Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato.

Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficileIMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of "germinate to decontaminate" strategy may thus become more practicable.

Aerosol and Surface Deposition Characteristics of Two Surrogates for Bacillus anthracis Spores.

Spores of an acrystalliferous derivative of Bacillus thuringiensis subsp. kurstaki, termed Btcry-, are morphologically, aerodynamically, and structurally indistinguishable from Bacillus anthracis spores. Btcry- spores were dispersed in a large, open-ended barn together with spores of Bacillus atrophaeus subsp. globigii, a historically used surrogate for Bacillus anthracis Spore suspensions (2 × 1012 CFU each of B. atrophaeus subsp. globigii and Btcry-) were aerosolized in each of five spray events using a backpack misting device incorporating an air blower; a wind of 4.9 to 7.6 m s-1 was also flowing through the barn in the same direction. Filter air samplers were situated throughout the barn to assess the aerosol density of the spores during each release. Trays filled with a surfactant in aqueous buffer were placed on the floor near the filter samplers to assess spore deposition. Spores were also recovered from arrays of solid surfaces (concrete, aluminum, and plywood) that had been laid on the floor and set up as a wall at the end of the barn. B. atrophaeus subsp. globigii spores were found to remain airborne for significantly longer periods, and to be deposited on horizontal surfaces at lower densities, than Btcry- spores, particularly near the spray source. There was a 6-fold-higher deposition of Btcry- spores than of B. atrophaeus subsp. globigii spores on vertical surfaces relative to the surrounding airborne density. This work is relevant for selecting the best B. anthracis surrogate for the prediction of human exposure, hazard assessment, and hazard management following a malicious release of B. anthracis IMPORTANCE: There is concern that pathogenic bacteria could be maliciously disseminated in the air to cause human infection and disruption of normal life. The threat from spore-forming organisms, such as the causative agent of anthrax, is particularly serious. In order to assess the extent of this risk, it is important to have a surrogate organism that can be used to replicate the dispersal characteristics of the threat agent accurately. This work compares the aerosol dispersal and deposition behaviors of the surrogates Btcry- and B. atrophaeus subsp. globigii Btcry- spores remained in the air for a shorter time, and were markedly more likely to adhere to vertical surfaces, than B. atrophaeus subsp. globigii spores.

Long-term antibiotic exposure in soil is associated with changes in microbial community structure and prevalence of class 1 integrons.

Antimicrobial resistance is one of the most significant challenges facing the global medical community and can be attributed to the use and misuse of antibiotics. This includes use as growth promoters or for prophylaxis and treatment of bacterial infection in intensively farmed livestock from where antibiotics can enter the environment as residues in manure. We characterised the impact of the long-term application of a mixture of veterinary antibiotics alone (tylosin, sulfamethazine and chlortetracycline) on class 1 integron prevalence and soil microbiota composition. Class 1 integron prevalence increased significantly (P < 0.005) from 0.006% in control samples to 0.064% in the treated plots. Soil microbiota was analysed using 16S rRNA gene sequencing and revealed significant alterations in composition. Of the 19 significantly different (P < 0.05) OTUs identified, 16 were of the Class Proteobacteria and these decreased in abundance relative to the control plots. Only one OTU, of the Class Cyanobacteria, was shown to increase in abundance significantly; a curiosity given the established sensitivity of this class to antibiotics. We hypothesise that the overrepresentation of Proteobacteria as OTUs that decreased significantly in relative abundance, coupled with the observations of an increase in integron prevalence, may represent a strong selective pressure on these taxa.

Identification of genes required by Bacillus thuringiensis for survival in soil by transposon-directed insertion site sequencing.

Transposon-directed insertion site sequencing was used to identify genes required by Bacillus thuringiensis to survive in non-axenic plant/soil microcosms. A total of 516 genetic loci fulfilled the criteria as conferring survival characteristics. Of these, 127 (24.6 %) were associated with uptake and transport systems; 227 loci (44.0 %) coded for enzymatic properties; 49 (9.5 %) were gene regulation or sensory loci; 40 (7.8 %) were structural proteins found in the cell envelope or had enzymatic activities related to it and 24 (4.7 %) were involved in the production of antibiotics or resistance to them. Eighty-three (16.1 %) encoded hypothetical proteins or those of unknown function. The ability to form spores was a key survival characteristic in the microcosms: bacteria, inoculated in either spore or vegetative form, were able to multiply and colonise the soil, whereas a sporulation-deficient mutant was not. The presence of grass seedlings was critical to colonisation. Bacteria labelled with green fluorescent protein were observed to adhere to plant roots. The sporulation-specific promoter of spo0A, the key regulator of sporulation, was strongly activated in the rhizosphere. In contrast, the vegetative-specific promoters of spo0A and PlcR, a pleiotropic regulator of genes with diverse activities, were only very weakly activated.

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.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis.

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season.

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ('vegetative isolates') with the majority possessing multiple delta-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified delta-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.

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.

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.