Global Genome Comparative Analysis Reveals Insights of Resistome and Life-Style Adaptation of Pseudomonas putida

Most Pseudomonas putida strains are environmental microorganisms exhibiting a wide range of metabolic capability but certain strains have been reported as rare opportunistic pathogens and some emerged as multidrug resistant P. putida. This study aimed to assess the drug resistance profile of, via whole genome analysis, P. putida strain T2-2 isolated from oral cavity. At the same time, we also compared the nonenvironmental strain with environmentally isolated P. putida. In silico comparative genome analysis with available reference strains of P. putida shows that T2-2 has lesser gene counts on carbohydrate and aromatic compounds metabolisms, which suggested its little versatility. The detection of its edd gene also suggested T2-2’s catabolism of glucose via ED pathway instead of EMP pathway. On the other hand, its drug resistance profile was observed via in silico gene prediction and most of the genes found were in agreement with drug-susceptibility testing in laboratory by automated VITEK 2. In addition, the finding of putative genes of multidrug resistance efflux pump and ATP-binding cassette transporters in this strain suggests a multidrug resistant phenotype. In summary, it is believed that multiple metabolic characteristics and drug resistance in P. putida strain T2-2 helped in its survival in human oral cavity

Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene

Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum-sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homlogue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl homoserine lactone and 3-hydroxy dodecanoyl homoserine lactone from induced E.coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Functional analysis of fibroblast-activating factor from porphyromonas gingivalis W50

Periodontal diseases are complex, multifactorial, polymicrobial infections characterized by the destruction of tooth-supporting tissues. The disease begins as acute inflammation of the gingival tissue and untreated infections can progress to formation of teeth pockets, and eventually loss of teeth. According to the World Health Organization, periodontal disease affects 10 - 15% of adult populations worldwide and it was shown that periodontitis also enhances the risk for cardiovascular diseases and diabetes. While human subgingival plaque harbors more than 500 bacterial species, considerable evidence points to Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, as the major etiologic agent of chronic periodontitis. This black-pigmented bacteria produces a myriad of virulence factors that apparently contribute to periodontal tissue destruction either directly or indirectly by modulating the host inflammatory response. Among them is fibroblast-activating factor (FAF) encoded by the gene, faf. This 24 kDa outer-membrane protein affects the supporting tissue of the periodontium, among which is the proliferative stimulating effect on human gingival fibroblasts (HGFs). It has been shown that FAF undergoes post-translational processing in which the N-terminal signal peptide is cleaved before the protein is secreted. Both full length faf and faf without the signal peptide sequence (designated δfaf) was amplified and cloned into pET28a for overexpression in E. coli BL21. SDS-PAGE analysis illustrated a higher expression level of δFAF than its full-length counterparts. An optimum expression level was obtained at 0.5 mM IPTG for 16 h at 25ºC. Both variants of purified recombinant FAF proteins at concentrations between 0.5 - 1.5 μg/ml significantly enhanced cell proliferation and DNA synthesis in mouse embryonic fibroblasts (NIH3T3), human dermal fibroblasts (HDFn) and HGF in a time- and dose-dependent manner. The biological activity of the proteins is suggestive of a specific target cell effect as several cancerous cell lines (MCF7 and HeLa) and T-lymphocytes (Jurkat) were not responsive to both recombinant proteins. The recombinant proteins were also not able to revive fibroblasts under 24 h starvation. On the other hand, both FAF variants and growth factors (EGF and FGF) showed a synergistic effect on the proliferation of fibroblasts. However, the biological activities of the proteins were abolished when heated to 60°C or in the presence of selective protease inhibitors. In addition, quantitative real-time PCR analysis demonstrated that both recombinant proteins induced a down-regulation of collagen synthesis in the fibroblasts while bone resorption assays showed an increase in calcium release in mouse calvarial culture. This ultimately causes destruction of the connective tissue attachment to the root surface and resorption of the bone. Meanwhile, treating the human fibroblasts HGF with both recombinant proteins triggered the secretion of proinflammatory cytokine, IL-8, and the synthesis was sustained for almost a week. However, the secondary messenger, cAMP, appeared not to be involved in the signal transduction and hence, the activity of both FAFC and δFAFN in the host cells. Immunohistochemical studies successfuly showed the binding of both recombinant proteins on the cell surface receptor of the fibroblasts. However, the isolation of the binding partner of δFAF protein from the host membrane fractions using pull-down assay was not successsful. To determine the localization of the functional domains of FAF, several truncation mutants were constructed. A mutant construct, FAF∆(121-139), showed that a deletion of a peptide fragment spanning residues 121 – 139 rendered the FAF inactive, suggesting that these residues are likely involved in modulating the protein’s biological activity

Functional analysis of fibroblast-activating factor from porphyromonas gingivalis W50

Periodontal diseases are complex, multifactorial, polymicrobial infections characterized by the destruction of tooth-supporting tissues. The disease begins as acute inflammation of the gingival tissue and untreated infections can progress to formation of teeth pockets, and eventually loss of teeth. According to the World Health Organization, periodontal disease affects 10 - 15% of adult populations worldwide and it was shown that periodontitis also enhances the risk for cardiovascular diseases and diabetes. While human subgingival plaque harbors more than 500 bacterial species, considerable evidence points to Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, as the major etiologic agent of chronic periodontitis. This black-pigmented bacteria produces a myriad of virulence factors that apparently contribute to periodontal tissue destruction either directly or indirectly by modulating the host inflammatory response. Among them is fibroblast-activating factor (FAF) encoded by the gene, faf. This 24 kDa outer-membrane protein affects the supporting tissue of the periodontium, among which is the proliferative stimulating effect on human gingival fibroblasts (HGFs). It has been shown that FAF undergoes post-translational processing in which the N-terminal signal peptide is cleaved before the protein is secreted. Both full length faf and faf without the signal peptide sequence (designated δfaf) was amplified and cloned into pET28a for overexpression in E. coli BL21. SDS-PAGE analysis illustrated a higher expression level of δFAF than its full-length counterparts. An optimum expression level was obtained at 0.5 mM IPTG for 16 h at 25ºC. Both variants of purified recombinant FAF proteins at concentrations between 0.5 - 1.5 μg/ml significantly enhanced cell proliferation and DNA synthesis in mouse embryonic fibroblasts (NIH3T3), human dermal fibroblasts (HDFn) and HGF in a time- and dose-dependent manner. The biological activity of the proteins is suggestive of a specific target cell effect as several cancerous cell lines (MCF7 and HeLa) and T-lymphocytes (Jurkat) were not responsive to both recombinant proteins. The recombinant proteins were also not able to revive fibroblasts under 24 h starvation. On the other hand, both FAF variants and growth factors (EGF and FGF) showed a synergistic effect on the proliferation of fibroblasts. However, the biological activities of the proteins were abolished when heated to 60°C or in the presence of selective protease inhibitors. In addition, quantitative real-time PCR analysis demonstrated that both recombinant proteins induced a down-regulation of collagen synthesis in the fibroblasts while bone resorption assays showed an increase in calcium release in mouse calvarial culture. This ultimately causes destruction of the connective tissue attachment to the root surface and resorption of the bone. Meanwhile, treating the human fibroblasts HGF with both recombinant proteins triggered the secretion of proinflammatory cytokine, IL-8, and the synthesis was sustained for almost a week. However, the secondary messenger, cAMP, appeared not to be involved in the signal transduction and hence, the activity of both FAFC and δFAFN in the host cells. Immunohistochemical studies successfuly showed the binding of both recombinant proteins on the cell surface receptor of the fibroblasts. However, the isolation of the binding partner of δFAF protein from the host membrane fractions using pull-down assay was not successsful. To determine the localization of the functional domains of FAF, several truncation mutants were constructed. A mutant construct, FAF∆(121-139), showed that a deletion of a peptide fragment spanning residues 121 – 139 rendered the FAF inactive, suggesting that these residues are likely involved in modulating the protein’s biological activity

Porphyromonas gingivalis:An overview of periodontopathic pathogen below the gum line

Periodontal disease represents a group of oral inflammatory infections initiated by oral pathogens which exist as a complex biofilms on the tooth surface and cause destruction to tooth supporting tissues. The severity of this disease ranges from mild and reversible inflammation of the gingiva (gingivitis) to chronic destruction of connective tissues, the formation of periodontal pocket and ultimately result in loss of teeth. While human subgingival plaque harbors more than 500 bacterial species, considerable research has shown that Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, is the major etiologic agent which contributes to chronic periodontitis. This black-pigmented bacterium produces a myriad of virulence factors that causes destruction to periodontal tissue either directly or indirectly by modulating the host inflammatory response. Here, this review provides an overview of P. gingivalis and how its virulence factors contribute to the pathogenesis with other microbiome consortium in oral cavity

Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity

Isolation and characterisation of a proanthocyanidin with antioxidative, antibacterial and anti-cancer properties from fern Blechnum orientale

BACKGROUND: Blechnum orientale Linn. (Blechnaceae), a fern, is traditionally used in the treatment of various ailments, such as skin diseases, stomach pain, urinary bladder complaints, and also as a female contraceptive. Previously, we reported a strong radical scavenging activity, antibacterial activity and cytotoxicity against HT29 colon cancer cells by aqueous extract of B. orientale. OBJECTIVE: In this study, we attempted to isolate and identify the active compound from the aqueous extract of B. orientale. MATERIALS AND METHODS: Aqueous extract of B. orientale was subjected to repeated MCI gel chromatography, Sephadex-LH-20, Chromatorex C18 and semi-preparative high performance liquid chromatography and was characterized using nuclear magnetic resonance and electrospray ionization mass-spectrometry spectroscopic methods. Antioxidant activity was determined using 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay. Antibacterial assays were conducted using disc diffusion whereas the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined using the broth microdilution assay. Cytotoxicity was assessed using thiazolylblue tetrazoliumbromide. RESULTS: A polymeric proanthocyanidin consisting of 2-12 epicatechin extension units and epigallocathecin terminal units linked at C4-C8 was elucidated. Bioactivity studies showed strong radical scavenging activity (IC(50) = 5.6 ± 0.1 µg/mL), antibacterial activity (MIC = 31.3-62.5 µg/mL) against five gram-positive bacteria and selective cytotoxicity against HT29 colon cancer cells (IC(50) = 7.0 ± 0.3 µg/mL). CONCLUSION: According to our results, the proanthocyanidin of B. orientale demonstrated its potential as a natural source of antioxidant with antibacterial and anti-cancer properties. SUMMARY: A bioactive proanthocyanidin was isolated from the aqueous extract of medicinal fern Blechnum orientale Linn and the structure was elucidated using NMR and ESI-MS spectral studies. The proanthocyanidin compound possessed strong radical scavenging activity (IC(50) 5.6 ± 0.1 µg/mL). The proanthocyaniding compound showed bactericidal activity against five gram-positive bacteria inclusive of MRSA (minimum inhibitory concentration, MIC and minimum bactericidal concentration, MBC 31.3-62.5 µg/mL). The proanthocyanidin compound is strongly cytotoxic towards cancer cells HT29 (IC(50) 7.0 ± 0.3 µg/mL), HepG2 (IC(50) 16 µg/mL) and HCT116 (IC(50) 20 µg/mL) while weakly cytotoxic towards the non-malignant Chang cells (IC(50) 48 µg/mL). Abbreviation used: CC: Column chromatography, DP: degree of polymerization, DPPH: 2,2-diphenyl-1-picrylhydrazyl, ESI-MS: electronsprayionisation mass-spectrometry, MBC: Minimum bactericidal concentration, MIC: Minimum inhibitory concentration, MTT: Thiazolyl Blue Tetrazolium Bromide, MRSA: methicillin-resistant Staphylococcus aureus, NMR: nuclear magnetic resonance, TLC: thin layer chromatography, PD: prodelphinidi