Cell viability assay as a tool to study activity and inhibition of hepatitis C p7 channels.

The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments and thus helping virus assembly and release. This activity is essential for virus infectivity, making viroporins an attractive target for drug development. The protein sequence and drug sensitivity of p7 vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Here, we investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a-b, 2a-b, 3a and 4a using patch-clamp electrophysiology and cell-based assays. We established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity and its sensitivity to inhibitors. The results from the cell viability assay were consistent with control measurements using established assays of haemadsorption and intracellular pH, and agreed with data from patch-clamp electrophysiology. Hexamethylene amiloride (HMA) was the most potent inhibitor of p7 activity, but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. The alkyl-chain iminosugars NB-DNJ, NN-DNJ and NN-DGJ were tested and their activity was found to be genotype-specific. In the current study, we introduce cell viability assays as a rapid and cost-efficient technique to assess viroporin activity and identify channel inhibitors as potential novel antiviral drugs.

A combination of silver nanoparticles and visible blue light enhances the antibacterial efficacy of ineffective antibiotics against methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Silver nanoparticles (AgNPs) are potential antimicrobials agents, which can be considered as an alternative to antibiotics for the treatment of infections caused by multi-drug resistant bacteria. The antimicrobial effects of double and triple combinations of AgNPs, visible blue light, and the conventional antibiotics amoxicillin, azithromycin, clarithromycin, linezolid, and vancomycin, against ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were investigated. METHODS: The antimicrobial activity of AgNPs, applied in combination with blue light, against selected isolates of MRSA was investigated at 1/2-1/128 of its minimal inhibitory concentration (MIC) in 24-well plates. The wells were exposed to blue light source at 460 nm and 250 mW for 1 h using a photon emitting diode. Samples were taken at different time intervals, and viable bacterial counts were determined. The double combinations of AgNPs and each of the antibiotics were assessed by the checkerboard method. The killing assay was used to test possible synergistic effects when blue light was further combined to AgNPs and each antibiotic at a time against selected isolates of MRSA. RESULTS: The bactericidal activity of AgNPs, at sub-MIC, and blue light was significantly (p < 0.001) enhanced when both agents were applied in combination compared to each agent alone. Similarly, synergistic interactions were observed when AgNPs were combined with amoxicillin, azithromycin, clarithromycin or linezolid in 30-40 % of the double combinations with no observed antagonistic interaction against the tested isolates. Combination of the AgNPs with vancomycin did not result in enhanced killing against all isolates tested. The antimicrobial activity against MRSA isolates was significantly enhanced in triple combinations of AgNPs, blue light and antibiotic, compared to treatments involving one or two agents. The bactericidal activities were highest when azithromycin or clarithromycin was included in the triple therapy compared to the other antibiotics tested. CONCLUSIONS: A new strategy can be used to combat serious infections caused by MRSA by combining AgNPs, blue light, and antibiotics. This triple therapy may include antibiotics, which have been proven to be ineffective against MRSA. The suggested approach would be useful to face the fast-growing drug-resistance with the slow development of new antimicrobial agents, and to preserve last resort antibiotics such as vancomycin.

Antifungal activity of amphotericin B and voriconazole against the biofilms and biofilm-dispersed cells of Candida albicans employing a newly developed in vitro pharmacokinetic model.

BACKGROUND: Candida albicans is a common cause of a variety of superficial and invasive disseminated infections the majority of which are associated with biofilm growth on implanted devices. The aim of the study is to evaluate the activity of amphotericin B and voriconazole against the biofilm and the biofilm-dispersed cells of Candida albicans using a newly developed in vitro pharmacokinetic model which simulates the clinical situation when the antifungal agents are administered intermittently. METHODS: RPMI medium containing 1-5 X 10(6) CFU/ml of C. albicans was continuously delivered to the device at 30 ml/h for 2 hours. The planktonic cells were removed and biofilms on the catheter were kept under continuous flow of RPMI medium at 10 ml/h. Five doses of amphotericin B or voriconazole were delivered to 2, 5 and 10 day-old biofilms at initial concentrations (2 and 3 µg/ml respectively) that were exponentially diluted. Dispersed cells in effluents from the device were counted and the adherent cells on the catheter were evaluated after 48 h of the last dose. RESULTS: The minimum inhibitory concentration of voriconazole and amphotericin B against the tested isolate was 0.0325 and 0.25 µg/ml respectively. Amphotericin B significantly reduced the dispersion of C. albicans cells from the biofilm. The log10 reduction in the dispersed cells was 2.54-3.54, 2.30-3.55, and 1.94-2.50 following addition of 5 doses of amphotericin B to 2-, 5- and 10-day old biofilms respectively. The number of the viable cells within the biofilm was reduced by 18 (±7.63), 5 and 4% following addition of the 5 doses of amphotericin B to the biofilms respectively. Voriconazole showed no significant effect on the viability of C. albicans within the biofilm. CONCLUSION: Both antifungal agents failed to eradicate C. albicans biofilm or stop cell dispersion from them and the resistance progressed with maturation of the biofilm. These findings go along with the need for removal of devices in spite of antifungal therapy in patients with device-related infection. This is the first study which investigates the effects of antifungal agents on the biofilm and biofilm-dispersion of C. albicans in an in vitro pharmacokinetic biofilm model.

Simulation of hydrogen sulphide absorption in alkaline solution using a packed column.

In this work, a simulation tool was developed for hydrogen sulphide (H2S) removal in an alkaline solution in packed columns working at countercurrent. Modelling takes into account the mass-transfer enhancement due to the reversible reactions between H2S and the alkaline species (CO(²â»)(3), HCO⁻(3), and HO⁻) in the liquid film. Many parameters can be controlled by the user such as the gas and liquid inlet H2S concentrations, the gas and liquid flow rates, the scrubbing liquid pH, the desired H2S removal efficiency, the temperature, the alkalinity, etc. Since the influence of the hydrodynamic and mass-transfer performances in a packed column is well known, the numerical resolutions performed were dedicated to the study of the influence of the chemical conditions (through the pH and the alkalinity), the temperature and the liquid-to-gas mass flow rate ratio (L/G). A packed column of 3 m equipped with a given random packing material working at countercurrent and steady state has been modelled. The results show that the H2S removal efficiency increases with the L/G, the pH, the alkalinity and more surprisingly with the temperature. Alkalinity has a very significant effect on the removal efficiency through the mass-transfer enhancement and buffering effect, which limits pH decreasing due to H2S absorption. This numerical resolution provides a tool for designers and researchers involved in H2S treatment to understand deeper the process and optimize their processes.

Recent progress on field-effect transistor-based biosensors: device perspective.

Over the last few decades, field-effect transistor (FET)-based biosensors have demonstrated great potential across various industries, including medical, food, agriculture, environmental, and military sectors. These biosensors leverage the electrical properties of transistors to detect a wide range of biomolecules, such as proteins, DNA, and antibodies. This article presents a comprehensive review of advancements in the architectures of FET-based biosensors aiming to enhance device performance in terms of sensitivity, detection time, and selectivity. The review encompasses an overview of emerging FET-based biosensors and useful guidelines to reach the best device dimensions, favorable design, and realization of FET-based biosensors. Consequently, it furnishes researchers with a detailed perspective on design considerations and applications for future generations of FET-based biosensors. Finally, this article proposes intriguing avenues for further research on the topology of FET-based biosensors.

A modern approach to the authentication and quality assessment of thyme using UV spectroscopy and chemometric analysis.

INTRODUCTION: Recently, the fields of chemometrics and multivariate analysis have been widely implemented in the quality control of herbal drugs to produce precise results, which is crucial in the field of medicine. Thyme represents an essential medicinal herb that is constantly adulterated due to its resemblance to many other plants with similar organoleptic properties. OBJECTIVE: To establish a simple model for the quality assessment of Thymus species using UV spectroscopy together with known chemometric techniques. The success of this model may also serve as a technique for the quality control of other herbal drugs. MATERIALS AND METHODS: The model was constructed using 30 samples of authenticated Thymus vulgaris and challenged with 20 samples of different botanical origins. The methanolic extracts of all samples were assessed using UV spectroscopy together with chemometric techniques: principal component analysis (PCA), soft independent modeling of class analogy (SIMCA) and hierarchical cluster analysis (HCA). RESULTS: The model was able to discriminate T. vulgaris from other Thymus, Satureja, Origanum, Plectranthus and Eriocephalus species, all traded in the Egyptian market as different types of thyme. The model was also able to classify closely related species in clusters using PCA and HCA. The model was finally used to classify 12 commercial thyme varieties into clusters of species incorporated in the model as thyme or non-thyme. CONCLUSION: The model constructed is highly recommended as a simple and efficient method for distinguishing T. vulgaris from other related species as well as the classification of marketed herbs as thyme or non-thyme.

Application of chemometrics in authentication of herbal medicines: a review.

INTRODUCTION: Herbal medicines (HM) and their preparations have been widely used for hundreds of years all over the world. However, they have not been officially recognised due to a lack of adequate or accepted research methodology for their evaluation. OBJECTIVE: To present a concise overview of the recent applications of chemometrics in solving the ambiguity of herbal medicine authentication during the last two decades. METHODOLOGY: Studies involving the applications of chemometric analysis in combination with different analytical methods were classified according to the method of analysis used including chromatographic (HPLC, GC and CE), spectroscopic (NMR, IR, UV and ICP) and genetic analysis (RAPD). The purpose of each of these studies was classified into one of three main categories: taxonomic discrimination, quality assessment or classification between plants of different geographic origins. RESULTS: This review comprises over 150 studies, covering the past two decades, emphasising the significance of chemometric methods in the discrimination of many herbs from closely related species and from adulterants, based on the principal bioactive components and phytochemical diversity. Furthermore, the differentiation between varieties and hybrids was achieved in addition to the prediction of the active components by quantitative methods of analysis. Discrimination according to geographical origin and localities, processing methods, DNA profiling and metabolomics were also efficiently investigated. CONCLUSION: Chemometric methods have provided an efficient and powerful tool for the quality control and authentication of different herbs.

Valorization and characterization of bio-oil from Salvadora persica seed for air pollutant adsorption.

Salvadora persica (SP) is an important medicinal plant. Numerous articles have been conducted on the leaf, the roots, and the stem of the plant, but there is little information about the seed. Thus, the present work tries to identify the chemical composition of SP seed bio-oil and investigates its use as an adsorbent for cyclohexane removal. This study extracted bio-oil from seeds using different polar and non-polar organic solvents. Two techniques have been used to determine the chemical composition of the bio-oil extracted: FTIR and GC-MS. Results show that the extracted bio-oil presented 13 new major organic bio-compounds in n-hexane and ethanol SP seed extracts. Moreover, the analytical results showed that the two extracts are complex and contained thiocyanic acid, benzene, 3-pyridine carboxaldehyde, benzyl nitrile, ethyl tridecanoate, ethyl oleate, and dodecanoic acid ethyl ester. Additionally, each technique of analysis showed that the extracted bio-oils from SP seeds are rich in non-polar compounds. Indeed, the major fatty acids obtained are pentadecylic acid, myristic acid, lauric acid, oleic acid, margaric acid, and tricosanoic acid. This work provides guidelines for identifying these compounds, among others, and offers a platform for using SP seeds as a herbal alternative for various chemical, industrial, and medical applications. Furthermore, the capacity of SP extracts for air pollution treatment, namely, the removal of cyclohexane in batch mode, was investigated. Results showed that cyclohexane adsorption could be a chemical process involving both monolayer and multilayer adsorption mechanisms. The pores and the grooves on the surface of the SP bio-oil extract helped in adsorbing the cyclohexane with an outstanding maximum removal capacity of about 674.23 mg/g and 735.75 mg/g, respectively, for the ethanol and hexane SP extracts, which is superior to many other recent adsorbents.

Antibacterial Activity of Some Medicinal Plants in Al Baha Region, Saudi Arabia, Against Carcinogenic Bacteria Related to Gastrointestinal Cancers.

BACKGROUND: Gastrointestinal cancers are the most dangerous cancers all over the world. The gut microbiota dysbiosis increases the risk of GI cancers and induces the host's susceptibility to carcinogenic bacteria. Antibiotic resistance is rising in these bacteria. Thus, discovering new safe and effective antibacterial agents is a worldwide concern. This study evaluates the antibacterial activity of six wild medicinal plants from the Al Bahah region in Saudi Arabia. METHODS: Arial parts of Cissus quadrangularis, Aloe castellorum, Psiadia punctulata, Aloe pseudorubroviolacea, Barbeya oleoides, Teucrium yemense were collected and dried for extraction with ethanol. The minimum inhibitory concentrations (MIC) of these ethanolic extracts against carcinogenic bacteria Bacteroides fragilis, Clostridium ssp., Cutibacterium acnes, Escherichia coli, Fusobacterium nucleatum, Helicobacter pylori, Mycoplasma spp., Neisseria gonorrhoeae, Porphyromonas gingivalis, Salmonella enterica, and Treponema pallidum were evaluated to determine its antibacterial activity. RESULTS: All extracts showed antibacterial activity with MIC lower than 1 mg/ml. Psiadia punctulata showed higher antibacterial activity, while the Aloe species showed the lowest antibacterial activity. CONCLUSION: The studied plants' extracts showed high effectiveness as antibacterial activity against the carcinogenic bacteria related to gastrointestinal cancers due to their high content of pharmaceutical components. These plants could be explored further for the development of new antibacterial products against these carcinogenic bacteria.

Chitosan-Functionalized Hydroxyapatite-Cerium Oxide Heterostructure: An Efficient Adsorbent for Dyes Removal and Antimicrobial Agent.

The current research intended to employ a facile and economical process, which is also ecofriendly to transform camel waste bones into novel heterostructure for cleansing of diverse waste waters. The bones of camel were utilized for preparation of hydroxyapatite by hydrothermal method. The prepared hydroxyapatite was applied to the synthesis of cerium oxide-hydroxyapatite coated with natural polymer chitosan (CS-HAP-CeO2) heterostructure. Being abundant natural polymer polysaccharide, chitosan possesses exceptional assets such as accessibility, economic price, hydrophilicity, biocompatibility as well as biodegradability, therefore style it as an outstanding adsorbent for removing colorant and other waste molecules form water. This heterostructure was characterized by various physicochemical processes such as XRD, SEM-EDX, TEM, and FT-IR. The CS-HAP-CeO2 was screened for adsorption of various industrially important dyes, viz., Brilliant blue (BB), Congo red (CR), Crystal violet (CV), Methylene blue (MB), Methyl orange (MO), and Rhodamine B (RB) which are collective pollutants of industrial waste waters. The CS-HAP-CeO2 demonstrated exceptional adsorption against CR dye. The adsorption/or removal efficiency ranges are BB (11.22%), CR (96%), CV (28.22%), MB (47.74%), MO (2.43%), and RB (58.89%) dyes. Moreover, this heterostructure showed excellent bacteriostatic potential for E. coli, that is liable for serious waterborne diseases. Interestingly, this work revealed that the incorporation of cerium oxide and chitosan into hydroxyapatite substantially strengthened antimicrobial and adsorption capabilities than those observed in virgin hydroxyapatite. Herein, we recycled the unwanted camel bones into a novel heterostructure, which assists to reduce water pollution, mainly caused by the dye industries.

The Superiority of Bacillus megaterium over Escherichia coli as a Recombinant Bacterial Host for Hyaluronic Acid Production.

(1) Background: Hyaluronic acid (HA) is a polyanionic mucopolysaccharide extensively used in biomedical and cosmetic industries due to its unique rheological properties. Recombinant HA production using other microbial platforms has received increasing interest to avoid potential toxin contamination associated with its production by streptococcal fermentation. In this study, the Gram-negative strains Escherichia coli (pLysY/Iq), E. coli Rosetta2, E. coli Rosetta (DE3) pLysS, E. coli Rosetta2 (DE3), E. coli Rosetta gammiB(DE3)pLysS, and the Gram-positive Bacillus megaterium (MS941) were investigated as new platforms for the heterologous production of HA. (2) Results: The HA biosynthesis gene hasA, cloned from Streptococcus equi subsp. zoopedemicus, was ligated into plasmid pMM1522 (MoBiTec), resulting in pMM1522 hasA, which was introduced into E. coli Rosetta-2(DE3) and B. megaterium (MS941). The initial HA titer by the two hosts in the LB medium was 5 mg/L and 50 mg/L, respectively. Streptococcal hasABC and hasABCDE genes were ligated into plasmid pPT7 (MoBiTec) and different E. coli host strains were then transformed with the resulting plasmids pPT7hasABC and pPT7hasABCDE. For E. coli Rosetta-gamiB(DE3)pLysS transformed with pPT7hasABC, HA production was 500 ± 11.4 mg/L in terrific broth (TB) medium. Productivity was slightly higher (585 ± 2.9 mg/L) when the same host was transformed with pPT7 carrying the entire HA operon. We also transformed B. megaterium (MS941) protoplasts carrying T7-RNAP with pPT7hasABC and pPT7hasABCDE. In comparison, the former plasmid resulted in HA titers of 2116.7 ± 44 and 1988.3 ± 19.6 mg/L in LB media supplemented with 5% sucrose and A5 medium + MOPSO, respectively; the latter plasmid boosted the titer final concentration further to reach 2476.7 ± 14.5 mg/L and 2350 ± 28.8 mg/L in the two media, respectively. The molecular mass of representative HA samples ranged from 105 − 106 Daltons (Da), and the polydispersity index (PDI) was <2. Fourier transform infrared spectroscopy (FTIR) spectra of the HA product were identical to those obtained for commercially available standard polymers. Finally, scanning electron microscopic examination revealed the presence of extensive HA capsules in E. coli Rosetta-gamiB(DE3)pLysS, while no HA capsules were produced by B. megaterium. (3) Conclusions: Our results suggested that Gram-positive bacteria are probably superior host strains for recombinant HA production over their Gram-negative counters. The titers and the molecular weight (MW) of HA produced by B. megaterium were significantly higher than those obtained by different E. coli host strains used in this study.

The Role of MicroRNAs in Lung Cancer Metabolism.

MicroRNAs (miRNAs) are short-strand non-coding RNAs that are responsible for post-transcriptional regulation of many biological processes. Their differential expression is important in supporting tumorigenesis by causing dysregulation in normal biological functions including cell proliferation, apoptosis, metastasis and invasion and cellular metabolism. Cellular metabolic processes are a tightly regulated mechanism. However, cancer cells have adapted features to circumvent these regulations, recognizing metabolic reprogramming as an important hallmark of cancer. The miRNA expression profile may differ between localized lung cancers, advanced lung cancers and solid tumors, which lead to a varying extent of metabolic deregulation. Emerging evidence has shown the relationship between the differential expression of miRNAs with lung cancer metabolic reprogramming in perpetuating tumorigenesis. This review provides an insight into the role of different miRNAs in lung cancer metabolic reprogramming by targeting key enzymes, transporter proteins or regulatory components alongside metabolic signaling pathways. These discussions would allow a deeper understanding of the importance of miRNAs in tumor progression therefore providing new avenues for diagnostic, therapeutic and disease management applications.

A Comprehensive Insight on the Health Benefits and Phytoconstituents of Camellia sinensis and Recent Approaches for Its Quality Control.

Tea, Camellia sinensis, which belongs to the family Theaceae, is a shrub or evergreen tree up to 16 m in height. Green tea is very popular because of its marked health benefits comprising its anticancer, anti-oxidant, and antimicrobial activities, as well as its effectiveness in reducing body weight. Additionally, it was recognized by Chinese people as an effective traditional drink required for the prophylaxis against many health ailments. This is due to the complex chemical composition of green tea, which comprises different classes of chemical compounds, such as polyphenols, alkaloids, proteins, minerals, vitamins, amino acids, and others. The beneficial health effects of green tea ultimately led to its great consumption and increase its liability to be adulterated by either low-quality or non-green tea products with concomitant decrease in activity. Thus, in this review, green tea was selected to highlight its health benefits and phytoconstituents, as well as recent approaches for its quality-control monitoring that guarantee its incorporation in many pharmaceutical industries. More research is needed to find out other more biological activities, active constituents, and other simple and cheap techniques for its quality assurance that ascertain the prevention of its adulteration.

Authentication and discrimination of green tea samples using UV-vis, FTIR and HPLC techniques coupled with chemometrics analysis.

Green tea is a popular beverage consumed worldwide. Its quality should be controlled adequately as the quality is influenced by several factors in addition to adulterations. This study aimed to develop a simple method for assessing the quality of green tea samples obtained from the South and the East Asian regions. The UV-vis, FTIR and HPLC data from 38 samples were subjected to multivariate analyses using the unsupervised recognition techniques comprising Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA). The model for their authentication was constructed and validated by applying the supervised recognition techniques as Soft Independent Modeling of Class Analogy (SIMCA) and Partial Least Square Discriminant Analysis (PLS-DA). The percentages of caffeine in the identified samples were determined using a validated HPLC assay in addition to in vitro determination of their antioxidant activity using DPPH radical-scavenging capacity assay. HCA and PCA based on UV data successfully distributed the tested samples into informative clusters. However, that obtained from visible data could only differentiate samples with respect to their powdered condition. On the contrary, PCA from FTIR and HPLC data could hardly discriminate any of the samples. The models constructed using SIMCA and PLS-DA showed a good class separation between the South and the East Asian samples. The percentages of caffeine in the identified samples and the IC50 in DPPH assay are greatly diverse among all the tested samples. Thus, UV spectroscopy and chemometrics have provided a simple and quick tool for the quality control of commercial green tea samples.

Enhancement of the in vitro activity of amphotericin B against the biofilms of non-albicans Candida spp. by rifampicin and doxycycline.

The in vitro activity of amphotericin B (AMB) alone and in combination with rifampicin (RIF) and doxycycline (DOX) was tested against the biofilms of 30 clinical isolates of non-albicans Candida (NAC) species namely, Candida parapsilosis, Candida krusei and Candida glabrata. The killing activity of AMB at 10 x MIC was significantly increased in combination with either antibiotic. With RIF, the killing activity increased by 20.6, 23.5 and 14 % against the biofilms of C. parapsilosis, C. krusei and C. glabrata, respectively; with DOX, the killing activity increased by 30.64, 35.28 and 31.13 %, respectively. Pre-exposure of the isolates to the same combinations significantly reduced the number of colonized cells in the biofilms by 20, 25.14 and 13.07 % with RIF for C. parapsilosis, C. krusei and C. glabrata, respectively, and by 18.94, 24.52 and 29.15 % with DOX, respectively. The data showed that combination of RIF or DOX with AMB enhanced the killing activity of the antifungal agent against biofilms of NAC species. Whether such an effect operates against biofilm-associated infections needs to be clarified by further in vivo studies.

Novel Microdilution Method to Assess Double and Triple Antibiotic Combination Therapy In Vitro.

An in vitro microdilution method was developed to assess double and triple combinations of antibiotics. Five antibiotics including ciprofloxacin, amikacin, ceftazidime, piperacillin, and imipenem were tested against 10 clinical isolates of Pseudomonas aeruginosa. Each isolate was tested against ten double and nine triple combinations of the antibiotics. A 96-well plate was used to test three antibiotics, each one alone and in double and triple combinations against each isolate. The minimum bacteriostatic and bactericidal concentrations in combination were determined with respect to the most potent antibiotic. An Interaction Code (IC) was generated for each combination, where a numerical value was designated based on the 2-fold increase or decrease in the MICs with respect to the most potent antibiotic. The results of the combinations were verified by time-kill assay at constant concentrations of the antibiotics and in a chemostat. Only 13% of the double combinations were synergistic, whereas 5% showed antagonism. Forty-three percent of the triple combinations were synergistic with no antagonism observed, and 100% synergism was observed in combination of ciprofloxacin, amikacin, and ceftazidime. The presented protocol is simple and fast and can help the clinicians in the early selection of the effective antibiotic therapy for treatment of severe infections.

Efficacy of ultraviolet C light at sublethal dose in combination with antistaphylococcal antibiotics to disinfect catheter biofilms of methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis in vitro.

BACKGROUND: Biofilm formation inside inserted medical devices leads to their failure and acts as a source of refractory infections. The ultraviolet C (UVC) light is a potential therapy that can be used against the biofilm of bacterial pathogens. OBJECTIVE: We evaluated the efficacy of sublethal dose of UVC light with anti-staphylococcal antibiotics against biofilms made from 30 isolates of methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus and S. epidermidis on vascular catheters. MATERIALS AND METHODS: A novel biofilm device was used to assess the combined approach. The biofilms on the catheters were irradiated with the UVC light at 254 nm and irradiance of 6.4 mW followed by treatment with vancomycin or quinupristin/dalfopristin at twice their minimum bactericidal concentrations or with linezolid at 64 µg/mL for 24 hours. The catheters were cut into segments and sonicated, and the number of the sessile cells was determined colorimetrically using XTT viable cells assay. The effect of UVC radiation followed by treatment with an antistaphylococcal antibiotic on the viability of the bacteria in the biofilm was visualized using LIVE/DEAD BacLight bacterial viability stain and confocal laser scanning microscopy. RESULTS: Exposure of the bacterial biofilms to the UVC light or each of the antibiotics alone was ineffective in killing the bacteria. Treatment of the biofilms with the antibiotics following their exposure to UVC light significantly (P<0.001) reduced the number of viable cells within the biofilms but did not completely eradicate them. CONCLUSION: To our knowledge, this combinatorial approach has not been investigated before. The combined approach can be used as a therapeutic modality for managing biofilm-associated infections by preventing the establishment of biofilms and/or disrupting the formed biofilms on the inserted medical devices with the goal of increasing their usefulness and preventing infectious complications. Further investigations are needed to assess the effectiveness of the combined approach in the clinical settings.

Efficacy of selected biocides in the decontamination of common nosocomial bacterial pathogens in biofilm and planktonic forms.

The efficacy and use of biocides to eliminate pathogens in the health care environment are based on their testing against planktonic bacteria. In the environment, bacteria exist in biofilms, as they do on medical devices, and as planktonic or viable non-culturable forms as well. This work aimed to evaluate the efficacy of four biocides against the biofilm and planktonic phases of nine common nosocomial bacteria. The bactericidal activity of the biocides against bacteria in the planktonic form was assessed using a broth microdilution technique. The killing activity of the biocides against biofilms was evaluated using cells grown on polyethylene tubes under a dynamic flow-cell system that was designed for biofilm growth. All biocides completely killed the planktonic bacteria at all concentrations; however, they did not eradicate the biofilms of the same pathogens. Our study highlights the need for an alternative strategy, one that utilizes chemicals that have been tested to disrupt or prevent biofilm growth, in order to enhance current disinfection practice.

A novel combination approach of human polyclonal IVIG and antibiotics against multidrug-resistant Gram-positive bacteria.

BACKGROUND: Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and enterococci, have shown a remarkable ability to develop resistance to antimicrobial agents. OBJECTIVE: We aimed to assess possible enhancement of the antimicrobial activity of vancomycin, amoxicillin, clarithromycin, and azithromycin by human polyclonal intravenous immunoglobulin G (IVIG) against 34 multidrug-resistant (MDR) bacterial isolates, including MRSA, Enterococcus faecium, and Enterococcus faecalis. MATERIALS AND METHODS: Double combinations of the antibiotics with the IVIG were assessed by checkerboard assay, where the interaction was evaluated with respect to the minimum inhibitory concentration (MIC) of the antibiotics. The results of the checkerboard assay were verified in vitro using time-kill assay and in vivo using an invasive sepsis murine model. RESULTS: The checkerboard assay showed that IVIG enhanced the antimicrobial activity of amoxicillin and clarithromycin against isolates from the three groups of bacteria, which were resistant to the same antibiotics when tested in the absence of IVIG. The efficacy of vancomycin against 15% of the tested isolates was enhanced when it was combined with the antibodies. Antagonism was demonstrated in 47% of the E. faecalis isolates when clarithromycin was combined with the IVIG. Synergism was proved in the time-kill assay when amoxicillin was combined with the antibodies; meanwhile, antagonism was not demonstrated in all tested combinations, even in combinations that showed such response in checkerboard assay. CONCLUSION: The suggested approach is promising and could be helpful to enhance the antimicrobial activity of not only effective antibiotics but also antibiotics that have been proven to be ineffective against MDR bacteria. To our knowledge, this combinatorial approach against MDR bacteria, such as MRSA and enterococci, has not been investigated before.

In vitro and in vivo antimicrobial activity of combined therapy of silver nanoparticles and visible blue light against Pseudomonas aeruginosa.

Silver nanoparticles (AgNPs) have been used as potential antimicrobial agents against resistant pathogens. We investigated the possible therapeutic use of AgNPs in combination with visible blue light against a multidrug resistant clinical isolate of Pseudomonas aeruginosa in vitro and in vivo. The antibacterial activity of AgNPs against P. aeruginosa (1×10(5) colony forming unit/mL) was investigated at its minimal inhibitory concentration (MIC) and sub-MIC, alone and in combination with blue light at 460 nm and 250 mW for 2 hours. The effect of this combined therapy on the treated bacteria was then visualized using transmission electron microscope. The therapy was also assessed in the prevention of biofilm formation by P. aeruginosa on AgNP-impregnated gelatin biopolymer discs. Further, in vivo investigations were performed to evaluate the efficacy of the combined therapy to prevent burn-wound colonization and sepsis in mice and, finally, to treat a real infected horse with antibiotic-unresponsive chronic wound. The antimicrobial activity of AgNPs and visible blue light was significantly enhanced (P<0.001) when both agents were combined compared to each agent alone when AgNPs were tested at MIC, 1/2, or 1/4 MIC. Transmission electron microscope showed significant damage to the cells that were treated with the combined therapy compared to other cells that received either the AgNPs or blue light. In addition, the combined treatment significantly (P<0.001) inhibited biofilm formation by P. aeruginosa on gelatin discs compared to each agent individually. Finally, the combined therapy effectively treated a horse suffering from a chronic wound caused by mixed infection, where signs of improvement were observed after 1 week, and the wound completely healed after 4 weeks. To our knowledge, this combinatorial therapy has not been investigated before. It was proved efficient and promising in managing infections caused by multidrug resistant bacteria and could be used as an alternative to conventional antibiotic therapy.