Bruguiera gymnorhiza (L.) Lam. at the Forefront of Pharma to Confront Zika Virus and Microbial Infections-An In Vitro and In Silico Perspective.

The recent emergence of Zika virus (ZIKV) in Brazil and the increasing resistance developed by pathogenic bacteria to nearly all existing antibiotics should be taken as a wakeup call for the international authority as this represents a risk for global public health. The lack of antiviral drugs and effective antibiotics on the market triggers the need to search for safe therapeutics from medicinal plants to fight viral and microbial infections. In the present study, we investigated whether a mangrove plant, Bruguiera gymnorhiza (L.) Lam. (B. gymnorhiza) collected in Mauritius, possesses antimicrobial and antibiotic potentiating abilities and exerts anti-ZIKV activity at non-cytotoxic doses. Microorganisms Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, Proteus mirabilis ATCC 25933, Bacillus cereus ATCC 11778 and Candida albicans ATCC 26555 were used to evaluate the antimicrobial properties. Ciprofloxacin, chloramphenicol and streptomycin antibiotics were used for assessing antibiotic potentiating activity. ZIKVMC-MR766NIID (ZIKVGFP) was used for assessing anti-ZIKV activity. In silico docking (Autodock 4) and ADME (SwissADME) analyses were performed on collected data. Antimicrobial results revealed that Bruguiera twig ethyl acetate (BTE) was the most potent extract inhibiting the growth of all nine microbes tested, with minimum inhibitory concentrations ranging from 0.19-0.39 mg/mL. BTE showed partial synergy effects against MRSA and Pseudomonas aeruginosa when applied in combination with streptomycin and ciprofloxacin, respectively. By using a recombinant ZIKV-expressing reporter GFP protein, we identified both Bruguiera root aqueous and Bruguiera fruit aqueous extracts as potent inhibitors of ZIKV infection in human epithelial A549 cells. The mechanisms by which such extracts prevented ZIKV infection are linked to the inability of the virus to bind to the host cell surface. In silico docking showed that ZIKV E protein, which is involved in cell receptor binding, could be a target for cryptochlorogenic acid, a chemical compound identified in B. gymnorhiza. From ADME results, cryptochlorogenic acid is predicted to be not orally bioavailable because it is too polar. Scientific data collected in this present work can open a new avenue for the development of potential inhibitors from B. gymnorhiza to fight ZIKV and microbial infections in the future.

Does washing medical devices before and after use decrease bacterial contamination?: An in vitro study.

ABSTRACT: Surface treatment of medical devices may be a way of avoiding the need for replacement of these devices and the comorbidities associated with infection. The aim of this study was to evaluate whether pre- and postcontamination washing of 2 prostheses with different textures can decrease bacterial contamination.The following microorganisms were evaluated: Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis and Enterococcus faecalis. Silicone and expanded polytetrafluoroethylene vascular prostheses were used and divided into 3 groups: prostheses contaminated; prostheses contaminated and treated before contamination; and prostheses contaminated and treated after contamination. Treatments were performed with antibiotic solution, chlorhexidine and lidocaine. After one week of incubation, the prostheses were sown in culture medium, which was incubated for 48 hours. The area of colony formation was evaluated by fractal dimension, an image analysis tool.The antibiotic solution inhibited the growth of S epidermidis and chlorhexidine decrease in 53% the colonization density for S aureus in for both prostheses in the pre-washing. In postcontamination washing, the antibiotic solution inhibited the growth of all bacteria evaluated; there was a 60% decrease in the colonization density of S aureus and absence of colonization for E faecalis with chlorhexidine; and lidocaine inhibited the growth of S aureus in both prostheses.Antibiotic solution showed the highest efficiency in inhibiting bacterial growth, especially for S epidermidis, in both washings. Lidocaine was able to reduce colonization by S aureus in post-contamination washing, showing that it can be used as an alternative adjuvant treatment in these cases.

Phytochemical, antimicrobial and time-kill kinetics potentials of Euphorbia nivulia Buch.-Ham.: A Cholistan desert medicinal plant.

Euphorbia nivulia a locally occurring plant species possesses antiseptic, analgesic and anti-inflammatory properties and is ethnopharmacologically used in various ailments like skin, ear disorders, boils, and worm infestation. Preliminary phytochemical screening showed presence of flavonoids, polyphenolics, glycosides, alkaloids, tannins and triterpenoids in (70% aqueous-ethanolic) Euphorbia nivulia crude extract (En cr) and its four fractions, i.e., hexane fraction (En hex), butanol fraction (En bt), chloroform fraction (En ch), and aqueous fraction (En aq). In current study, Agar well diffusion and time-kill kinetic assays were performed for antimicrobial activity. 300 mg/ml concentration showed maximum inhibitory zone. Highest zone of inhibition (15.5mm) was demonstrated by En ch fraction against Proteus mirabilis. Staphyllococcus aureus was the most sensitive bacteria against whom all fractions except En aq fraction were active. Maximum MIC (15.3 mg/ml) was shown by En ch fraction against Proteus mirabilis. Similarly, En ch fraction showed (15.1 mg/ml) remarkable MIC against Candida albicans. Significant higher antibacterial and antifungal activity was revealed in high concentration. Time-kill kinetics studies revealed bacteriostatic action. Noteworthy antimicrobial activity may be due to bioactive compounds of extract which may be a potential antibacterial and antifungal agent.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

Molecular ß-lactamase characterization of Gram-negative pathogens recovered from patients enrolled in the ceftazidime-avibactam phase 3 trials (RECAPTURE 1 and 2) for complicated urinary tract infections: Efficacies analysed against susceptible and resistant subsets.

This study characterized the ß-lactamase content of baseline pathogens recovered from patients with complicated urinary tract infections (cUTI), including acute pyelonephritis, who were enrolled in two phase 3 clinical trials of ceftazidime-avibactam (RECAPTURE 1 and 2), and correlated the clinical efficacy of ceftazidime-avibactam and the comparator doripenem according to resistance mechanisms. A total of 26.2% (93/355) ceftazidime-avibactam and 26.8% (101/377) doripenem patients had baseline isolates that met the MIC screening criteria. The majority of Enterobacteriaceae (87.5%; 154/176) carried blaCTX-M. This pattern was mainly observed in Escherichia coli (96.8%; 92/95) and Klebsiella pneumoniae (96.0%; 48/50), whereas most Proteus mirabilis (80.0%; 8/10) carried plasmid AmpC genes. Two K. pneumoniae and 1 Klebsiella oxytoca carried blaOXA-48 and 1 K. pneumoniae carried blaNDM-1. Five (13/35; 37.1%) Pseudomonas aeruginosa isolates were screened, and 2 carbapenemase producers (IMP-18 and VIM-2) were detected. Among patients enrolled in the ceftazidime-avibactam arm who were infected by MIC screen-positive Enterobacteriaceae, clinical cure occurred in 85.7-95.5%, regardless of ß-lactamase content; the respective rate in the doripenem arm was 82.1-92.5%. A total of 75.0% in the ceftazidime-avibactam arm and 100.0% in the doripenem arm of patients infected by P. aeruginosa with MIC screen-positive criteria were clinically cured. Ceftazidime-avibactam efficacy was comparable to doripenem efficacy for treating cUTI caused by uropathogens producing extended-spectrum and/or AmpC ß-lactamases.

Activity of Salvia dolomitica and Salvia somalensis Essential Oils against Bacteria, Molds and Yeasts.

Essential oils (EOs) from Salvia dolomitica and Salvia somalensis, widely employed in the cosmetic and perfume industry, were analyzed for composition and tested against bacterial and fungal pathogens isolated from clinical and environmental specimens. The analyses were carried out against Staphylococcus aureus, Staphylococcus pseudointermedius, Pseudomonas aeruginosa, Escherichia coli, Streptococcus canis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus mirabilis, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Aspergillus niger, Aspergillus flavus, Candida albicans, Candida krusei, Mucor sp. and Trichothecium roseum. Both EOs showed similar percentages of total monoterpenes and sesquiterpene hydrocarbons. The main constituents were 1,8-cineole and ß-caryophyllene in S. dolomitica and bornyl acetate and camphor in S. somalensis. The selected EOs have no relevant antifungal or antibacterial activities if compared to conventional drugs.

Evaluation of the in vitro growth of urinary tract infection-causing gram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU) medium.

Bacteriuria is a hallmark of urinary tract infection (UTI) and asymptomatic bacteriuria (ABU), which are among the most frequent infections in humans. A variety of gram-negative and gram-positive bacteria are associated with these infections but Escherichia coli contributes up to 80% of cases. Multiple bacterial species including E. coli can grow in human urine as a means to maintain colonization during infections. In vitro bacteriuria studies aimed at modeling microbial growth in urine have utilized various compositions of synthetic human urine (SHU) and a Composite SHU formulation was recently proposed. In this study, we sought to validate the recently proposed Composite SHU as a medium that supports the growth of several bacterial species that are known to grow in normal human urine and/or artificial urine. Comparative growth assays of gram-negative and gram-positive bacteria E. coli, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus agalactiae, Staphylococcus saprophyticus and Enterococcus faecalis were undertaken using viable bacterial count and optical density measurements over a 48h culture period. Three different SHU formulations were tested in various culture vessels, shaking conditions and volumes and showed that Composite SHU can support the robust growth of gram-negative bacteria but requires supplementation with 0.2% yeast extract to support the growth of gram-positive bacteria. Experiments are also presented that show an unexpected but major influence of P. mirabilis towards the ability to measure bacterial growth in generally accepted multiwell assays using absorbance readings, predicted to have a basis in the release of volatile organic compound(s) from P. mirabilis during growth in Composite SHU medium. This study represents an essential methodological validation of a more chemically defined type of synthetic urine that can be applied to study mechanisms of bacteriuria and we conclude will offer a useful in vitro model to investigate the basis of some of the most common infections of humans.

Cranberry derivatives enhance biofilm formation and transiently impair swarming motility of the uropathogen Proteus mirabilis HI4320.

Proteus mirabilis is a major cause of catheter-associated urinary tract infection (CAUTI), emphasizing that novel strategies for targeting this bacterium are needed. Potential targets are P. mirabilis surface-associated swarming motility and the propensity of these bacteria to form biofilms that may lead to catheter blockage. We previously showed that the addition of cranberry powder (CP) to lysogeny broth (LB) medium resulted in impaired P. mirabilis swarming motility over short time periods (up to 16 h). Herein, we significantly expanded on those findings by exploring (i) the effects of cranberry derivatives on biofilm formation of P. mirabilis, (ii) whether swarming inhibition occurred transiently or over longer periods more relevant to real infections (∼3 days), (iii) whether swarming was also blocked by commercially available cranberry juices, (iv) whether CP or cranberry juices exhibited effects under natural urine conditions, and (v) the effects of cranberry on medium pH, which is an indirect indicator of urease activity. At short time scales (24 h), CP and commercially available pure cranberry juice impaired swarming motility and repelled actively swarming bacteria in LB medium. Over longer time periods more representative of infections (∼3 days), the capacity of the cranberry material to impair swarming diminished and bacteria would start to migrate across the surface, albeit by exhibiting a different motility phenotype to the regular "bull's-eye" swarming phenotype of P. mirabilis. This bacterium did not swarm on urine agar or LB agar supplemented with urea, suggesting that any potential application of anti-swarming compounds may be better suited to settings external to the urine environment. Anti-swarming effects were confounded by the ability of cranberry products to enhance biofilm formation in both LB and urine conditions. These findings provide key insights into the long-term strategy of targeting P. mirabilis CAUTIs.

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated.

Antibiotic resistance pattern among biofilm producing and non producing Proteus strains isolated from hospitalized patients; matter of hospital hygiene and antimicrobial stewardship.

A retrospective study on antimicrobial susceptibility and biofilm production were carried out for eighty eight strains of Proteus strains isolated from UTI and other hospital samples during April 2011-April 2012. The antibiotic susceptibility was carried out by Kirby-Bauer disk diffusion and MIC by E-test. Biofilm production was measured by microtiter method and confirmed by Scanning electron microscopy. Plasmids from biofilm producing isolates were detected by alkaline lysis technique. From 88 patients infected by proteus species, 58% were female and 42% were mail. The most frequent age range was 20-29 (77.39%) and the least were 60-69 years old (3.4%) (p = 0.05). Eighty one isolates were identified as P. mirabilis while, 7 identified as P. vulgaris. 67.04% [n = 59] of the isolates showed MIC range (16-32 +/- 0.05 microg mL(-1)) to ceftriaxone, 46.59% [n = 41] exhibited least MIC range to chloramphenicol (8-64 +/- 0.08 microg mL(-1)). 31% [n = 28] of the isolates also exhibited MIC range 1-4 microg mL(-1) to ciprofloxacin. 17% [n = 15] of the isolates exhibited strong biofilm while, 6% [n = 6] did not show any biofilm (p < or = 0.05). Plasmid isolation from biofilm producing isolates revealed that stains number 19, 24 and 87' that produced strong biofilm carried similar high M. Wt. plasmid. From above results it can be concluded that the majority of Proteus isolated from UTI patients were belong to P. mirabilis. Ciprofloxacin was the most effective antibiotic for treatment of the infected patients. Limited number of the isolates could produce strong biofilm that were bearing plasmids. Majority of the biofilm producing isolates were also resistance at least to 4 antibiotics routinely prescribed in our hospital.

Inhibition of development, swarming differentiation and virulence factors in Proteus mirabilis by an extract of Lithrea molleoides and its active principle (Z,Z)-5-(trideca-4',7'-dienyl)-resorcinol.

Antibacterial activity of Lithrea molleoides extract against Proteus mirabilis has been previously reported by our group. In the present study, the compound (Z,Z)-5-(trideca-4',7'-dienyl)-resorcinol (1) was isolated as its responsible active principle. The effects of the compound obtained and of L. molleoides extract on P. mirabilis growth and virulence factors were evaluated. Compound 1 showed MIC and MBC values of 4000 µg/ml. It was found that the extract, at four times the MIC, produced complete killing of the uropathogen at 2h from the beginning of the experiment, while the alkylresorcinol, at four times the MIC, produced the same effect after 24 h. Hemolysis was adversely affected in treatments with both products at 8 µg/ml, while hemagglutination was not altered. The whole extract induced complete autoaggregation of P. mirabilis at 2000 µg/ml, while compound 1 at the same concentration did not show this property. Swarming motility was delayed in treatments with the extract and with 1 at 1000 and 8 µg/ml, respectively, at 8h from the beginning of the assay. Complete inhibition of the phenomenon was still observed after 24 h when compound 1 was added at 125 µg/ml. These findings offer the possibility of new classes of antimicrobial medicines to tackle infections caused by P. mirabilis.

Antiquorum sensing and antibiofilm potential of Capparis spinosa.

BACKGROUND: Emergence of antibiotic resistance among bacterial pathogens often leads to the failure of existing antibiotics to treat bacterial infections; thus, there is a need to seek alternative treatment measures. The aim of this study was to evaluate the anti-quorum sensing (anti-QS) and antibiofilm potential of Capparis spinosa to prevent the onset of bacterial infections as an alternate to antibiotics. METHODS: The methanolic extract of the dried fruits of C. spinosa was assessed for its activity in inhibiting QS-depedent phenomenon such as violacein pigment production in Chromobacterium violaceum, biosurfactant production in Pseudomonas aeruginosa PAO1, swimming and swarming motility, exopolysaccharide production (EPS) and biofilm formation in Escherichia coli, Proteus mirabilis, Serratia marcescens and PAO1. RESULTS: Extract of C. spinosa showed a higher degree of anti-QS activity in a dose dependent manner without affecting the bacterial growth. At 2 mg/mL, this extract significantly (p ≤0.005) inhibited the biofilm formation to 79, 75, 73, 70% and EPS production to 58, 46, 66 and 67% in S. marcescens, PAO1, E. coli and P. mirabilis, respectively. It also exhibited inhibition in swimming and swarming motility of bacterial pathogens. The non-enzymatic nature of the anti-QS compound in C. spinosa was confirmed by proteinase K and heat treatment. CONCLUSIONS: Because the methanolic extract of C. spinosa demonstrated anti-QS and antibiofilm activity at 0.5-2 mg/mL, it could be further exploited for novel molecules to treat the emerging infections of antibiotic resistant bacterial pathogens.

Effect of Ibicella lutea on uropathogenic Proteus mirabilis growth, virulence, and biofilm formation.

BACKGROUND: Proteus mirabilis, an important uropathogen that can cause complicated urinary tract infections (UTI), has emerged as a therapeutic problem following mutations that compromise the use of antimicrobial drugs. Due to the serious effects associated with uropathogenic P. mirabilis and the problems related to the use of antibiotics, it is necessary to develop alternative strategies for its control. The objective of this study was to assess the effect of Ibicella lutea extract, a South American indigenous plant, on growth, virulence and biofilm production of uropathogenic P. mirabilis. METHODOLOGY: This study was based on the extract generation and the assessment of its effect on bacterial features related to virulence. These assays involved determination of antibacterial activity, swarming motility, Western blot to assess expression of fimbriae and flagella, biofilms formation, haemagglutination, haemolysis, and electron microscopy. RESULTS AND CONCLUSIONS: I. lutea extract had an effect on bacterial growth rate and bacterial morphology. It also affected P. mirabilis swarming differentiation, hemagglutination and biofilm formation on glass and polystyrene. These findings suggest that I. lutea may have a role as an agent for the control of P. mirabilis UTI.

Antibacterial activity of Quercus ilex bark's extracts.

The antibacterial activity of different extracts of Quercus ilex bark (Fagaceae) was studied in vitro against seven reference strains of bacteria by using a disc-diffusion method and agar-dilution method. The ethyl acetate extract (QE), n-butanol extract (QB) and final aqueous layer (QA) were effective against all bacterial strains tested at MICs ranging from 128 to 512 microg/ml. The n-hexane extract (QH) and dichloromethane extract (QD) showed no activity.

Bulk acoustic wave bacterial growth sensor applied to analysis of antimicrobial properties of tea.

A bulk acoustic wave (BAW) bacterial growth sensor has been proposed for study of inhibitory effects of tea by continuous monitoring of disturbances in Proteus growth in the aqueous extracts of various teas, e.g. green tea, Fuzhuan brick tea, Oolong tea, Kudin tea, and black tea. The kinetic parameters, e.g. asymptote (A), maximum specific growth rate (microm), lag time (lambda), and generation time (g), accurately estimated by using the growth response model, have been first used to characterize antimicrobial properties of tea. All of the parameters were changed via the inhibitory effects by tea. Green tea gives the weakest inhibitory action while others show stronger inhibitory actions. These inhibitory effects have also been examined by using the pour plate count technique. Both of the results show that, in addition to the antimicrobial properties of tea polyphenols and catechins, etc., the inhibitory effects may be attributed to the metabolites produced during the fermentation processing of these teas except green tea. The conventional disk diffusion test has been used to determine the minimum inhibitory concentration (MIC) against P. rettgeri. The MICs of green tea, Fuzhuan brick tea, Oolong tea, and black tea were found to be 1113, 818.0, 681.2, and 510.4 microg/mL, respectively. The BAW bacterial growth sensor has shown to have advantages over other techniques, including the disk diffusion test, photometry, and the impedance method.

Secretory IgA does not enhance the bacteriostatic effects of iron-binding or vitamin B12-binding proteins in human colostrum.

Human milk contains an unsaturated iron-binding protein (lactoferrin) and an unsaturated vitamin B12-binding protein. Lactoferrin has bacteriostatic properties, and a bacteriostatic role for the B12-binding protein has been postulated. In this study the bacteriostatic effect of lactoferrin was confirmed for strains of Escherichia coli, Pseudomonas and Proteus. Growth inhibition attributable to the unsaturated B12-binding protein could be demonstrated only with a known vitamin B12-dependent E. coli. It has previously been shown that the bacteriostatic effect of lactoferrin is potentiated by horse IgG antibody, and a similar potentiating effect of secretory IgA antibody in colostrum and milk would have obvious importance. An attempt was therefore made to demonstrate potentiation of bacteriostatic effects by naturally occurring secretory IgA antibody to E. coli. The results obtained indicate that secretory IgA antibody does not enhance the growth-inhibiting effects of either lactoferrin or the vitamin B12-binding protein.

Evidence against the involvement of chemotaxis in swarming of Proteus mirabilis.

Nonswarming and nonchemotactic mutants of Proteus mirabilis were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light. These mutants were used in experiments to determine if chemotaxis is involved in the swarming of P. mirabilis. Nonchemotactic mutants failed to form chemotactic bands in a semisolid casein hydrolysate medium, yet they swarmed on the same medium containing 1.5% agar. Nonswarming mutants were attracted towards individual amino acids and components of tryptose. In cross-feeding experiments, no evidence was obtained to indicate the production of a diffusable chemical repellent. In studies with the wild-type P. mirabilis, no clear-cut negative chemotaxis was seen even though three different assays were used and numerous chemicals were tested. Additional evidence against the involvement of chemotaxis in swarming comes from finding that dialysis does not interfere with swarming; swarm cells will swarm immediately when transferred to fresh media, and swarm cells will swarm on an agar-water medium supplemented with a surfactant. These data indicate that chemotaxis is not involved in the swarming of P. mirabilis.