Silversol® (a Colloidal Nanosilver Formulation) Inhibits Growth of Antibiotic-Resistant Staphylococcus aureus by Disrupting Its Physiology in Multiple Ways.

Antibiotic-resistant strains of Staphylococcus aureus are being viewed as a serious threat by various public health agencies. Identifying novel targets in this important pathogen is crucial to the development of new effective antibacterial formulations. We investigated the antibacterial effect of a colloidal nanosilver formulation, Silversol®, against an antibiotic-resistant strain of S. aureus using appropriate in vitro assays. Moreover, we deciphered the molecular mechanisms underlying this formulation's anti-S. aureus activity using whole transcriptome analysis. Lower concentrations of the test formulation exerted a bacteriostatic effect against this pathogen, and higher concentrations exerted a bactericidal effect. Silversol® at sub-lethal concentration was found to disturb multiple physiological traits of S. aureus such as growth, antibiotic susceptibility, membrane permeability, efflux, protein synthesis and export, biofilm and exopolysaccharide production, etc. Transcriptome data revealed that the genes coding for transcriptional regulators, efflux machinery, transferases, ß-lactam resistance, oxidoreductases, metal homeostasis, virulence factors, and arginine biosynthesis are expressed differently under the influence of the test formulation. Genes (argG and argH) involved in arginine biosynthesis emerged among the major targets of Silversol®'s antibacterial activity against S. aureus.

Duration of fermentation affects microbiome composition and biological activity of an Indian traditional formulation - Panchagavya.

OBJECTIVE: This study aimed at investigating whether the duration of fermenting Panchagavya (PG) preparation in copper vessel affects its biological activity and microbiome composition. MATERIALS AND METHODS: Prophylactic potential of PG against bacterial infection was assessed through an in vivo assay employing the nematode worm Caenorhabditis elegans as a model host. Bacterial diversity of the PG samples was revealed through metagenomic analysis. RESULTS: Duration of fermentation was found to affect biological activity as well as microbiome composition of the PG samples. PG-samples fermented ≥60 min lost their prophylactic potential, and develop anthelmintic activity. Bacterial phyla whose relative abundance was significantly different between the prophylactic and anthelmintic PG samples were Planctomycetota, Proteabacteria, Bacteroidota, Verrucomicrobiota, Patescibacteria, Acidobacteriota, Chloroflexi, Firmicutes and Campilobacterota. CONCLUSION: This study validates the prophylactic potential of Panchagavya against bacterial pathogens, and shows that duration of the fermentation time while preparing PG can have profound effect on its biological activities. Biological activities of PG samples seem to have a correlation with their inherent microbial community. Metagenomic profiling can be an effective tool for standardization of PG formulations.

Sub-lethal concentration of a colloidal nanosilver formulation (Silversol®) triggers dysregulation of iron homeostasis and nitrogen metabolism in multidrug resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a notorious pathogen. Its multidrug resistant strains are listed among priority pathogens against whom discovery of novel antibacterial agents and, elucidation of new anti-pathogenicity mechanisms are urgently warranted. This study describes multiple antibacterial effects of a colloidal nano-silver formulation- Silversol® against a multi-drug resistant strain of P. aeruginosa. RESULTS: Minimum inhibitory concentration (MIC) of Silversol® against P. aeruginosa was found to be 1.5 ppm; and at sub-MIC of 1 ppm, it was able to alter quorum-sensing regulated pigmentation (pyocanin 82%↓; pyoverdine 48%↑), exopolysaccharide synthesis (76%↑) and biofilm formation, susceptibility to antibiotics (streptomycin and augmentin), protein synthesis and export (65%↑), nitrogen metabolism (37%↑ nitrite accumulation), and siderophore production in this pathogen. Network analysis of the differentially expressed genes in the transcriptome of the silversol-treated bacterium identified ten genes as the potential molecular targets: norB, norD, nirS, nirF, nirM, nirQ, nosZ, nosY, narK1, and norE (all associated with nitrogen metabolism or denitrification). Three of them (norB, narK1, and norE) were also validated through RT-PCR. CONCLUSIONS: Generation of nitrosative stress and disturbance of iron homeostasis were found to be the major mechanisms associated with anti-Pseudomonas activity of Silversol®.

Identification of anti-pathogenic activity among in silico predicted small-molecule inhibitors of Pseudomonas aeruginosa LasR or nitric oxide reductase (NOR).

Introduction: Antibiotic-resistant Pseudomonas aeruginosa strains cause considerable morbidity and mortality globally. Identification of novel targets in this notorious pathogen is urgently warranted to facilitate discovery of new anti-pathogenic agents against it. This study attempted to identify small-molecule inhibitors of two important proteins LasR and nitric oxide reductase (NOR) in P. aeruginosa. 'Las' system can be said to be the 'master' regulator of quorum sensing in P. aeruginosa, whose receptor protein is LasR. Similarly, NOR is crucial to detoxification of reactive nitrogen species. Methods: In silico identification of potential LasR or NOR inhibitors was attempted through a virtual screening platform AtomNet® to obtain a final subset of <100 top scoring compounds. These compounds were evaluated for their in vivo anti-pathogenic activity by challenging the model host Caenorhabditis elegans with P. aeruginosa in the presence or absence of test compounds. Survival of the worm population in 24-well assay plates was monitored over a period of 5 days microscopically. Results: Of the 96 predicted LasR inhibitors, 11 exhibited anti-Pseudomonas activity (23%-96% inhibition of bacterial virulence as per third-day end-point) at 25-50 µg/mL. Of the 85 predicted NOR inhibitors, 8 exhibited anti-Pseudomonas activity (40%-85% inhibition of bacterial virulence as per second-day end-point) at 25-50 µg/mL. Conclusion: Further investigation on molecular mode of action of compounds found active in this study is warranted. Virtual screening can be said to be a useful tool in narrowing down the list of compounds requiring actual wet-lab screening, saving considerable time and efforts for drug discovery.

Potent anthelmintic activity of a colloidal nano-silver formulation (Silversol®) against the model worm Caenorhabditis elegans.

OBJECTIVE: In the background of a very small number of effective anthelmintics available today with a narrow activity spectrum, and a rise in resistance against them among parasitic helminths, there is an urgent need for discovery of novel broad-spectrum anthelmintics displaying no or minimal toxicity towards the host. Silver being used since centuries for therapeutic purposes and considered safe for human consumption, we investigated anthelmintic activity of a colloidal nanosilver formulation Silversol®. Anthelmintic efficacy of the test formulation was assayed employing the nematode Caenorhabditis elegans as a model worm through a live-dead count. RESULTS: Silversol® exerted anthelmintic action superior to one of the positive controls (Benzimidazole), and almost at par to another positive control (Ivermectin). At concentrations ≥ 2 ppm, it could kill all the worms present in the experimental well. Lower concentrations of silver were found to have a cuticle-damaging action on worms. Further investigation is warranted to assess whether Silversol® can exert similar potent activity against different species of parasitic helminths, and elucidate the underlying molecular mechanisms of action.

Network analysis for identifying potential anti-virulence targets from whole transcriptome of Pseudomonas aeruginosa and Staphylococcus aureus exposed to certain anti-pathogenic polyherbal formulations.

Introduction: Antimicrobial resistance (AMR) is a serious global threat. Identification of novel antibacterial targets is urgently warranted to help antimicrobial drug discovery programs. This study attempted identification of potential targets in two important pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Methods: Transcriptomes of P. aeruginosa and S. aureus exposed to two different quorum-modulatory polyherbal formulations were subjected to network analysis to identify the most highly networked differentially expressed genes (hubs) as potential anti-virulence targets. Results: Genes associated with denitrification and sulfur metabolism emerged as the most important targets in P. aeruginosa. Increased buildup of nitrite (NO2) in P. aeruginosa culture exposed to the polyherbal formulation Panchvalkal was confirmed through in vitro assay too. Generation of nitrosative stress and inducing sulfur starvation seemed to be effective anti-pathogenic strategies against this notorious gram-negative pathogen. Important targets identified in S. aureus were the transcriptional regulator sarA, immunoglobulin-binding protein Sbi, serine protease SplA, the saeR/S response regulator system, and gamma-hemolysin components hlgB and hlgC. Conclusion: Further validation of the potential targets identified in this study is warranted through appropriate in vitro and in vivo assays in model hosts. Such validated targets can prove vital to many antibacterial drug discovery programs globally.

Prevalence of anemia in pre-school tribal children with reference to parasitic infections and nutritional impact.

Objectives: Anemia is a global health problem and has very high prevalence in developing as well as developed countries, particularly in children and women. The present study evaluates hematological predictors, nutrition deficiency, parasitic infections and their association with the prevalence of anemia. This analysis will help to identify the anemic status of tribal preschool children. Methods: This was a cross-sectional study conducted in 300 children (age: 6 months to 5 years) in Santrampur village, Gujarat. Blood was collected and used to determine complete blood count (CBC); we also performed ELISA (enzyme-linked immunoassay) for the estimation of ferritin, transferrin, sTfR (soluble transferrin receptor), vitamin B12 and vitamin B9 (folate). Stool samples were also collected and assessed by ELISA for Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum. Microscopy was used to screen samples for malaria. Results: Of the 300 children analyzed, 87.7% were anemic, 239 children were mildly anemic, 20 were moderately anemic and 4 were severely anemic. Mean Hb level was 9.49 ± 1.47 g/dL; males and females had an Hb level of 9.39 ± 1.59 g/dL and 9.58 ± 1.34 g/dL, respectively. Twenty-six children had sickle cell anemia and five had thalassemia. Over 50% of the children had vitamin B12 and B9 deficiency and 16% had abnormalities in CRP (C-reactive protein) levels. Parasitic infection by C. parvum was positively associated the anemia followed by the prevalence of G. lamblia and E. histolytica. Conclusion: An increased awareness of parents in the improvement of sanitary facilities and nutritional counselling with regards to iron-rich food consumption is recommended to if we are to prevent anemia among pre-school children. To reduce parasitic infestation, effective periodic deworming measures are also recommended.

Identifying the Molecular Targets of an Anti-pathogenic Hydroalcoholic Extract of Punica granatum Peel Against Multidrug-resistant Serratia marcescens.

BACKGROUND: Antibiotic-resistant members of the family Enterobacteriaceae are among the serious threats to human health globally. This study reports the anti-pathogenic activity of Punica granatum peel extract (PGPE) against a multi-drug resistant, beta-lactamase producing member of this family i.e. Serratia marcescens. OBJECTIVE: This study aimed at assessing the anti-pathogenic activity of PGPE against the gramnegative bacterial pathogen S. marcescens and identifying the molecular targets of this extract in the test bacterium. METHODS: Effect of PGPE on S. marcescens growth and quorum sensing (QS)-regulated pigment production was assessed through broth dilution assay. In vivo anti-infective and prophylactic activity of PGPE was assessed employing the nematode worm Caenorhabditis elegans as a model host. Differential gene expression in PGPE-exposed S. marcescens was studied through a whole transcriptome approach. RESULTS: PGPE was able to modulate QS-regulated pigment production in S. marcescens without exerting any heavy growth-inhibitory effect at concentrations as low as ≥2.5 µg/mL. It could attenuate the virulence of the test bacterium towards the worm host by 22-42% (p≤0.01) at even lower concentrations (≥0.5 µg/mL). PGPE also exerted a post-extract effect on S. marcescens. This extract was found to offer prophylactic benefit too, to the host worm, as PGPE-pre-fed worms scored better (34-51%; p≤0.001) survival in face of subsequent bacterial attack. Differential gene expression analysis revealed that PGPE affected the expression of a total of 66 genes in S. marcescens by ≥1.5 fold. CONCLUSION: The anti-virulence effect of PGPE against S. marcescens is multifaceted, affecting stress-response machinery, efflux activity, iron homeostasis, and cellular energetics of this bacterium notably. Among the major molecular targets identified in this study are LPS export transporter permease (LptF), t-RNA pseudouridine synthase (TruB), etc.

Anti-infective potential of a quorum modulatory polyherbal extract (panchvalkal) against certain pathogenic bacteria.

BACKGROUND: In view of the gravity of the problem of antimicrobial resistance among pathogenic bacteria against conventional bactericidal agents, investigation on alternative approaches to combat bacterial infections is warranted. OBJECTIVE: Current study aimed at investigating anti-infective potential of a polyherbal ayurvedic formulation namely panchvalkal against three different pathogenic bacteria. MATERIALS AND METHODS: The panchvalkal formulation available as Pentaphyte P5® was tested for its possible in vitro quorum-modulatory potential against Chromobacterium violaceum, Serratia marcescens, and Staphylococcus aureus through broth dilution assay. Invivo efficacy was demonstrated employing Caenorhabditis elegans as the model host for test pathogens. RESULTS: This formulation was found to exert quorum-modulatory effect on C. violaceum, S. marcescens, and S. aureus at 250-750 µg/ml. Besides altering production of the quorum sensing-regulated pigments in these bacteria, the test formulation also had in vitro effect on antibiotic susceptibility, catalase activity and haemolytic potential of the pathogens. Invivo assay confirmed the protective effect of this panchvalkal formulation on C. elegans, when challenged with the pathogenic bacteria. Repeated exposure of S. aureus to panchvalkal did not induce resistance in this bacterium. CONCLUSION: To the best of our awareness, this the first report on quorum-modulatory potential of panchvalkal formulation, validating the anti-infective potential and moderate prebiotic property of this polyherbal preparation.

The Anti-infective Potential of Hydroalcoholic Extract of Phyllanthus emblica Seeds Against Selected Human-pathogenic Bacteria.

INTRODUCTION: In the context of the global threat of antimicrobial resistance (AMR) among bacterial pathogens against conventional bactericidal antibiotics, investigation on complementary/ alternative approaches to manage bacterial infections is warranted. The present study aimed at investigating the anti-pathogenic potential of Phyllanthus emblica seed extract (PESE) against four different pathogenic bacteria. METHODS: Hydroalcoholic extract of P. emblica seeds was tested for its possible in vitro quorummodulatory potential against Chromobacterium violaceum, Serratia marcescens, Pseudomonas aeruginosa, and Staphylococcus aureus through broth dilution assay. In vivo efficacy of PESE was assayed employing Caenorhabditis elegans as the model host for these four pathogens. RESULTS: PESE was found to exert in vitro quorum-modulatory effect on C. violaceum, S. marcescens, P. aeruginosa, and S. aureus at ≥50 µg/mL. This extract could curb the haemolytic activity of all the four test bacteria by 23-65%, inhibit biofilm formation, and was also able to modulate their antibiotic susceptibility (AS) and catalase activity. Susceptibility of P. aeruginosa and S. aureus to lysis by human serum was enhanced under the influence of this extract by 23% and 49%, respectively. Repeated exposure of both these notorious pathogens to PESE did not induce resistance in them. In vivo assay confirmed the anti-virulence effect of this extract in the C. elegans host, wherein the nematode host challenged with the PESE-treated pathogenic bacteria scored better survival. PESE also displayed notable prebiotic potential by promoting the growth of three probiotic strains. CONCLUSION: To the best of our awareness, this is the first report on the quorum-modulatory potential of P. emblica seed extract, validating its anti-infective potential and prebiotic property.

Supernatants of the Probiotic Bacterial Cultures at sub-MIC Levels Attenuate Virulence of Pathogenic Bacteria Towards the Model Host Caenorhabditis elegans.

OBJECTIVE: This study assessed the anti-pathogenic potential of culture supernatants from three probiotic strains viz. Lactobacillus plantarum, Bifidobacterium bifidum, and Enterococcus faecium against five different gram-positive and gram-negative pathogenic bacteria. METHODS: The effect of probiotic culture supernatants on growth and production of quorum sensing (QS)-regulated pigments was assessed through broth dilution assay. In vivo anti-infective efficacy of these supernatants was evaluated by challenging the model nematode host Caenorhabditis elegans with pathogenic bacteria pre-treated with probiotic culture supernatants, and worm survival was monitored over a period of 8 days. RESULTS: All the probiotic culture supernatants (at 5-10 %v/v) exhibited bactericidal activity against four of the test pathogens (including three multi-drug resistant gram-negative bacteria) and they (at ≥ 1%v/v) could also modulate QS in pathogenic bacteria. Pre-treatment with culture supernatants from all the three probiotic strains were able to attenuate virulence of four of the test pathogens towards C. elegans. Worms challenged with supernatant-pre-treated pathogens recorded 75-100% better survival than those challenged with pathogens receiving no such pretreatment. Virulence-attenuating effect of the culture supernatants was also carried forward to daughter populations of the supernatant-treated parent pathogens. Lactic acid seemed to contribute notably towards anti-virulence activity of the culture supernatants, but acidic pH did not seem to be important for such activity. CONCLUSION: Culture supernatants of probiotic strains used in this study can be said to possess appreciable virulence-attenuating potential. Further investigation for characterization of these supernatants and identification of the bioactive constituents is warranted. Elucidation of the molecular basis associated with the anti-pathogenic potential of these supernatants may lead to the identification of novel targets in pathogenic bacterial populations.

Anti-infective efficacy of Psidium guajava L. leaves against certain pathogenic bacteria.

Water extracts of Psidium guajava leaves prepared by three different extraction methods were compared with respect to their anti-infective activity against Pseudomonas aeruginosa and Staphylococcus aureus in the nematode host Caenorhabditis elegans. The water extract prepared by Microwave Assisted Extraction method was found to have better anti-infective activity, and its activity was further compared with hydroalcoholic extract prepared using the same extraction method against five different pathogenic bacteria. Both these extracts could attenuate virulence of P. aeruginosa, S. aureus, Serratia marcescens, and Chromobacterium violaceum, towards C. elegans. Anti-infective efficacy of P. guajava leaf extract seems partly to stem from its quorum-modulatory property, as it could modulate production of quorum sensing-regulated pigments in all the susceptible bacteria.

Anti-infective potential of hydroalcoholic extract of Punica granatum peel against gram-negative bacterial pathogens.

Background:Punica granatum extracts have been prescribed in traditional medicine for management of a variety of disease conditions including microbial infections. Generation of scientific evidence for validation of P. granatum peel extract's anti-pathogenic efficacy is required. Methods: Hydroalcoholic extract of P. granatum peel (PGPE), prepared by microwave assisted extraction method was evaluated for its quorum-modulatory potential against two different human-pathogenic bacteria viz. Chromobacterium violaceum and Pseudomonas aeruginosa. Results: This extract was able to modulate in vitro production of quorum sensing-regulated pigments in both these test bacteria at ≥5 µg/ml. Virulence traits of P. aeruginosa like haemolytic activity, and biofilm formation were negatively affected by the test extract, and it also made P. aeruginosa more susceptible to lysis by human serum. Antibiotic susceptibility of both test bacteria was modulated owing to pre-treatment with PGPE. Exposure of these test pathogens to PGPE (≥0.5 µg/ml) effectively reduced their virulence towards the nematode Caenorhabditis elegans. Repeated subculturing of P. aeruginosa on PGPE-supplemented growth medium did not induce resistance to PGPE in this notorious pathogen, and this extract was also found to exert a post-extract effect on P. aeruginosa. Individual constituent phytocompounds of PGPE were found to be less efficacious than the whole extract. PGPE seemed to interfere with the signal-response machinery of P. aeruginosa and C. violaceum. PGPE also exhibited notable prebiotic potential by promoting growth of probiotic strains- Bifidobacterium bifidum and Lactobacillus plantarum at ≤50 µg/ml. Conclusions: This study indicates PGPE to be an effective antipathogenic and prebiotic preparation, and validates its therapeutic use mentioned in traditional medicine. This study also emphasizes the need for testing any bioactive extract at broadest possible concentration range, particularly in vivo, so that an accurate picture of dose-response relationship can emerge.

Antipathogenic Potential of a Polyherbal Wound-Care Formulation (Herboheal) against Certain Wound-Infective Gram-Negative Bacteria.

This study investigated antipathogenic efficacy of a polyherbal wound-healing formulation Herboheal against three multidrug-resistant strains of gram-negative bacterial pathogens associated with wound infections. Herboheal was evaluated for its quorum-modulatory potential against three different human-pathogenic bacteria, first in vitro through the broth dilution assay and then in vivo in the model host Caenorhabditis elegans. Herboheal at ≥0.1% v/v was able to inhibit (19-55%) in vitro production of quorum sensing-regulated pigments in all these bacteria and seemed to interfere with bacterial quorum sensing by acting as a signal-response inhibitor. This formulation could compromise haemolytic activity of all three bacteria by ∼18-69% and induced their catalase activity by ∼8-21%. Herboheal inhibited P. aeruginosa biofilm formation up to 40%, reduced surface hydrophobicity of P. aeruginosa cells by ∼9%, and also made them (25%) more susceptible to lysis by human serum. Antibiotic susceptibility of all three bacteria was modulated owing to pretreatment with Herboheal. Exposure of these test pathogens to Herboheal (≥0.025% v/v) effectively reduced their virulence towards the nematode Caenorhabditis elegans. Repeated subculturing of P. aeruginosa on the Herboheal-supplemented growth medium did not induce resistance to Herboheal in this mischievous pathogen, and this polyherbal extract was also found to exert a post-extract effect on P. aeruginosa, wherein virulence of the Herboheal-unexposed daughter cultures, of the Herboheal-exposed parent culture, was also found to be attenuated. Overall, this study indicates Herboheal formulation to be an effective antipathogenic preparation and validates its indicated traditional therapeutic use as a wound-care formulation.

Validation of the anti-infective potential of a polyherbal 'Panchvalkal' preparation, and elucidation of the molecular basis underlining its efficacy against Pseudomonas aeruginosa.

BACKGROUND: A Panchvalkal formulation (Pentaphyte P-5®) mentioned in ancient texts of Indian traditional medicine was investigated for its anti-infective potential against Pseudomonas aeruginosa. METHODS: Effect of the test formulation on bacterial growth and pigment production was evaluated by broth dilution assay. In vivo efficacy was evaluated using Caenorhabditis elegans as the model host. Whole transcriptome approach was taken to study the effect of test formulation on bacterial gene expression. RESULTS: This formulation in vitro was found to be capable of affecting quorum sensing (QS)-regulated traits (pyocyanin, pyoverdine, biofilm) of Pseudomonas aeruginosa. In combination with antibiotics, it enhanced susceptibility of the test bacterium to antibiotics like cephalexin and tetracycline. Effect of Panchvalkal formulation (PF) on QS-regulated traits of P. aeruginosa was not reversed even after repeated exposure of the bacterium to PF. In vivo efficacy of PF was demonstrated employing Caenorhabditis elegans as the model host, wherein PF-treated bacteria were able to kill lesser worms than their extract-unexposed counterparts. Whole transcriptome study revealed that approximately 14% of the P. aeruginosa genome was expressed differently under the influence of PF. CONCLUSIONS: Major mechanisms through which Panchvalkal seems to exert its anti-virulence effect are generation of nitrosative and oxidative stress, and disturbing iron and molybdenum homeostasis, besides interfering with QS machinery. This study is a good demonstration of the therapeutic utility of the 'polyherbalism' concept, so common in ayurved. It also demonstrates utility of the modern 'omics' tools for validating the traditional medicine i.e. ayuromics.

Anti-pathogenic potential of a classical ayurvedic Triphala formulation.

A classical ayurvedic polyherbal formulation namely Triphala was assessed for its anti-pathogenic potential against five different pathogenic bacteria. Virulence of four of them towards the model host Caenorhabditis elegans was attenuated (by 18-45%) owing to pre-treatment with Triphala (≤20 µg/ml). Triphala could also exert significant therapeutic effect on worms already infected with Chromobacterium violaceum, Serratia marcescens or Staphylococcus aureus. Prophylactic use of Triphala allowed worms to score 14-41% better survival in face of subsequent pathogen challenge. Repeated exposure to this formulation induced resistance in S. marcescens, but not in P. aeruginosa. It also exerted a post-extract effect (PEE) on three of the test pathogens. Triphala was able to modulate production of quorum sensing (QS)-regulated pigments in three of the multidrug-resistant gram-negative test bacteria. Haemolytic activity of S. aureus was heavily inhibited under the influence of this formulation. P. aeruginosa's lysozyme-susceptibility was found to increase by ~25-43% upon Triphala-pretreatment. These results validate therapeutic potential of one of the most widely used polyherbal ayurvedic formulations called Triphala.