Mahmoud Y. Alkawareek

Eradication of Pseudomonas aeruginosa biofilms by atmospheric pressure non-thermal plasma.

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (∼10′s s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.

Synergistic phage-antibiotic combinations for the control of Escherichia coli biofilms in vitro.

The potential application of phage therapy for the control of bacterial biofilms has received increasing attention as resistance to conventional antibiotic agents continues to increase. The present study identifies antimicrobial synergy between bacteriophage T4 and a conventional antibiotic, cefotaxime, via standard plaque assay and, importantly, in the in vitro eradication of biofilms of the T4 host strain Escherichia coli 11303. Phage-antibiotic synergy (PAS) is defined as the phenomenon whereby sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterias production of virulent phage. Increasing sub-lethal concentrations of cefotaxime resulted in an observed increase in T4 plaque size and T4 concentration. The application of PAS to the T4 one-step growth curve also resulted in an increased burst size and reduced latent period. Combinations of T4 bacteriophage and cefotaxime significantly enhanced the eradication of bacterial biofilms when compared to treatment with cefotaxime alone. The addition of medium (10(4) PFU mL(-1)) and high (10(7) PFU mL(-1)) phage titres reduced the minimum biofilm eradication concentration value of cefotaxime against E. coli ATCC 11303 biofilms from 256 to 128 and 32 μg mL(-1), respectively. Although further investigation is needed to confirm PAS, this study demonstrates, for the first time, that synergy between bacteriophage and conventional antibiotics can significantly improve biofilm control in vitro.

Application of atmospheric pressure nonthermal plasma for the in vitro eradication of bacterial biofilms.

The use of atmospheric pressure nonthermal plasma represents an interesting and novel approach for the decontamination of surfaces colonized with microbial biofilms that exhibit enhanced tolerance to antimicrobial challenge. In this study, the influence of an atmospheric pressure nonthermal plasma jet, operated in a helium and oxygen gas mixture under ambient pressure, was evaluated against biofilms of Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. Within < 4 min of plasma exposure, complete eradication of the two gram-positive bacterial biofilms was achieved. Although gram-negative biofilms required longer treatment time, their complete eradication was still possible with 10 min of exposure. Whilst this study provides useful proof of concept data on the use of atmospheric pressure plasmas for the eradication of bacterial biofilms in vitro, it also demonstrates the critical need for improved understanding of the mechanisms and kinetics related to such a potentially significant approach.

Potential cellular targets and antibacterial efficacy of atmospheric pressure non-thermal plasma

Atmospheric pressure non-thermal plasma (APNTP) has been gaining increasing interest as a new alternative antibacterial approach. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. Mechanistic elucidation of the antimicrobial activity will facilitate development and rational optimisation of this approach for potential medical applications. In this study, the antibacterial efficacy of an in-house-built APNTP jet was evaluated alongside an investigation of the interactions between APNTP and major cellular components in order to identify the potential cellular targets involved in plasma-mediated bacterial destruction mechanisms. The investigated plasma jet exhibited excellent, rapid antibacterial activity against a selected panel of clinically significant bacterial species including Bacillus cereus, meticillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa, all of which were completely inactivated within 2 min of plasma exposure. Plasma-mediated damaging effects were observed, to varying degrees, on all of the investigated cellular components including DNA, a model protein enzyme, and lipid membrane integrity and permeability. The antibacterial efficacy of APNTP appears to involve a multiple-target mechanism, which potentially reduces the likelihood of emergence of microbial resistance towards this promising antimicrobial approach. However, cellular membrane damage and resulting permeability perturbation was found to be the most likely rate-determining step in this mechanism.

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity

The main aim of this study was to determine the virucidal inactivation efficacy of an in‐house‐designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus.

Eradication and phenotypic tolerance of Burkholderia cenocepacia biofilms exposed to atmospheric pressure non-thermal plasma.

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.

The In Vitro Susceptibility of Biofilm Forming Medical Device Related Pathogens to Conventional Antibiotics

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) and kill kinetics were established for vancomycin, rifampicin, trimethoprim, gentamicin, and ciprofloxacin against the biofilm forming bacteria Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus (ATCC 29213), Methicillin Resistant Staphylococcus aureus (MRSA) (ATCC 43300), Pseudomonas aeruginosa (PAO1), and Escherichia coli (NCTC 8196). MICs and MBCs were determined via broth microdilution in 96-well plates. MBECs were studied using the Calgary Biofilm Device. Values obtained were used to investigate the kill kinetics of conventional antimicrobials against a range of planktonic and biofilm microorganisms over a period of 24 hours. Planktonic kill kinetics were determined at 4xMIC and biofilm kill kinetics at relative MBECs. Susceptibility of microorganisms varied depending on antibiotic selected and phenotypic form of bacteria. Gram-positive planktonic isolates were extremely susceptible to vancomycin (highest MBC: 7.81 mg L−1: methicillin sensitive and resistant S. aureus) but no MBEC value was obtained against all biofilm pathogens tested (up to 1000 mg L−1). Both gentamicin and ciprofloxacin displayed the broadest spectrum of activity with MIC and MBCs in the mg L−1 range against all planktonic isolates tested and MBEC values obtained against all but S. epidermidis (ATCC 35984) and MRSA (ATCC 43300).

The Synthesis and Characterization of Fatty Acid Salts of Chitosan as Novel Matrices for Prolonged Intragastric Drug Delivery

The aim of this study was to prepare fatty acid salts of chitosan (CS) and to evaluate the salts as matrices for sustained drug release and prolonged gastric retention. CS-laurate and CS-palmitate were formed by mixing saturated CS solution and aqueous solutions of sodium laurate and sodium palmitate, respectively, and collected by centrifugation. They were characterized using Fourier-transform infrared spectroscopy and differential scanning calorimetry. Different matrices as effervescent tablets were prepared using each of these CS-salts, CS and the corresponding physical mixtures of CS and the fatty acids. Sodium bicarbonate as an effervescent agent and ranitidine HCl as a model drug were incorporated into these matrices. In vitro buoyancy and drug dissolution were studied for the matrices in 0.1 M HCl. Tablets with fatty acid salts of CS showed both rapid and prolonged buoyancy (> 8 h). Comparatively, CS tablets exhibited a short floatation period (< 2 h) and tablets were completely disintegrated within 1 h of soaking. In addition, slow and prolonged drug release was achieved from tablets of fatty acid salts of CS with average drug release of 80.1 and 71.8% for CS-laurate and CS-palmitate, respectively. Rapid drug release (> 80% at 1 h) was exhibited by tablets with CS or the physical mixtures.

Evaluation of paromomycin sulphate permeation using ex vivo human skin model

Abstract Ex vivo evaluation of drug release and skin permeation from topical formulations of antileishmanial drug paromomycin sulphate was carried out using intact full thickness human skin. Potency-based microbiological assay was used for the analysis of paromomycin concentrations. A total percentage drug recovery of 86 ± 26% was obtained. Incubation periods of 1 and 3 h resulted in percentage drug permeation into deep skin layers ranging from 1.3 ± 0.04% to 5.3 ± 2.0% with paraffin-based ointment and from 1.6 ± 0.8% to 3.9 ± 1% with microemulsion-based emulgel. Although a small percentage, this is still significantly higher than those previously reported using animal skin models.

Preparation of Aqueous Core-Poly(d,l-Lactide-co-Glycolide) Shell Microcapsules With Mononuclear Cores by Internal Phase Separation: Optimization of Formulation Parameters

Previously, our group employed the internal phase separation method to produce aqueous core-PLGA [poly(d,l-lactide-co-glycolide)] shell microcapsules with polynuclear core morphologies. This report describes the preparation of the more desired and challenging architecture with mononuclear cores. Optimization of formulation parameters including (1) varying the composition of the internal phase and (2) incorporating selected organic solvents (dichloromethane, chloroform, methanol, and acetonitrile) into the internal phase was systematically evaluated. Varying the composition of the internal phase (i.e., PLGA and water levels) failed to produce mononuclear microcapsules. However, incorporating methanol or acetonitrile into the internal phase produced microcapsules with mononuclear cores as confirmed by phase-contrast microscopy, transmission electron microscopy, and scanning electron microscopy. Stability of the prepared emulsions (internal phase of PLGA, acetone, acetonitrile, and water) was optimized by evaluating different types of surfactants with varying concentrations. Among them, lecithin in the range of 0.5%-5% wt/wt provided the best emulsion stability. Interestingly, increasing lecithin concentrations led to the production of microcapsules with smaller sizes (from 2.4 ± 1.6 to 1.1 ± 0.8 μm) and higher percentage of mononuclear cores. The resulting aqueous core-PLGA shell microcapsules are expected to have interesting applications in drug delivery systems with controlled release for hydrophilic drugs and proteins.