Defence Science and Technology Laboratory

Dental plaque forms naturally on teeth and is of benefit to the host by helping to prevent colonization by exogenous species. The bacterial composition of plaque remains relatively stable despite regular exposure to minor environmental perturbations. This stability (microbial homeostasis) is due in part to a dynamic balance of both synergistic and antagonistic microbial interactions. However, homeostasis can break down, leading to shifts in the balance of the microflora, thereby predisposing sites to disease. For example, the frequent exposure of plaque to low pH leads to inhibition of acid-sensitive species and the selection of organisms with an aciduric physiology, such as mutans streptococci and lactobacilli. Similarly, plaque accumulation around the gingival margin leads to an inflammatory host response and an increased flow of gingival crevicular fluid. The subgingival microflora shifts from being mainly Gram-positive to being comprised of increased levels of obligately anaerobic, asaccharolytic Gram-negative organisms. It is proposed that disease can be prevented or treated not only by targeting the putative pathogens but also by interfering with the processes that drive the breakdown in homeostasis. Thus, the rate of acid production following sugar intake could be reduced by fluoride, alternative sweeteners, and low concentrations of antimicrobial agents, while oxygenating or redox agents could raise the Eh of periodontal pockets and prevent the growth and metabolism of obligately anaerobic species. These views have been incorporated into a modified hypothesis (the "ecological plaque hypothesis") to explain the relationship between the plaque microflora and the host in health and disease, and to identify new strategies for disease prevention.

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.

The LysR family of transcriptional regulators represents the most abundant type of transcriptional regulator in the prokaryotic kingdom. Members of this family have a conserved structure with an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. Despite considerable conservation both structurally and functionally, LysR-type transcriptional regulators (LTTRs) regulate a diverse set of genes, including those involved in virulence, metabolism, quorum sensing and motility. Numerous structural and transcriptional studies of members of the LTTR family are helping to unravel a compelling paradigm that has evolved from the original observations and conclusions that were made about this family of transcriptional regulators.

Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals. In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal. The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent. The diagnosis of disease is not straightforward. F. tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel. Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia. Little is known about the virulence mechanisms of F. tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages. An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia. Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F. tularensis. In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia.

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die. Here we report the complete genome of B. pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them. The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins. A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome. Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B. mallei. We propose that variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

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.

The 2013–2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic ‘gravity’ model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics. Frequent dispersal and short-lived local transmission clusters fuelled the 2013–2016 Ebola virus epidemic in Guinea, Liberia and Sierra Leone. Understanding how and why viruses spread during epidemics is crucial for planning how to prevent and respond to future threats. Andrew Rambaut and colleagues provide an overview of the genetic epidemiology of the 2013–2016 epidemic caused by Ebola virus in West Africa. By analysing more than 1,600 Ebola virus genomes, the authors determine the factors that were important in the spread of the epidemic and also explain why the virus did not spread into neighbouring countries.

The search for new antifouling (AF) coatings that are environmentally benign has led to renewed interest in the ways that micro-organisms colonize substrates in the marine environment. This review covers recently published research on the global species composition and dynamics of marine biofilms, consisting mainly of bacteria and diatoms found on man-made surfaces including AF coatings. Marine biofilms directly interact with larger organisms (macrofoulers) during colonization processes; hence, recent literature on understanding the basis of the biofilm/macrofouling interactions is essential and will also be reviewed here. Overall, differences have been identified in species composition between biofilm and planktonic forms for both diatoms and bacteria at various exposure sites. In most studies, the underlying biofilm was found to induce larval and spore settlement of macrofoulers; however, issues such as reproducibility, differences in exposure sites and biofilm composition (natural multispecies vs. monospecific species) may influence the outcomes.

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogenous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.

The quest for advanced designer adsorbents for air filtration and monitoring hazardous trace gases has recently been more and more driven by the need to ensure clean air in indoor, outdoor, and industrial environments. How to increase safety with regard to personal protection in the event of hazardous gas exposure is a critical question for an ever-growing population spending most of their lifetime indoors, but is also crucial for the chemical industry in order to protect future generations of employees from potential hazards. Metal-organic frameworks (MOFs) are already quite advanced and promising in terms of capacity and specific affinity to overcome limitations of current adsorbent materials for trace and toxic gas adsorption. Due to their advantageous features (e.g., high specific surface area, catalytic activity, tailorable pore sizes, structural diversity, and range of chemical and physical properties), MOFs offer a high potential as adsorbents for air filtration and monitoring of hazardous trace gases. Three advanced topics are considered here, in applying MOFs for selective adsorption: (i) toxic gas adsorption toward filtration for respiratory protection as well as indoor and cabin air, (ii) enrichment of hazardous gases using MOFs, and (iii) MOFs as sensors for toxic trace gases and explosives.

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g. by NAD(P)H:quinone oxidoreductase, does not. The actions of menadione on ROS production and cell fate were compared with those of a non-cycling analogue (2,4-dimethoxy-2-methylnaphthalene (DMN)) using real-time confocal microscopy of isolated perfused murine pancreatic acinar cells. Menadione generated ROS with a concomitant decrease of NAD(P)H, consistent with redox cycling. The elevation of ROS was prevented by the antioxidant N-acetyl-l-cysteine but not by the NADPH oxidase inhibitor diphenyliodonium. DMN produced no change in reactive oxygen species per se but significantly potentiated menadione-induced effects, probably via enhancement of one-electron reduction, since DMN was found to inhibit NAD(P)H:quinone oxidoreductase detoxification. Menadione caused apoptosis of pancreatic acinar cells that was significantly potentiated by DMN, whereas DMN alone had no effect. Furthermore, bile acid (taurolithocholic acid 3-sulfate)-induced caspase activation was also greatly increased by DMN, whereas DMN had no effect per se. These results suggest that acute generation of ROS by menadione occurs via redox cycling, the net effect of which is induction of apoptotic pancreatic acinar cell death. Two-electron detoxifying enzymes such as NAD(P)H:quinone oxidoreductase, which are elevated in pancreatitis, may provide protection against excessive ROS and exert an important role in determining acinar cell fate.

ABSTRACT. This paper describes an experimental method for simulating an odour plume in the field so that its fine‐scale characteristics may be determined. It was found that the ‘odour’ arrived at a series of fixed points up to 15 m from the source in a series of discrete bursts, which were widely distributed in time but were typically 0.1 s long and 0.5 s apart. The strengths of the bursts were also found to be widely distributed and some contained considerable fluctuations. Thus an odour plume is not continuous, but intermittent, and appears at a fixed point downwind of the odour source as a series of bursts of odour which are variable in strength and duration. The distribution of the length of bursts and the time between them does not greatly vary with distance from source. Although concentration does decay with distance from source, instantaneous measurement would not be a reliable guide to the mean concentration and hence to position relative to source. To obtain reliable positional information from the odour plume an insect would have to average received stimuli over many seconds. More information is therefore required about the dynamics of insects' olfactory responses.

Vibrational spectroscopy can provide rapid, label-free, and objective analysis for the clinical domain. Spectroscopic analysis of biofluids such as blood components (e.g. serum and plasma) and others in the proximity of the diseased tissue or cell (e.g. bile, urine, and sputum) offers non-invasive diagnostic/monitoring possibilities for future healthcare that are capable of rapid diagnosis of diseases via specific spectral markers or signatures. Biofluids offer an ideal diagnostic medium due to their ease and low cost of collection and daily use in clinical biology. Due to the low risk and invasiveness of their collection they are widely welcomed by patients as a diagnostic medium. This review underscores recent research within the field of biofluid spectroscopy and its use in myriad pathologies such as cancer and infectious diseases. It highlights current progresses, advents, and pitfalls within the field and discusses future spectroscopic clinical potentials for diagnostics. The requirements and issues surrounding clinical translation are also considered.

ABSTRACT Inflammation is the body's first line of defense against infection or injury, responding to challenges by activating innate and adaptive responses. Microbes have evolved a diverse range of strategies to avoid triggering inflammatory responses. However, some pathogens, such as the influenza virus and the Gram-negative bacterium Francisella tularensis , do trigger life-threatening “cytokine storms” in the host which can result in significant pathology and ultimately death. For these diseases, it has been proposed that downregulating inflammatory immune responses may improve outcome. We review some of the current candidates for treatment of cytokine storms which may prove useful in the clinic in the future and compare them to more traditional therapeutic candidates that target the pathogen rather than the host response.

Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic diversity with >300 unique "genomic islands" identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

With recent advances in battery capacity and the development of hydrogen fuel cells, autonomous underwater vehicles (AUVs) are being used to undertake longer missions that were previously performed by manned or tethered vehicles. As a result, more advanced navigation systems are needed to maintain an accurate position over a larger operational area. The accuracy of the navigation system is critical to the quality of the data collected during survey missions and the recovery of the AUV. Many different methods for navigation in different underwater environments have been proposed in the literature. In this correspondence paper, the state of the art in navigation technologies for AUVs is investigated for theoretical and operational systems. Their suitability for use in different environments is compared and current limitations of these methods are identified. In addition, new approaches to address these current problems and areas for future research are suggested. Finally, it is concluded that only geophysically referenced methods will enable AUVs to navigate accurately over large areas and that advances in underwater feature recognition are required before these methods can be implemented in operational AUVs.

ATP-binding cassette (ABC) transporters are widespread among living organisms and comprise one of the largest protein families. For example, components of ABC transporters are encoded by approximately 5% of the Escherichia coli and Bacillus subtilis genomes ([47][1], [80][2]). These transporters are