A disclosure gel for visual detection of live Bacillus anthracis spores.

AIMS: To develop a gel formulation to trigger a visual signal for rapid disclosure of the location and extent of surface contamination with viable Bacillus anthracis spores. METHODS AND RESULTS: Methylumbelliferyl-α-d-glucopyranoside was combined with hyaluronic acid to produce a gel that could be applied to a surface as a coating. It remained hydrated for a sufficient time for α-glucosidase activity present in intact B. anthracis spores to cleave the substrate and release the fluorescent product, methylumbelliferone. The presence of B. anthracis spores could be disclosed at 5 × 104 CFU per reaction test well (0·32 cm2 ) both visually and using fluorescence detection equipment. CONCLUSIONS: The disclosure gel provides a rapid, visual response to the presence of B. anthracis spores on a surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The disclosure gel demonstrates the first steps towards the development of a formulation that can provide nonspecialist users with a visual alert to the presence of B. anthracis spores on a surface. It is envisioned that such a formulation would be beneficial in scenarios where exposure to spore release is a risk, and could be used in the initial assessment of equipment to aid prioritization and localized execution of a decontamination strategy.

The signatures of microorganisms and of human and environmental biomes can now be used to provide evidence in legal cases.

The microorganisms with which we share our world go largely unnoticed. We are, however, beginning to be able to exploit their apparently silent presence as witnesses to events that are of legal concern. This information can be used to link forensic samples to criminal events and even perpetrators. Once dead, our bodies are rapidly colonised, internally and externally. The progress of these events can be charted to inform how long and even by what means a person has died. A small number of microbial species could actually be the cause of such deaths as a result of biocrime or bioterrorism. The procedures and techniques to respond to such attacks have matured in the last 20 years. The capability now exists to identify malicious intent, characterise the threat agent to isolate level and potentially link it to perpetrators with a high level of confidence.

Re-aerosolization of Bacillus thuringiensis spores from concrete and turf.

Spores of Bacillus anthracis deposited on surfaces can become airborne again as a result of air currents and mechanical forces. As such, they are a potential source of infection by inhalation. Spores of Bacillus thuringiensis were used to quantify this phenomenon in a simulation of outdoor conditions. Concrete and turf surfaces were inoculated by aerosol to produce high spore densities (greater than 1 × 109  CFU per m2 ) which were then subjected to the passage of air at 10 ms-1 with and without simulated walking. Re-aerosolized spores were sampled by wetted wall cyclone air samplers. The mean total re-aerosolization rate from concrete (m-2  min-1 ) was 1·16 × 10-3 for wind alone and 3·2 × 10-3 for wind and simulated walking while for turf the respective values were 2·7 × 10-4 and 6·7 × 10-4 . SIGNIFICANCE AND IMPACT OF THE STUDY: Following the malicious and/or accidental release of an aerosol of Bacillus anthracis spores, the immediate risk of human inhalation would decrease as the spores were deposited on surfaces or diluted by wind flow. There is, however, a concern that the deposited spores could become re-aerosolized and so present an ongoing hazard. Using an accurate simulant for B. anthracis spores a method is reported here that allowed the enumeration of re-aerosolized spores from concrete and turf by wind flow and footfall. Under the conditions used, the rates of re-aerosolization were low. These findings will need to be verified under real outdoor conditions before the true significance in terms of secondary exposure to pathogenic spores can be assessed.

Germination and persistence of Bacillus anthracis and Bacillus thuringiensis in soil microcosms.

AIMS: Decontaminating large, outdoor spaces of Bacillus anthracis spores presents significant problems, particularly in soil. Proof was sought that the addition of germinant chemicals could cause spores of B. anthracis and Bacillus thuringiensis, a commonly used simulant of the threat agent, to convert to the less resistant vegetative form in a microcosm. METHODS AND RESULTS: Nonsterile plant/soil microcosms were inoculated with spores of B. thuringiensis and two nonpathogenic strains of B. anthracis. A combination of L-alanine (100 mmol l(-1)) and inosine (10 mmol l(-1)) resulted in a 6 log decrease in spore numbers in both strains of B. anthracis over 2 weeks at 22°C; a 3 log decrease in B. anthracis Sterne spore numbers was observed after incubation for 2 weeks at 10°C. Negligible germination nor a decrease in viable count occurred in either strain when the concentration of L-alanine was decreased to 5 mmol l(-1). Germinated spores of B. thuringiensis were able to persist in vegetative form in the microcosms, whereas those of B. anthracis rapidly disappeared. The pleiotropic regulator PlcR, which B. anthracis lacks, does not contribute to the persistence of B. thuringiensis in vegetative form in soil. CONCLUSIONS: The principle of adding germinants to soil to trigger the conversion of spores to vegetative form has been demonstrated. Bacillus anthracis failed to persist in vegetative form or resporulate in the microcosms after it had been induced to germinate. SIGNIFICANCE AND IMPACT OF THE STUDY: The large scale, outdoor decontamination of B. anthracis spores may be facilitated by the application of simple, defined combinations of germinants.

Expression of prtA from Photorhabdus luminescens in Bacillus thuringiensis enhances mortality in lepidopteran larvae by sub-cutaneous but not oral infection.

The prtA gene from Photorhabdus luminescens encodes the virulence factor Protease A. When P. luminescens is injected into the hemocoel of insects by entomopathogenic nematodes, PrtA is a key component of pathogenicity thought to help degrade the immune system. The prtA gene was cloned and introduced on a plasmid into Bacillus thuringiensis. PrtA was shown to be actively expressed in vitro by cleavage of a specific Dabcyl-Edans heptapeptide substrate. There was no difference in the speed or level of mortality when spores and δ-endotoxins crystals of the transformed strain were fed to larvae of Pieris brassicae, as compared to the wild-type strain. When vegetative cells were injected into the hemocoel of larvae of Galleriamellonella, however, there was a significant increase in the rate and level of mortality over the wild type. The yield of B. thuringiensis per cadaver was a hundred-fold greater in the PrtA-secreting strain. The increased pathogenicity from intrahemocoelic infection may have been due to a greater ability to overcome the immune response of G. mellonella while other factors such as resident gut bacteria may have negated this advantage after oral dosage.

Bacillus thuringiensis HD-1 Cry- : development of a safe, non-insecticidal simulant for Bacillus anthracis.

AIMS: A representative simulant for spores of Bacillus anthracis is needed for field testing. Bacillus thuringiensis is gaining recognition as a suitable organism. A strain that does not form the insecticidal, parasporal crystals that are characteristic of this species is a more accurate physical representative of B. anthracis spores. We developed noninsecticidal derivatives of two isolates of B. thuringiensis HD-1. METHODS AND RESULTS: Two plasmid-cured derivatives of B. thuringiensis HD-1, unable to make crystal toxins ('Cry(-) '), were isolated. These isolates and the existing Cry(-) strain, B. thuringiensis Al Hakam, were probed with PCR assays against the known insecticidal genes cry, vip and cyt. Their genomic DNA was sequenced to demonstrate a lack of insecticidal genes. This was confirmed by bioassays against a number of invertebrate species. Real-time PCR assays were developed to identify the B. thuringiensis HD-1 Cry(-) derivatives and an effective differential and selective medium was assessed. CONCLUSIONS: All three Cry(-) isolates are devoid of known insecticidal determinants. The B. thuringiensis HD-1 Cry(-) derivatives can easily be recovered from soil and identified by PCR with some selectivity. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. thuringiensis HD-1 Cry(-) derivatives represent accurate, nongenetically manipulated simulants for B. anthracis with excellent human and environmental safety records.

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

Transposon-directed insertion site sequencing was used to identify genes required by Burkholderia thailandensis to survive in plant/soil microcosms. A total of 1,153 genetic loci fulfilled the criteria as being likely to encode survival characteristics. Of these, 203 (17.6 %) were associated with uptake and transport systems; 463 loci (40.1 %) coded for enzymatic properties, 99 of these (21.4 %) had reduction/oxidation functions; 117 (10.1 %) were gene regulation or sensory loci; 61 (5.3 %) encoded structural proteins found in the cell envelope or with enzymatic activities related to it, distinct from these, 46 (4.0 %) were involved in chemotaxis and flagellum, or pilus synthesis; 39 (3.4 %) were transposase enzymes or were bacteriophage-derived; and 30 (2.6 %) were involved in the production of antibiotics or siderophores. Two hundred and twenty genes (19.1 %) encoded hypothetical proteins or those of unknown function. Given the importance of motility and pilus formation in microcosm persistence the nature of the colonization of the rhizosphere was examined by confocal microscopy. Wild type B. thailandensis expressing red fluorescent protein was inoculated into microcosms. Even though the roots had been washed, the bacteria were still present but they were motile with no attachment having taken place, perhaps being retained in a biofilm.

Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP.

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE-MLST method emerged as a superior method to T-RFLP-MLST for rapid typing of bacterial communities.

Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica.

Several strains of Bacillus thuringiensis were previously isolated from soil in Antarctica and appeared to have physiological adaptations to this cold, nutrient-poor environment. In spite of this they could produce abnormally large, parasporal crystals under laboratory conditions. Here, they have been further characterised for toxin genes and invertebrate pathogenicity. All of the strains were positive in PCR assays for the cry1Aa and cry2 genes. This was confirmed by sequence analysis and the parasporal crystals of all strains contained polypeptides of about 130kDa. This potential for lepidopteran toxicity was borne out in bioassays of purified δ-endotoxins against larvae of Pieris brassicae: the LD(50) values of B2408 (288µg) were comparable to that of the reference strain, HD-12 (201µg). There was no activity against the nematode Caenorhabditis elegans in spite of the fact that all strains appeared to possess the cry6 gene. PCR screening for genes encoding other nematode-toxic classes of toxins (Cry5, 4 and 21) was negative. B. thuringiensis has never previously been shown to be toxic to Collembola (springtails) but the purified δ-endotoxins of one of the Antarctic strains showed some activity against Folsomia candida and Seira domestica (224µg and 238µg, respectively). It seems unlikely that the level of toxicity demonstrated against springtails would support a pathogenic life-style in nature. All of the strains were positive for genes encoding Bacillus cereus-type enterotoxins. In the absence of higher insects and mammals the ecological value of retaining the toxic capability demonstrated here is uncertain.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

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 ecology of Bacillus thuringiensis on the Phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae.

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

Multiple-locus sequence typing analysis of Bacillus thuringiensis recovered from the phylloplane of clover (Trifolium hybridum) in vegetative form.

Abstract The chromosomal genotype, as judged by multi locus sequence typing, and the episomal genotype, as judged by plasmid profile and cry gene content, were analyzed for a collection of strains of Bacillus thuringiensis. These had been recovered in vegetative form over a period of several months from the leaves of a small plot of clover (Trifolium hybridum). A clonal population structure was indicated, although greater variation in sequence types (STs) was discovered than in previous collections of B. cereus/B. thuringiensis. Isolates taken at the same time had quite different genotypes, whereas those of identical genotypes were recovered at different times. The profiles of plasmid content and cry genes generally bore no relation to each other nor to the STs. Evidently, although relatively little recombination was occurring in the seven chromosomal genes analyzed, a great deal of conjugal transfer, and perhaps recombination, was occurring involving plasmids. A clinical diarrheal isolate of B. cereus and the commercial biopesticide strain HD-1 of B. thuringiensis, both included as out-groups, were found to have very similar STs. This further emphasizes the role of episomal elements in the characteristics and differentiation of these two species.

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium.

AIM: To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis. METHODS AND RESULTS: Four strains of B. thuringiensis, which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce delta-endotoxins; loss of ability to produce beta-exotoxin and disruption of vip3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The delta-endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content. CONCLUSION: Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.

The analysis of microorganisms by microcalorimetry in the pharmaceutical industry.

Many features of microorganisms make them pre-eminently suitable for study by microcalorimetry. They have thus, in the past, been the basis of fundamental studies in metabolism and cellular physiology. In this review we look at the application of calorimetry to the impact of bacteria and fungi on the pharmaceutical industry both in the exploitation of useful microorganisms and the fight against harmful ones. Obviously they are of great relevance to the pharmaceutical industry as agents of human disease, with more antimicrobial products registered for production than for any other type of human affliction. Microcalorimetry offers the opportunity to study microorganisms in real time and in heterogeneous systems, allowing for more descriptive and representative analysis. Other advantages that microcalorimetry confers over traditional microbiological techniques are reductions in time, better reproducibility and simplicity. Also the manufacture of all pharmaceutical products requires the exclusion of microorganisms to a greater or lesser degree. The enumeration and identification of such contaminants is of great importance for the well-being of patients and to maintain the integrity of the product. New techniques are required to increase the reliability and sensitivity over conventional methods. Finally, as our understanding of biology develops, the sophistication of therapeutic agents available, such as vaccines, cytokines and engineered antibodies, is increasing. Necessarily, prokaryotic and eukaryotic cells, possibly transformed with the appropriate genes, are the producers of such proteins. Microcalorimetry offers a sensitive means of developing the conditions for optimum production of such products in active form since it gives instantaneous information on the physiology of the producer cell.

Effects of media composition of delta-endotoxin production and morphology of Bacillus thuringiensis in wild types and spontaneously mutated strains.

Bacillus thuringiensis strains HD-73 and 4412, and two spontaneous mutants termed 4412aa-ind and 4412sph-cry were studied for the ability to produce crystals of different size and shape when grown in a rich medium and in an appropriate minimal medium defined during this study. Strain 4412aa-ind showed medium-dependent variation in the crystal phenotype. Scanning electron microscopy was utilized in order to show crystal variations in size and shape. B. thuringiensis strains 4412aa-ind and 4412sph-cry grown in rich and in minimal media produced differences in crystal morphology, and SDS-PAGE gel indicated that crystal variation was only at the morphological level and not in composition of the amino acids. A further nineteen B. thuringiensis strains were tested for the ability to sporulate in two simple defined media. Of these strains thirteen were able to complete sporulation with crystal production in one or both media. All strains grew and sporulated in a medium containing the usual twenty amino acids, and no vitamins or other growth factors were required.

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.

A philosophical interpretation of nursing.

The authors discuss their interpretation of nursing as the practice of caring. Through interpretive phenomenology, they lift out the essential meaning of nursing as practiced, and through generative interpretation of philosophical treatments of relationships, practice, and caring, they enlighten nursing practice. They discuss the implications of interpreting nursing as the practice of caring for nursing scholarship, education, and ethics.

Prevalence of beta-exotoxin, diarrhoeal toxin and specific delta-endotoxin in natural isolates of Bacillus thuringiensis.

Of newly isolated colonies with the appearance of Bacillus thuringiensis, 47.5% were found to produce the parasporal crystals characteristic of this species. These positive isolates were screened using the polymerase chain reaction for their possession of a gene encoding a specific protoxin type, CryIB. Strains with and without this gene were screened for their ability to produce beta-exotoxin and Bacillus cereus-type enterotoxin. It was found that 35% of the isolates possessed the cryIB gene; of these 83% also produced enterotoxin and 58% produced beta-exotoxin. No statistical significance was found for linkage between any of these characteristics. The probability, therefore, of isolating a strain of B. thuringiensis which specifically possessed the cryIB gene but did not produce either of the other, undesired, toxins, from the soil sample used, was 1.2%.