Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D,L-lactide-co-glycolide) nanoparticles

Abstract

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7?nm, 26.1?mv) showed time- and concentration-dependent activity against MRSA growth, killing ～ 90% of planktonic bacterial cells in 3?h at 400?μg ml^?1, and completely inhibiting biofilm formation at 1000?μg ml^?1. Moreover, CNPs at 400?μg ml^?1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (～ 25%) and biofilm formation (～ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

The effect of gold and silver nanoparticles,chitosan and their combinations on bacterial biofilms of food-borne pathogens

Abstract

The antimicrobial activity of gold and silver nanoparticles (AuNPs, AgNPs), chitosan (CS) and their combinations was established by determining the minimum inhibitory concentration for planktonic (MICPC₈₀) and biofilm growth (MICBC₈₀), for biofilm formation (MICBF₈₀), metabolic activity (MICBM₈₀) and reduction (MICBR₈₀), and for the metabolic activity of preformed biofilm (MICMPB₈₀). Biofilms were quantified in microtitre plates by crystal violet staining and metabolic activity was evaluated by the MTT assay. Chitosan effectively suppressed biofilm formation (0.31–5?mg ml^?1) in all the tested strains, except Salmonella enterica Infantis (0.16–2.5?mg ml^?1) where CS and its combination with AgNPs induced biofilm formation. Nanoparticles inhibited biofilm growth only when the highest concentrations were used. Even though AuNPs, AgNPs and CS were not able to remove biofilm mass, they reduced its metabolic activity by at least 80%. The combinations of nanoparticles with CS did not show any significant positive synergistic effect on the tested target properties.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Antimicrobial activity and antibiotic synergy of a biphosphinic ruthenium complex against clinically relevant bacteria

Abstract

The aim of this study was to investigate the antibacterial activity, antibiotic-associated synergy, and anti-biofilm activity of the ruthenium complex, cis-[RuCl₂ (dppb) (bqdi)]²⁺ (RuNN). RuNN exhibited antimicrobial activity against Gram-positive bacteria with minimum inhibitory concentration (MIC) values ranging from 15.6 to 62.5?µg ml^?1 and minimum bactericidal concentration (MBC) values ranging from 62.5 to 125?µg ml^?1. A synergistic effect against Staphylococcus spp. was observed when RuNN was combined with ampicillin, and the range of associated fractional inhibitory concentration index (FICI) values was 0.187 to 0.312. A time-kill curve indicated the bactericidal activity of RuNN in the first 1–5?h. In general, RuNN inhibited biofilm formation and disrupted mature biofilms. Furthermore, RuNN altered the cellular morphology of S. aureus biofilms. Further, RuNN did not cause hemolysis of erythrocytes. The results of this study provide evidence that RuNN is a novel therapeutic candidate to treat bacterial infections caused by Staphylococcus biofilms.     

Impediment to growth and yeast-to-hyphae transition in Candida albicans by copper oxide nanoparticles

Abstract

The effects of two prominent copper oxide nanoparticles (CuO-NP and Cu₂O-NP), with the oxidation state of Cu⁺⁺ (cupric) and Cu⁺ (cuprous), on Candida albicans were evaluated. CuO-NP and Cu₂O-NP were synthesized and characterized by XRD, FESEM, HR-TEM and Zeta potential. At sub-MIC (50?µg ml^?1), both cupric and cuprous oxide NPs prevented yeast-to-hyphae switching and wrinkling behaviour in C. albicans. The mechanism for the antifungal action of the two NPs differed; CuO-NP significantly elicited reactive oxygen species, whereas membrane damage was more pronounced with Cu₂O-NP. Real time PCR analysis revealed that CuO-NP suppressed the morphological switching of yeast-to-hyphae by down-regulating cph1, hst7 and ras1 and by up-regulation of the negative regulator tup1. In comparison, Cu₂O-NP resulted in down-regulation of ras1 and up-regulation of the negative regulators nrg1 and tup1. Between the two NPs, CuO exhibited increased antifungal activity due to its stable oxidation state (Cu⁺⁺) and its smaller dimensions compared with Cu₂O-NP.     

Prospects for using a new sequential chemiluminescence strategy for monitoring the caffeine content in soft and energy drinks via the catalytic activities of different nano‐metal oxides

This article suggests a new sequential injection analysis chemiluminescence (SIA‐CL) strategy for monitoring the caffeine (CAF) content in soft and energy drinks using the catalytic activities of different nano‐metal oxides. The present study describes three different SIA‐CL systems (luminol–ferricyanide (III) coupled with Fe₂O₃ or ZnO nanoparticles (NPs), and luminol–H₂O₂ coupled with CuONPs. All experimental conditions were optimized and the linear concentration ranges of pure CAF were evaluated using the calibration graphs. The selectivity of the developed SIA‐CL systems was studied under the influence of various interfering species that may be present in soft or energy drinks such as sodium ions, sucrose, glucose, sodium benzoate, sodium citrate, riboflavin, niacin, citric, phosphoric and ascorbic acids. International Council for Harmonization (ICH) guidelines were obeyed for the validation of the suggested CL methods. The developed SIA‐CL systems displayed linear relationships over the concentration ranges 1.0–350, 5.0–400 and 10.0–400 μg ml^?1 with Fe₂O₃ NPs, ZnO NPs and CuO NPs, respectively. The recorded lower limits of detection and quantification were 0.7, 2.7 and 7.8 μg ml^?1, and 1.0, 5.0 and 10.0 μg ml^?1 for the previously mentioned SIA‐CL systems. The results revealed high selectivity for CAF determination and were in good agreement with those obtained by other reported methods.     

Oxidative stress response to aluminum oxide (Al<Subscript>2</Subscript>O<Subscript>3</Subscript>) nanoparticles in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The development of nanotechnologies has increased the amount of manufactured metal oxide nanoparticles in the environment. In the view of nanoparticle dispersion to the environment, assessment of their toxicity becomes very crucial. Aluminum oxide (Al₂O₃) nanoparticles have wide range of use in industry as well as personal care products. The aim of this study was to evaluate the dose dependent effects of 13-nm-sized Al₂O₃ nanoparticles on wheat correlating with the appearance of enzymatic and non-enzymatic antioxidant defense response. Wheat roots were exposed to different concentrations of Al₂O₃ nanoparticles (5, 25 and 50 mg mL^?1) for 96 h. The effects of Al₂O₃ nanoparticles were studied using different parameters such as H₂O₂ content, superoxide dismutase and catalase activity, lipid peroxidation, total proline, photosynthetic pigment and anthocyanin content. The results indicated that while Al₂O₃ nanoparticles caused a dose dependent increase in H₂O₂ content, superoxide dismutase activity, lipid peroxidation and proline contents, the catalase activity was decreased in compare the control. Moreover, total chlorophyll, chlorophyll a, carotenoids and anthocyanin contents reduced in the highest concentration 50 mg mL^?1. In conclusion, Al₂O₃ nanoparticles caused oxidative stress in wheat after 96 h.     

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation

Abstract

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250?µg ml^?1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.     

Study of the major essential oil compounds of Coriandrum sativum against Acinetobacter baumannii and the effect of linalool on adhesion,biofilms and quorum sensing

Acinetobacter baumannii is a pathogen that has the ability to adhere to surfaces in the hospital environment and to form biofilms which are increasingly resistant to antimicrobial agents. The aim of this work was to study the antimicrobial activity of the major oil compounds of Coriandrum sativum against A. baumannii. The effect of linalool on planktonic cells and biofilms of A. baumannii on different surfaces, as well as its effect on adhesion and quorum sensing was evaluated. From all the compounds evaluated, linalool was the compound with the best antibacterial activity, with minimum inhibitory concentration values between 2 and 8 μl ml^?1. Linalool also inhibited biofilm formation and dispersed established biofilms of A. baumannii, changed the adhesion of A. baumannii to surfaces and interfered with the quorum- sensing system. Thus, linalool could be a promising antimicrobial agent for controlling planktonic cells and biofilms of A. baumannii.     

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.     

Disturbance of firefly luciferase-based bioassays by different aluminum species

Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al⁰ nanoparticles (Al⁰–NPs) but not Al₂O₃–NPs decreased luminescence, correlated to high absorbance of Al⁰–NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al–NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results.     

Ecotoxicological impacts of exposure to copper oxide nanoparticles on the gill of the Swan mussel,Anodonta cygnea (Linnaeus, 1758)

This study was conducted to investigate the ecotoxicological effects of exposure to copper oxide nanoparticles (CuO NPs) on the gill of the swan mussel Anodonta cygnea using several approaches including qualitative and quantitative histopathology, ultra-morphology (scanning electron microscopy [SEM]) and measures of clearance rate (CR) and bioaccumulation of CuO NPs. Histological alterations in mussels exposed to 0.25 (T1), 2.5 (T2) and 25.0?µg L^?1 (T3) CuO NPs for 12 days include changes in the length and form of gill lamellae, changes in inter-lamellar spaces, epithelial hyperplasia, atrophy and tissue rupture. Ultra-morphological changes following CuO NP exposure included epithelial hyperplasia and hypertrophy, epithelial lifting, tissue rupture (water channel fusion) and extensive necrosis of the gill surfaces. I_Gill (gill damage severity) index values for both histopathological and ultra-morphological data were significantly (P?0.05) higher in T3. The CR of mussels was significantly (P??1 g^?1 dry weight]) in comparison to controls (CR?=?108?±?47.14 [L min^?1 g^?1 dry weight]). CuO NPs accumulated in exposed mussels at all exposure concentrations until day 4, but there was no further change in accumulation levels by the end of the exposure period. The accumulated content of CuO NPs was significantly (P??1 exposure concentration. Based on these results, significant accumulation of CuO NPs in the gills of swan mussel could affect histological and ultra-structural characteristics of this organ and consequently have deleterious impacts on its filtration activity.     

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml^?1 for kaempferol and 0.5-16 µg ml^?1 for quercetin. Kaempferol and quercetin decreased (P?<?0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.     

Fouling deterrent chemical defence in three muricid gastropod egg masses from the Southeast coast of India

Abstract

The egg masses of the marine muricid gastropod molluscs Chicoreus virgineus, Chicoreus ramosus and Rapana rapiformis were studied for antifouling activities. The minimum inhibitory concentrations of crude extracts for the inhibition of byssal production and attachment of the brown mussel Perna indica were 650 μg ml^?1, 1150 μg ml^?1 and 925 μg ml^?1 from the three muricid gastropods, respectively. Higher LC₅₀ values than EC₅₀ values and 100% recovery of the mussels in the toxicity assay indicated the non-toxic nature of the extracts. The gradient partitioning of the egg mass extracts and subsequent antimicrofouling screening against 40 biofilm bacteria showed wide-spectrum antibacterial activity of the medium polar fraction from C. virgineus; the non-polar fraction from R. rapiformis and both non-polar and medium polar fractions from C. ramosus. The antimicrofouling activity from extracts of the three egg masses was found to be more prominent than antimacrofouling activity. This may be attributed to the targeting of a defence strategy against microbes in order to protect the developing mollusc embryos.     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Inhibitory effect of α-mangostin on Acinetobacter baumannii biofilms – an in vitro study

The present study was designed to investigate the anti-biofilm potential of alpha-mangostin (α-MG) against Acinetobacter baumannii (AB). The biofilm inhibitory concentration (BIC) of α-MG against AB was found to be 2 μg ml^?1. α-MG (0.5, 1 and 2 μg ml^?1) exhibited non-bactericidal concentration-dependent anti-biofilm activities against AB. However, α-MG failed to disintegrate the mature biofilms of AB even at a 10-fold increased concentration from its BIC. Results from qRT-PCR and in vitro bioassays further demonstrated that α-MG downregulated the expression of bfmR, pgaA, pgaC, csuA/B, ompA, bap, katE, and sodB genes, which correspondingly affects biofilm formation and its associated virulence traits. The present study suggests that α-MG exerts its anti-biofilm property by interrupting initial biofilm formation and the cell-to-cell signaling mechanism of AB. Additional studies are required to understand the mode of action responsible for the anti-biofilm property.     

Antibacterial and antibiofilm efficacy of Ag NPs,Ni NPs and Al2O3 NPs singly and in combination against multidrug-resistant Klebsiella pneumoniae isolates

BackgroundAlthough traditional antibiotic therapy provided an effective approach to combat pathogenic bacteria, the long-term and widespread use of antibiotic results in the evolution of multidrug-resistant bacteria. Recent progress in nanotechnology offers an alternative opportunity to discover and develop novel antibacterial agents.MethodsA total of 51 K. pneumoniae strains were collected from several specimens of hospitalized patients and identified by two parallel methods (biochemical tests and Vitek-2 system). The antibiotic sensitivity of isolates was evaluated by disk diffusion antibiogram and Vitek-2 system. The biofilms formation ability of antibiotic-resistant strains was examined by microtiter plate and tube methods based on crystal violet staining. The molecular technique was used to determine key genes responsible for biofilms formation of clinical isolates. The antibacterial and antibiofilm activities of Ag NPs, Ni NPs, Al₂O₃ NPs singly (NPs) and in combination (cNPs) were investigated against selected strains using standard methods. Moreover, the cytotoxicity of NPs was evaluated on mouse neural crest-derived (Neuro-2A) cell line.ResultsThe results of bacterial studies revealed that more than 80 % of the isolates were resistant to commonly used antibiotics and about 95 % of them were able to form biofilms. Moreover, the presence of fimA and mrkA genes were determined in all biofilm-producing strains. The results of antibacterial and antibiofilm activities of NPs and cNPs demonstrated the lower MIC and MBEC values for Al₂O₃ NPs singly as well as for Ag/Ni cNPs and Ag/Al₂O₃ cNPs in combination, respectively. Overall, the inhibitory effects of cNPs were superior to NPs against all strains. Furthermore, the results of the checkerboard assays showed that Ag NPs act synergistically with two other NPs against multidrug-resistant Klebsiella pneumoniae (MDR-K. pneumoniae) isolates. The in vitro cytotoxicity assay revealed no significant toxicity of NPs against Neuro-2A cells.ConclusionIn the present study, the combination of Ag NPs, Ni NPs, and Al₂O₃ NPs were used against MDR-K. pneumoniae strains and antibacterial and antibiofilm activities were observed for Ag/Ni cNPs and Ag/Al₂O₃ cNPs.     

High synergistic antibacterial,antibiofilm, antidiabetic and antimetabolic activity of Withania somnifera leaf extract-assisted zinc oxide nanoparticle

Nanotechnology is currently gaining immense attention to combat food borne bacteria, and biofilm. Diabetes is a common metabolic disease affecting majority of people. A better therapy relies on phytomediated nanoparticle synthesis. In this study, W. somnifera leaf extract-assisted ZnO NPs (Ws-ZnO NPs) was synthesized and characterized. From HR-TEM analysis, it has been found that the hexagonal wurtzite particle is 15.6 nm in size and − 12.14 mV of zeta potential. A greater antibacterial effect of Ws-ZnO NPs was noticed against E. faecalis and S. aureus at 100 µg mL⁻¹. Also, the biofilm of E. faecalis and S. aureus was greatly inhibited at 100 µg mL⁻¹ compared to E. coli and P. aeruginosa. The activity of α-amylase and α-glucosidase enzyme was inhibited at 100 µg mL⁻¹ demonstrating its antidiabetic potential. The larval and pupal development was delayed at 25 µg mL⁻¹ of Ws-ZnO NPs. A complete mortality (100%) was recorded at 25 µg mL⁻¹. Ws-ZnO NPs showed least LC₅₀ value (9.65 µg mL⁻¹) compared to the uncoated ZnO NPs (38.8 µg mL⁻¹) and leaf extract (13.06 µg mL⁻¹). Therefore, it is concluded that Ws-ZnO NPs are promising to be used as effective antimicrobials, antidiabetic and insecticides to combat storage pests.

     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.