Microneedle Arrays Allow Lower Microbial Penetration Than Hypodermic Needles In Vitro

Methods: In this study we determined, for the first time, the ability of microorganisms to traverse microneedle-induced holes using two different in vitro models.

Results: When employing Silescol® membranes, the numbers of Candida albicans, Pseudomonas aeruginosa and Staphylococcus epidermidis crossing the membranes were an order of magnitude lower when the membranes were punctured by microneedles rather than a 21G hypodermic needle. Apart from the movement of C. albicans across hypodermic needle-punctured membranes, where 40.2% of the microbial load on control membranes permeated the barrier over 24 h, the numbers of permeating microorganisms was less than 5% of the original microbial load on control membranes. Experiments employing excised porcine skin and radiolabelled microorganisms showed that the numbers of microorganisms penetrating skin beyond the stratum corneum were approximately an order of magnitude greater than the numbers crossing Silescol® membranes in the corresponding experiments. Approximately 103?cfu of each microorganism adhered to hypodermic needles during insertion. The numbers of microorganisms adhering to MN arrays were an order of magnitude higher in each case.

Conclusion: We have shown here that microneedle puncture resulted in significantly less microbial penetration than did hypodermic needle puncture and that no microorganisms crossed the viable epidermis in microneedle—punctured skin, in contrast to needle-punctured skin. Given the antimicrobial properties of skin, it is, therefore, likely that application of microneedle arrays to skin in an appropriate manner would not cause either local or systemic infection in normal circumstances in immune-competent patients. In supporting widespread clinical use of microneedle-based delivery systems, appropriate animal studies are now needed to conclusively demonstrate this in vivo. Safety in patients will be enhanced by aseptic or sterile manufacture and by fabricating microneedles from self-disabling materials (e.g. dissolving or biodegradable polymers) to prevent inappropriate or accidental reuse.

Anti-adherent and antifungal activities of surfactant-coated poly (ethylcyanoacrylate) nanoparticles

Application of non-drug-loaded poly(ethylcyanoacrylate) nanoparticles (NP) to buccal epithelial cells (BEC) imparted both anti-adherent and antifungal effects. NP prepared using emulsion polymerisation and stabilised using cationic, anionic and non-ionic surfactants decreased Candida albicans blastospore adhesion, an effect attributable to the peripheral coating of surfactant. Cetrimide and Pluronic (R) P 123 were shown to be most effective, producing mean percentage reductions in blastospore adherence of 52.7 and 37.0, respectively. Resultant zeta potential matched the polarity of the surfactant, with those stabilised using cetrimide being especially positive (+31.3 mV). Preparation using anionic surfactants was shown to be problematic, with low yield and wide particle size distribution. Evaluation of the antifungal effect of the peripheral coat was evaluated using zones of inhibition and viable counts assays. The former test revealed poor surfactant diffusion through agar, but did show evidence of limited kill. However, the latter method showed that cationic surfactants associated with NP produced high levels of kill, in contrast to those coated with anionic surfactants, where kill was not evident. Non-ionic surfactant-coated NP produced intermediate kill rates. Results demonstrate that surfactant-coated NP, particularly the cationic types, form the possible basis of a prophylactic formulation that primes the candidal target (BEC) against fungal adhesion and infection. (c) 2007 Elsevier B.V. All rights reserved.

Antimicrobial peptides from the skin secretion of the Wuyi Mountain torrent frog, Amolops wuyiensis: An inhabitant of a pristine aquatic environment

The antimicrobial peptides of amphibian skin secretions are proposed to aid survival in microbe-rich environments. While many amphibians inhabit such environments, other such as the Wuyi Mountain torrent frog, Amolops wuyiensis, live in pristine waters flowing from underground mountain springs. This species thus represents an interesting model in which to study antimicrobial peptides. “Shotgun” cloning of a skin-derived cDNA library from this species identified transcripts encoding a brevinin-1 and a ranatuerin-2. Peptides with coincident molecular masses to both predicted mature peptides were identified in HPLC fractions of skin secretion. Synthetic replicates of both peptides were generated by solid-phase peptide synthesis and tested for activity using Staphylococcus aureus, Escherichia coli and Candida albicans. The brevinin was found to be broad-spectrum and potent with minimum inhibitory concentrations (MICs) of 24 µM (Sa), 5 µM (Ec) and 20 µM (Ca). In contrast, the ranatuerin was less effective and of narrower spectrum with an MIC > 200 µM for Sa, 40 µM (Ec) and 120 µM (Ca). Thus this species of amphibian that lives in a pristine environment does indeed possess at least one potent and broad-spectrum antimicrobial peptide in its skin secretion arsenal. This phenomenon could be explained in several ways. Firstly, it may represent an ancestral peptide required when the stem species inhabited microbe-rich environments. However, there is mounting evidence for the second reason, that suggests the function of such peptides is not primarily in antimicrobial defence.

PRELIMINARY INVESTIGATIONS CONCERNING THE ANTI-ADHERENCE PROPERTIES OF POLYHEXAMETHYLENEBIGUANIDE (VANTOCIL IB(TM))

The effect of polyhexamethylenebiguanide (PHMB) on adherence of Candida albicans blastospores to human buccal epithelial cells (BEG) was examined in vitro. Treatments of either blastospores or BEC with PHMB (50 and 1000 mu g ml(-1)) significantly reduced the number of adherent blastospores per BEC and increased the number of BEC devoid of blastospores.

THE EFFECTS OF 3 NON-ANTIBIOTIC, ANTIMICROBIAL AGENTS ON THE SURFACE HYDROPHOBICITY OF CERTAIN MICROORGANISMS EVALUATED BY DIFFERENT METHODS

The effects of three non-antibiotic, antimicrobial agents (taurolidine, chlorhexidine acetate and providone-iodine) on the surface hydrophobicity of the clinical strains Escherichia coli, Staphylococcus saprophyticus, Staphylococcus epidermidis and Candida albicans were examined. Three recognized techniques for hydrophobicity measurements, Bacterial Adherence to Hydrocarbons (BATH), the Salt Aggregation Test (SAT) and Hydrophobic Interaction Chromatography (HIC) were compared. At concentrations reported to interfere with microbial-epithelial cell adherence, all three agents altered the cell surface hydrophobicity. However, these effects failed to exhibit a uniform relationship. Generally, taurolidine and povidone-iodine treatments decreased the hydrophobicity of the strains examined whereas chlorhexidine acetate effects depended upon the micro-organism treated. Subsequently, the exact contribution of altered cell surface hydrophobicity to the reported microbial anti-adherence effects is unclear. Comparison of the three techniques revealed a better correlation between the results obtained with the BATH test and HIC than the results obtained with the BATH and SAT or SAT and HIC. However, these differences may be due to the inaccuracy associated with the visual assessment of results employed by the SAT.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Trends in the epidemiology of Candida bloodstream infections in Northern Ireland between January 1984 and December 2000

Objective: To describe the epidemiology of Candida bloodstream infections (BSI) in Northern Ireland. Methods: Retrospective collation of data relating to all clinically significant BSI in a university teaching hospital, which had been recorded prospectively, between 1984 and 2000. Results: One hundred and forty five episodes of candidaemia occurred in 144 patients (of mean age 56.6 years). The contribution of Candida spp. towards all significant BSI increased from 2.00% to 2.5%. C. albicans was the most frequently isolated species, however, its incidence fell from 70% to 53% during the study period. The greatest increase in incidence was seen with C. glabrata which was the most common non-albicans species. Twenty-nine per cent of isolates occurred in patients from an intensive care unit and, surprisingly, a further 25.5% occurred in patients from a surgical service. Conclusion: There appears to be several subtle differences in the epidemiology of candidal BSI between Northern Ireland and other countries. © 2002 The British Infection Society.

Improving molecular detection of Candida DNA in whole blood:comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Trends in candidemia and antifungal susceptibility in a university hospital in Northern Ireland 2001-2006

Objectives: To describe the species distribution and antifungal susceptibility trends for documented episodes of candidemia at the Royal Hospitals, Belfast, 2001-2006. Methods: Laboratory-based retrospective observational study of all episodes of candidemia. Results: There were 151 episodes of candidemia. The species recovered were: 96 C. albicans; 26 C. glabrata; 18 C. parapsilosis; five C. tropicalis; four C. guilliermondii; one C. famata and one C. dubliniensis. We separated the data into two periods 2001-2003 and 2004-2006; contrary to the findings of other investigators, there was a notable trends toward increasing frequency of C. albicans and decreasing frequency of non-albicans species over time. Although the proportion of C. albicans, C. parapsilosis and C. tropicalis isolates susceptible to fluconazole was unchanged over time, a trend of decreased susceptibility of C. glabrata to fluconazole was noted over the six-year period. Overall, 73% and 7.7% of C. glabrata isolates had susceptible-dose-dependent and resistant phenotypes, respectively. The percentage of C. glabrata isolates susceptible to fluconazole (MIC

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 1 8S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100% concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ~72 to

Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog, (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group were named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH2) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate
displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50µM) and Candida albicans (MIC 150µM) but was nonhaemolytic at concentrations up to and including 150µM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9nM) and a dosedependent relaxation of rat tail artery smooth muscle (EC50 37.7nM). Senegalin thus represents a prototype biologically-active amphibian skin peptide and illustrates the fact thatamphibian skin secretion peptidomes continue to be unique sources of such molecules.

IQ-motif peptides as novel anti-microbial agents

The IQ-motif is an amphipathic, often positively charged, a-helical, calmodulin binding sequence found in a number of eukaryote signalling, transport and cytoskeletal proteins. They share common biophysical characteristics with established, cationic a-helical antimicrobial peptides, such as the human cathelicidin LL-37. Therefore, we tested eight peptides encoding the sequences of IQ-motifs derived from the human cytoskeletal scaffolding proteins IQGAP2 and IQGAP3. Some of these peptides were able to inhibit the growth of Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) comparable to LL-37. In addition some IQ-motifs had activity against the fungus Candida albicans. This antimicrobial activity is combined with low haemolytic activity (comparable to, or lower than, that of LL-37). Those IQ-motifs with anti-microbial activity tended to be able to bind to lipopolysaccharide. Some of these were also able to permeabilise the cell membranes of both Gram positive and Gram negative bacteria. These results demonstrate that IQ-motifs are viable lead sequences for the identification and optimisation of novel anti-microbial peptides. Thus, further investigation of the anti-microbial properties of this diverse group of sequences is merited.

Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities

Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160µM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5µM) and the yeast, Candida albicans (10µM). Haemolytic activity of TsAP-1 was low (4% at 160µM) and in contrast, that of TsAP-2 was considerably higher (18% at 20µM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5µM for S.aureus/C.albicans and 5µM for E.coli but with an associated large increase in haemolytic activity (30% at 5µM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E.coli lowering this from >320µM to 5µM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 µM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity.