Alistair H. Bishop

Antimicrobial properties of silver-containing wound dressings: a microcalorimetric study

The studies reported here have been undertaken to assess the potential use of isothermal microcalorimetry in studying the antimicrobial efficacy of wound dressings that contain antimicrobial agents. The microcalorimetric technique allows non-invasive and non-destructive analysis to be performed directly on a test sample, regardless of whether it is homogeneous or heterogeneous in nature. Microcalorimetry is an established procedure that offers quantitative measurements and has the distinct advantage over traditional antimicrobial test methodologies in that calorimetric measurements are made continuously over real-time, thus the dynamic response of microorganisms to an antimicrobial agent is observed in situ. The results described in this paper are for interaction of two silver-containing wound care products AQUACEL Ag Hydrofiber (ConvaTec, Deeside, UK) and Acticoat 7 with SILCRYST (Smith and Nephew Healthcare, UK) with the wound pathogenic organisms Staphylococcus aureus and Pseudomonas aeruginosa. Both dressings are shown, microcalorimetrically, to have the capacity to kill these common wound pathogens within 1-2 h of contact. A dose-response study was conducted with the AQUACEL Ag dressing. Microcalorimetry is shown to be rapid, simple and effective in the study of the antimicrobial properties of gel forming wound dressings.

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings.

Treatment with silver-containing wound dressings is becoming an increasingly popular strategy to eliminate growth of opportunistic wound pathogens during the healing process. However, there are concerns over the possible side-effects of silver to the patient; coupled to the cost of silver as an ingredient there is a desire to ensure that wound dressings contain the least quantity of active ingredient to ensure the minimum bactericidal concentration (MBC) of silver is maintained in the wound environment. This requires the ability to determine the efficacy of silver directly within the wound environment; an extremely complicated task that is difficult using classical (plate counting) microbiological assays because these cannot be conducted in situ. Here, we report a quantitative method for determining the efficacy of silver in wound dressings using an isothermal calorimetric method. The growth curves of P. aeruginosa (NCIMB 8628) were recorded in growth medium and in growth medium containing AQUACEL Ag Hydrofiber dressing. It was found that 10 mg of dressing was sufficient to ensure no detectable growth of organism in 2.5 mL of medium inoculated to 10(6) cfu/mL. This corresponded to a silver load of 1.1x10(-6) moles (equivalent to 4.4x10(-4) M, in the volume of medium used in the experiment). Experiments conducted with silver nitrate rather than dressing indicated the MBC of silver against P. aeruginosa was 1x10(-4) M. The results suggested that not all of the silver in the dressing was bioavailable, at least over the lifetime of the experiment. One advantage of this effect would be the lack of excess availability of the silver, which allays fears of potential toxicity to the patient and may provide an extended period of time over which the dressing is bactericidal.

The safety of Bacillus thuringiensis to mammals investigated by oral and subcutaneous dosage

Six strains of Bacillus thuringiensis were tested with two commercially available kits for their ability to produce Bacillus cereus-type enterotoxin and by dipteran bioassay for the production of β-exotoxin. All of the strains were positive for enterotoxin production including three which have been used world-wide for many years to control pest insects. Rats given oral doses totalling 1 × 1012 spores ( +crystals), over three weeks, or a single subcutaneous dose of 1 × 106 spores ( +crystals) showed no ill-effects in terms of their condition or in the pathology of their internal organs: this was in spite of the strain of B. thuringiensis used (13B) being an active producer of both β-exotoxin and enterotoxin. A commercial insecticide containing B. thuringiensis was sprayed onto spinach leaves. After normal food preparation regimes some leaves retained residual spore loads sufficient for a strongly enterotoxic strain to cause food poisoning in humans. These findings suggest that the agricultural use of some, previously unvalidated, strains of B. thuringiensis could give rise to cases of food poisoning and that rodents are unsuitable for testing the safety to humans of oral exposure to this organism.

Phylogenetic analysis and confirmation of the endospore-forming nature of Pasteuria penetrans based on the spo0A gene

Pasteuria penetrans is an obligate parasite of plant parasitic nematodes and has yet to be grown in vitro. We have cloned the pivotal sporulation gene, spo0A, which is the first whole gene yet to come from this organism. Partial spo0A sequences were also obtained from the related bacteria, Pasteuria ramosa and Alicyclobacillus acidocaldarius. Phylogenetic analyses using the spo0A sequence data from this and previous studies confirmed the closeness of the genera Pasteuria and members of the supergenus Bacillus. A segment of the spo0A gene was also used to show that genetic heterogeneity exists within and between populations of P. penetrans. This may explain, partly at least, the variability of P. penetrans as a biological control agent of nematodes.

Amplification of fluorescently labelled DNA within Gram-positive and acid-fast bacteria

Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA. The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell. Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls. A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only. Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei. Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L. lactis and M. phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.

Effect of Bacillus thuringiensis naturally colonising Brassica campestris var. chinensis leaves on neonate larvae of Pieris brassicae.

The feeding of neonate larvae of Pieris brassicae (Order Lepidoptera) on leaves of brassica plants that had been colonised by Bacillus thuringiensis resulted in the death of 35% of the population within 72h. The bacteria multiplied in the cadavers, resulting in an increase of about 50-fold compared to the living insects. Surviving insects showed no ill effects during the time of the study. There was negligible multiplication of B. thuringiensis in the frass.

The polypeptide components of the parasporal fibres of Pasteuria penetrans

A variety of treatments were tested for their ability to solubilize the parasporal fibres from Pasteuria penetrans, a parasite of some plant–parasitic nematodes. Selective solubilization of the parasporal fibres resulted from some of the extraction procedures tested. Subsequent acrylamide gel electrophoresis and Western blotting of the resolved polypeptides, using polyclonal sera against the spores, disclosed up to 15 distinct bands, ranging in size from 12 to 195 kDa. An N-terminal amino acid sequence was obtained from a 50 kDa polypeptide and an oligonucleotide primer deduced from it. A whole cell, fluorescent, primed in situ labelling (PRINS) technique was adapted to be applicable to spores of P. penetrans and P. ramosa, a parasite of water fleas. Positive responses were obtained using the parasporal fibre primer on spores of the former but not of the latter organism, implying that this 50 kDa polypeptide is produced by P. penetrans but not by P. ramosa.

The isolation of a single spore isolate of Pasteuria penetrans and its pathogenicity on Meloidogyne javanica

We have obtained a single spore isolate of Pasteuria penetrans, derived by allowing a single spore to attach to a second-stage juvenile (J2) of the root-knot nematode Meloidogyne javanica. By analysing DNA sequences at three different loci we have obtained evidence that the isolate is, indeed, genetically pure. We compared the ability of the single spore isolate and the parent population from which it was selected to attach to and parasitise both the original population of M. javanica on which it was isolated and a single egg mass line derived from it. There was no difference in the attachment of spores of the single spore isolate to juveniles compared to the parental population, although there were higher numbers of both attaching to J2 of the single egg mass line compared to its parental population. Judging from the numbers of egg masses and Pasteuria-infected females, the single spore isolate was less pathogenic to the parental population of M. javanica than was the parental spore population.