods for measuring bacterial protease activity in milk are insensitive and too slow to be used in routine testing in dairy processing plants. We have designed a novel bioluminescence resonance energy transfer (BRET)-based biosensor to measure the activity of proteases secreted by bacteria in milk. The BRET-based biosensor is highly selective for bacterial protease activity compared with other proteases tested, notably including plasmin, which is abundant in milk. It incorporates a novel peptide linker that is selectively cleaved by P. fluorescens AprX proteases. The peptide linker is flanked by green fluorescent protein (GFP2) at the N-terminus and a variant Renilla luciferase (RLuc2) at the C-terminus. Complete cleavage of the linker by bacterial proteases from Pseudomonas fluorescens strain 65, leads to a 95% decrease in the BRET ratio. We applied an azocasein-based calibration method to the AprX biosensor using standard international enzyme activity units. In a 10-min assay, the detection limit for AprX protease activity in buffer was equivalent to 40 pg/mL (≈0.8 pM, 22 μU/mL) and 100 pg/mL (≈2pM, 54 μU/mL) in 50% (v/v) full fat milk. The EC50 values were 1.1 ± 0.3 ng/mL (87 μU/mL) and 6.8 ± 0.2 ng/mL (540 μU/mL), respectively. The biosensor was approximately 800x more sensitive than the established FITC-Casein method in a 2-h assay, the shortest feasible time for the latter method. The protease biosensor is sensitive and fast enough to be used in production settings. It is suitable for measuring bacterial protease activity in raw and processed milk, to inform efforts to mitigate the effects of heat-stable bacterial proteases and maximise the shelf-life of dairy products.

Dietary Lactobacillus helveticus and Gum Arabic improves growth indices, digestive enzyme activities, intestinal microbiota, innate immunological parameters, antioxidant capacity, and disease resistance in common carp

The present study aimed at determining the effects of Lactobacillus helveticus (LH), Gum Arabic (GA; natural prebiotic), and their combination as synbiotic on growth performance, digestive enzymes activity, gut microbiota, innate immunity status, antioxidant capacity, and disease resistance against Aeromonas hydrophyla in common carp, Cyprinus carpio for 8 weeks. For this, 735 common carp juveniles (Mean ± standard deviation; 22.51 ± 0.40 g) were fed with 7 different diets including basal diet (C), LH1 (1 × 107 CFU/g), LH2 (1 × 109 CFU/g), GA1 (0.5%), GA2 (1%), LH1+GA1 (1 × 107 CFU/g + 0.5%), and LH2+GA2 (1 × 109 CFU/g + 1%) for 8 weeks. Dietary supplementation with GA and/or LH significantly increased growth performance, WBC, serum total immunoglobulin, superoxide dismutase and catalase activities, skin mucus lysozyme and total immunoglobulin and intestinal lactic acid bacteria. Whereas there were significant improvements in various parameters tested in different treatments, the highest improvement in growth performance, WBC, monocyte/neutrophil percentages, serum lysozyme, alternative complement, glutathione peroxidase and malondialdehyde, skin mucosal alkaline phosphatase, protease, and immunoglobulin, intestinal total bacterial count, protease and amylase activities were observed in the synbiotic treatments, particularly LH1+GA1. After an experimental infection with Aeromonas hydrophila, all experimental treatments exhibited significantly higher survival, compared to the control treatment. The highest survival was related to the synbiotic (particularly LH1+GA1), followed by prebiotic, and probiotic treatments. Overall, synbiotic containing 1 × 107 CFU/g LH + 0.5% GA can improve growth rate and feed efficiency in common carp. Moreover, the synbiotic can improve the antioxidant/innate immune systems and dominate lactic acid bacteria in the fish intestine that may be the reasons of the highest resistance against A. hydrophila infection.

A GntR family transcription factor in Porphyromonas gingivalis regulates bacterial growth, acylpeptidyl oligopeptidase, and gingipains activity.

Porphyromonas gingivalis is a keystone pathogen for periodontitis. The function of the GntR family transcription factor is poorly studied in P. gingivalis. Numerous processes govern bacterial growth. The survival and pathogenicity of P. gingivalis depend heavily on its capacity to acquire amino acids as nutritional sources. In this investigation, a GntR transcription factor, pg1007, was identified in P. gingivalis, the deletion of which significantly inhibited bacterial growth. The mutant strain also exhibited an increased extracellular activity of gingipains and acylpeptidyl oligopeptidase (AOP). Global gene expression profiling revealed that the expression levels of 59 genes were significantly altered in the Δpg1007 mutant, with an upregulation in gene expression for AOP, ABC transporters, and some membrane proteins. In addition, His-PG1007 protein was purified as a recombinant protein from Escherichia coli, and the conserved DNA sequence bound by it was determined using electrophoretic mobility shift assays and DNase I footprinting assays. Consequently, this study demonstrated that pg1007 is a crucial transcription factor in P. gingivalis and regulates the bacterial growth and activity of gingipains and AOP. These findings may enhance our understanding of the regulation of bacterial proliferation and protease activity in P. gingivalis.

Probiotic Characteristics of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus as Influenced by Carao (Cassia grandis)

Carao is considered a functional ingredient since its bioactive compounds are meaningful in nutritional, pharmacological, and medicinal applications. The objective of this study was to determine the effects of carao pulp powder on the bacterial viability, acid tolerance, bile tolerance, and protease activity of S. thermophilus STI-06 and L. bulgaricus LB-12. M17 broth with 0.5% lactose and MRS broth were used for S. thermophilus and L. bulgaricus, respectively, for determining bacterial viability, acid tolerance, and bile tolerance. Skim milk was used to study the protease activity of both bacteria. The carao was added at 0 (control), 1.3, 2.6, and 5.3 (g/L) into the broths and skim milk. The broths were enumerated for bacterial viability (every 2 h), bile tolerance (every 4 h), and acid tolerance (every 30 min), and the skim milk was analyzed for protease activity (every 12 h). The General Linear Model (PROC GLM) was used to analyze the data. The 2.6 g/L and 5.3 g/L usage level of carao improved the acid tolerance of S. thermophilus. Carao did not affect the acid tolerance of L. bulgaricus. The usage of 5.3 g/L of carao significantly improved the bile tolerance and protease activity of both bacteria. However, carao did not affect the viability of either bacteria. Overall, 5.3 g/L of carao with these probiotics could be recommended in fermentation processes.

Halogenated Indoles Decrease the Virulence of Vibrio campbellii in a Gnotobiotic Brine Shrimp Model.

ABSTRACTIndole signaling is viewed as a potential target for antivirulence therapy against antibiotic-resistant pathogens because of its link with the production of virulence factors. This study examined the antimicrobial and antivirulence properties of 44 indoles toward Vibrio campbellii. Based on the results, 17 halogenated indole analogues were selected, as they significantly improved the survival of brine shrimp larvae challenged with V. campbellii. Specifically, 6-bromoindole, 7-bromoindole, 4-fluoroindole, 5-iodoindole, and 7-iodoindole showed a high protective effect, improving the survival of brine shrimp to over 80% even at a low concentration of 10 μM. To explore the impact of selected indole analogues on bacterial virulence phenotypes, swimming motility, biofilm formation, protease activity, and hemolytic activity of V. campbellii were determined. The results showed that all of the 17 selected indole analogues decreased swimming motility at both 10 μM and 100 μM. Most of the indole analogues decreased biofilm formation at a concentration of 100 μM. In contrast, only a slightly decreased protease activity and no effect on hemolytic activity were observed at both concentrations. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system, and the results demonstrate the potential of these compounds in applications aiming at the protection of shrimp from vibriosis, a major disease in aquaculture.IMPORTANCE Bacterial diseases are a major problem in the aquaculture industry. In order to counter this problem, farmers have been using antibiotics, and this has led to the evolution and spread of antibiotic resistance. In order for the aquaculture industry to further grow in a sustainable way, novel and sustainable methods to control diseases are needed. We previously reported that indole signaling is a valid target for the development of novel therapies to control disease caused by Vibrio campbellii and related bacteria, which are among the major bacterial pathogens in aquaculture. In the present study, we identified indole analogues that are more potent in protecting brine shrimp (a model organism for shrimp) from V. campbellii. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system.

Protease Inhibitors from Solanum chacoense Inhibit Pectobacterium Virulence by Reducing Bacterial Protease Activity and Motility.

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato (Solanum tuberosum) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Oak bark (Quercus sp. cortex) protects plants through the inhibition of quorum sensing mediated virulence of Pectobacterium carotovorum.

Bacterial intercellular communication mediated by small diffusible molecules, known as quorum sensing (QS), is a common mechanism for regulating bacterial colonisation strategies and survival. Influence on QS by plant-derived molecules is proposed as a strategy for combating phytopathogens by modulating their virulence. This work builds upon other studies that have revealed plant-derived QS inhibitors extracted from oak bark (Quercus sp.). It was found that co-incubation of Pectobacterium carotovorum VKM-B-1247 with oak bark extract (OBE) reduced the production of acyl-HSL. This was accompanied by a dose-dependent decrease in the bacterial cellulolytic and protease activity. At the transcriptomic level, the OBE treatment suppressed the main QS-related genes expR/expI. Potato tubers pre-treated with OBE showed resistance to a manifestation of soft-rot symptoms. Analysis of the component composition of the OBE identified several biologically active molecules, such as n-hexadecanoic acid, 2,6-di-tert-butyl-4-methylphenol, butylated hydroxytoluene (BHT), gamma-sitosterol, lupeol, and others. Molecular docking of the binding energy between identified molecules and homology models of LuxR-LuxI type proteins allow to identify potential inhibitors. Collectively, obtained results figure out great potential of widely distributed oak-derived plant material for bacterial control during storage of potato.

Effectiveness of testing hard-to-heal wounds for bacterial protease activity: a randomised clinical trial.

The study aimed to evaluate whether using a point-of-care test for bacterial protease activity (BPA) to target antimicrobial dressing use can improve outcomes for hard-to-heal wounds and reduce cost. Wounds asymptomatic for infection and testing positive for BPA were randomly assigned to two weeks' treatment with a silver antimicrobial dressing in addition to standard of care (SoC) (intervention group) or to SoC only (control group). The patient's outcomes were monitored for 12 weeks. The study included 100 wounds. A reduction in annualised nursing resource of 29.0% (95% confidence interval (CI): 1.9-34.1) for hard-to-heal wounds was predicted for the intervention versus control group (44±25.10 intervention group nurse/clinic visits versus 62±31.23 control group nurse/clinic visits; p=0.034). The percentage of patients reporting problems reduced for all EQ5D-3L dimensions for the intervention group, with the largest reductions in 'pain/discomfort' (-36.2%) and 'anxiety/depression' (-19.1%). Prescription of antibiotics fell by 45% for wound-related infections in the intervention group compared with the control group. In the intervention group the number of patients who did not receive a prescription was 37/50 (74%), nine (18%) patients received one prescription and four (8%) patients received two or more prescriptions. In the control group 29/50 (58%) patients did not receive a prescription, 12 (24%) received one prescription and nine (18%) patients received two or more prescriptions; p=0.068. The utility of the BPA test to reduce predicted annualised nursing time was demonstrated. The strong trend towards reduced antibiotic prescribing and improved quality of life for patients with wounds treated for BPA deserves further study.

Bacterial protease activity as a biomarker to assess the risk of non-healing in chronic wounds: Results from a multicentre randomised controlled clinical trial.

Millions worldwide suffer from chronic wounds challenging clinicians and burdening healthcare systems. Bacteria impede wound healing; however, the diagnosis of excessive bacterial burden or infection is elusive. Clinical signs and symptoms of infection are inaccurate and unreliable. This trial evaluated a novel, point-of-care, lateral flow diagnostic designed to detect virulence factors released by the most common bacteria found in chronic wounds. A multicentre prospective cohort clinical trial examined the efficacy of a diagnostic test in detecting bacterial proteases taken from swab samples of chronic venous, arterial, pressure and mixed aetiology chronic wounds. Two hundred and sixty six wounds were included in the analysis of the study. The wounds were tested at the start of the study after which investigators were permitted to use whatever dressings they desired for the next 12 weeks. Healing status at 12 weeks was assessed. The presence of elevated bacterial protease activity decreased the probability of wound healing at 12 weeks. In contrast, a greater proportion of wounds were healed at 12 weeks if they had little or no bacterial protease activity at study start. In addition, the presence of elevated bacterial protease activity increased the time it takes for a wound to heal and increased the risk that a wound would not heal, when compared to the absence of bacterial protease activity. The results of this clinical trial indicate that bacterial protease activity, as detected by this novel diagnostic test, is a valid clinical marker for chronicity in wounds. The diagnostic test offers a tool for clinicians to detect clinically significant bacteria in real time and manage bacteria load before the clinical signs and symptoms of infection are evident.

Novel pathomechanism for spontaneous bacterial peritonitis: disruption of cell junctions by cellular and bacterial proteases

ObjectiveSpontaneous bacterial peritonitis (SBP) is a life-threatening complication of liver cirrhosis with a 1-year mortality of 66%. Bacterial translocation (BT) from the intestine to the mesenteric lymph nodes is crucial...

Evaluation of the combination of a biofilm-disrupting agent and negative pressure wound therapy: a case series

Approximately three million people in the US have hard-to-heal pressure ulcers (PUs), including 10% of hospitalised patients. Healing depends on ulcer stage and patient comorbidities. Despite advances in nutrition and wound care, PUs can take months or years to reach complete closure. To date, clinical studies have focused on single modality therapy. However, there is no one therapy that can address all of the deficits in these complex, hard-to-heal wounds. A commonly used treatment for PUs, negative pressure wound therapy (NPWT), has demonstrated improved healing in Stage 3 and 4 PUs. NPWT entails applying suction to a porous sponge fitted into the wound cavity and sealed with an occlusive dressing. Negative pressure facilitates wound healing by removing wound fluid containing harmful proteases, stimulating the formation of granulation tissue and promoting wound contracture. However, it does not affect biofilm formation. We hypothesised that adding an antibiofilm agent might increase the effectiveness of NPWT in recalcitrant PUs. A prospective case series was conducted in outpatient wound care centres and a skilled nursing facility to examine the combination of a biofilm-disrupting antimicrobial agent (Blast-X, Next Science, US) in combination with NPWT (VAC, 3M, US) in healing and reducing bacterial burden in treatment-resistant pressure ulcers. Patients consented to application of the antibiofilm agent and NPWT three times per week for four weeks. The wounds were measured, imaged for bacteria and tested for host and bacterial protease activity weekly. Of the 10 patients, four dropped out of the study before the end of the four weeks. Of the remaining six, four patients experienced a reduction in wound surface area and volume, reduced protease activity and lower bacterial levels. The results of this study showed that multimodal therapy, including NPWT and biofilm disruption, may restart the healing of stagnant treatment-resistant PUs.

Principles of Best Diagnostic Practice in Tissue Repair and Wound Healing: An Expert Consensus.

Chronic wound treatment currently relies heavily on visual assessment by clinicians; however, the clinical signs and symptoms of infection and inflammation are unreliable in chronic wounds. The specialty of wound care has witnessed the advent of advanced interventions, such as cellular and/or tissue based products (CTP). The success of advanced therapies relies on preparing the wound bed by reducing bacterial burden and inflammation. The lack of diagnostics in chronic wound care leads to uncertainty in the adequacy of wound bed preparation. Recent research suggests that two novel point-of-care diagnostic tests can assist in the detection of chronic inflammation known as elevated neutrophil derived protease activity (EPA) and bacterial pathogenesis known as bacterial protease activity(BPA) in chronic wounds. Despite the evidence, however, clinicians report that incorporating diagnostics into every day practice is challenging and across the globe, they have requested guidance on their use. Methods and Recommendations: A panel of wound care experts, experienced with these tests, met to develop guidelines on their use in wound care practice. The consensus panel concluded that the clinician should test for BPA first. The panel maintained that the risk of invasive infection resulting from the presence of pathogenic bacteria was the greatest threat to the patient’s health. If the BPA test is negative, the panel recommended testing for EPA. In addition, it was suggested that if the wound failed to progress after the elevated BPA was treated and subsequent testing was negative for BPA, the clinician should consider testing for EPA. Conclusions: In this manuscript, the consensus panel suggests pathways for testing, treating, and retesting for EPA and BPA. The panel expects that following the algorithm has the potential to improve healing outcomes, result in more cost-effective use of advanced therapies, and improve antimicrobial stewardship by guiding antimicrobial use.

Diagnostics for Wound Infections

Significance: Infections can significantly delay the healing process in chronic wounds, placing an enormous economic burden on health care resources. Identification of infection biomarkers and imaging modalities to observe and quantify them has seen progress over the years. Recent Advances: Traditionally, clinicians determine the presence of infection through visual observation of wounds and confirm their diagnosis through wound culture. Many laboratory markers, including C-reactive protein, procalcitonin, presepsin, and bacterial protease activity, have been quantified to assist diagnosis of infection. Moreover, imaging modalities like plain radiography, computed tomography, magnetic resonance imaging, ultrasound imaging, spatial frequency domain imaging, thermography, autofluorescence imaging, and biosensors have emerged for real-time wound infection diagnosis and showed their unique advantages in deeper wound infection diagnosis. Critical Issues: While traditional diagnostic approaches provide valuable information, they are time-consuming and depend on clinicians' experiences. There is a need for noninvasive wound infection diagnostics that are highly specific, rapid, and accurate, and do not require extensive training. Future Directions: While innovative diagnostics utilizing various imaging instrumentation are being developed, new biomarkers have been investigated as potential indicators for wound infection. Products may be developed to either qualitatively or quantitatively measure these biomarkers. This review summarizes and compares all available diagnostics for wound infection, including those currently used in clinics and still under development. This review could serve as a valuable resource for clinicians treating wound infections as well as patients and wound care providers who would like to be informed of the recent developments.

Unraveling the anti-biofilm potential of green algal sulfated polysaccharides against Salmonella enterica and Vibrio harveyi.

One of the main reasons for the bacterial resistance to antibiotics is caused by biofilm formation of microbial pathogens during bacterial infections. Salmonella enterica and Vibrio harveyi are known to form biofilms and represent a major health concern worldwide, causing human infections responsible for morbidity and mortality. The current study aims to investigate the effect of purified sulfated polysaccharides (SPs) from Chlamydomonas reinhardtii (Cr) on planktonic and biofilm growth of these bacteria. The effect of Cr-SPs on bacterial planktonic growth was assessed by using the agar well diffusion method, which showed clear zones ranging from 13 to 26mm in diameter from 0.5 to 8mg/mL of Cr-SPs against both the bacteria. Time-kill activity and reduction in clonogenic propagation further help to understand the anti-microbial potential of Cr-SPs. The minimum inhibitory concentration of Cr-SPs against S. enterica and V. harveyi was as low as 440μg/mL and 490μg/mL respectively. Cr-SPs inhibited bacterial cell attachment up to 34.65-100% at 0.5-8mg/mL in S. enterica and V. harveyi respectively. Cr-SPs also showed 2-fold decrease in the cell surface hydrophobicity, indicating their potential to prevent bacterial adherence. Interestingly, Cr-SPs efficiently eradicated the preformed biofilms. Increased reduction in total extracellular polysaccharide (EPS) and extracellular DNA (eDNA) content in a dose-dependent manner demonstrates Cr-SPs ability to interact and destroy the bacterial EPS layer. SEM analysisshowed that Cr-SPs effectively distorted preformed biofilms and also induced morphological changes. Furthermore, Cr-SPs also showed anti-quorum-sensing potential by reducing bacterial urease and protease activities. These results indicate the potential of Cr-SPs as an anti-biofilm agent and will help to develop them as alternative therapeutics against biofilm-forming bacterial infections. KEY POINTS: • Cr-SPs not only inhibited biofilm formation but also eradicated preformed biofilms. • Cr-SPs altered bacterial cell surface hydrophobicity preventing biofilm formation. • Cr-SPs efficiently degraded eDNA of the EPS layer disrupting mature biofilms. • Cr-SPs reduced activity of quorum-sensing-mediated enzymes like protease and urease.

MEDIUM OPTIMIZATION FOR PROTEASE PRODUCTION VIA MODERATELY HALOTOLERANT VIRGIBACILLUS PANTOTHENTICUS USING RESPONSE SURFACE METHODOLOGY

Virgibacillus pantothenticus is an industrially promising, yet scarcely studied, moderately halotolerant microorganism (up to 10% NaCl) with high activity protease production potential. Following Response Surface Methodology, we employed a Central Composite Design for the experiments and constructed a second order polynomial model to represent the resulting data. For medium optimization for protease production we optimized the resulting model. 32 experiments (following the central composite design scheme) where, carbon (glucose), nitrogen (ammonium sulfate), potassium (potassium phosphate monobasic) and magnesium (magnesium sulfate) sources were studied in the media. Bacterial growth, residual glucose and protease activities were determined at the 48th hour of each experiment. The model was optimized and the concentrations were found for each parameter. Under the optimum conditions, the predicted protease activity is also experimentally verified and the model prediction was in very good agreement with experimental results. The interactions between medium components and their effect on cell growth and protease production are also sought. This work reports the improvements on protease production of a potentially interesting industrial host, Virgibacillus pantothenticus.

Human and Bacterial Protease Activity as Prognostic Tool of Foot Infections in Diabetic Patients

Human and Bacterial Protease Activity as Prognostic Tool of Foot Infections in Diabetic Patients

The Lord of the Bacteria: The Fellowship of the Leader and Other Serine Protease Inhibitors.

Since antibiotics use is currently limited due to undesired side effects and the increasing antibiotic resistance of various bacteria strains, there is a pressing need to develop new strategies and methods preventing epidemic outbreaks. The virulent potency of bacteria relies on a number of different extracellularly secreted factors among which proteases considered as promising, novel drug targets are of special interest. The first evidence that bacterial cysteine, serine and metalloproteinases contributed to the progression of infection was found in the early 70's. This extracellular proteolytic system allows bacteria to penetrate into tissues, escape detection by the host's immune mechanisms and grow despite limited access to nutrition. A molecule able to selectively inhibit the activity of bacterial proteases in the spread of infection may lead to designing novel therapeutics. Moreover, due to their mechanism of action, bacterial protease inhibitors can be used to fight antibiotic-resistant strains. Herein, we undertake a review of various bacterial proteases together with the design and development of their inhibitors (excluding β-lactams) for the last ten years, and introduce the reader to a brief history of the subject.