Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Medicine & Surgery
Non Technical Summary
Anthrax is a worldwide zoonotic disease that primarily infects domestic and wild herbivorous animals. The causal agent is the spore-forming bacterium Bacillus anthracis. The spore is the infectious form of this pathogen and animals are infected through contact with environmental spores through grazing or drinking water. The disease is rapidly fatal and bacteria from the dead animals sporulate and the spores contaminate the soil around the carcass. Anthrax and spores are also a concern as a bioterrorism agent. Spores can persist for decades in infected environments. While many saprophytic Bacillus species replicate and sporulate in soil, the question of whether B. anthracis can do so remains controversial. The B. anthracis life cycle is thought to be that of an obligate animal parasite, with replication occurring within the infected animal and sporulation occurring during postmortem decomposition. However, there have been a few studies over the past 76 years that concluded that B. anthracis spores can germinate and replicate in soil. If true, what is the contribution of this soil replication cycle on persistence of spores and with outbreaks of disease?This project involves an investigation of germination of B. anthracis spores in a controlled laboratory soil environment, subsequent bacterial cell replication in soil, and the efficiency of the newly replicated cells to undergo the sporulation process. The presence of spores and vegetative cells will be assessed by viable plate counts and through imaging of engineered strains that are fluorescent either as spores or during the vegetative phase of growth. The involvement of blood contamination of soil on the ability of B. anthracis cells to replicate and sporulate in soil will be examined. The contribution of bodily fluids to the initial events promoting the survival of the organism in soil has not yet been investigated. The results of this study will have important implications in the design of remediation efforts to disinfect heavily spore-contaminated sites on farms, wildlife areas of concern, and sites of anthrax bioterrorism events.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Goals / Objectives
The long-term goals of my laboratory and this project are to better understand the ecology of B. anthracis infections. My laboratory has been involved in molecular studies of spore maturation. This pilot study will examine the ability of B. anthracis spores to germinate in soil environments and, importantly, to determine if they can subsequently re-sporulate and thus amplify spore populations. With the spore being the infectious form of this pathogen, the spore numbers are critical if outbreaks are to occur. My hypothesis is that although B. anthracis spore germination and some replication of the bacterial cells can occur in soil environments, the levels of bacterial replication and/or subsequent sporulation are inadequate to substantially increase spore numbers in contaminated soils. This would imply that the load of spores in a particular location is a reflection of the initial contamination from infected animals or bioterrorism event and not the result of later replicative events. To test this hypothesis, the following specific objectives are proposed.Specific Objectives:1. To determine if spores can germinate in soil and the resultant bacteria replicate in soil under controlled laboratory conditions.2. To determine if vegetative bacterial cells can sporulate in soil and how efficient is this process.3. To determine if bovine blood contamination of soil, as would be the case following death of a cow with anthrax, promotes growth and sporulation of B. anthracis in soil.
Project Methods
For objective 1: To determine if spores can germinate in soil and the resultant bacteria replicate in soil under controlled laboratory conditions. Spores (at doses of 104 to 106 spores) will be inoculated onto the soil samples and the tubes incubated at 30ºC. At timed intervals, a tube will be removed and the number of spores and heat-sensitive cells will be determined as described above. Samples with be taken immediately postinoculation and at weekly intervals thereafter for three months. Heat resistant and total viable counts will be determined. At the end of this experiment, it will be repeated with either shorter sampling intervals or extended beyond 3 months depending on the results obtained. At times corresponding to substantial heat-sensitive forms being present, the presence of vegetative bacteria will be evaluated by microscopy. The spores to be utilized will be from the strains expressing GFP or mCh driven by the inhA promoter. The fluorescence is only associated with viable vegetative cells. Spores produced by these strains are not fluorescent. Actively growing B. anthracis tend to be filamentous, and this was observed by Saile and Koehler (6). However, even unit length B. anthraciscells (found in poorer nutrient quality environments or less actively growing bacilli) are easily imaged.Anticipated results and alternative approaches. Based on previous studies and my hypothesis, I expect that spore germination will occur and we will observe reduced heatresistant titers in the soil. If the spore titers drop and do not later recover, it would suggest that the bacterial cells do not efficiently sporulate in this soil environment. This could be due to poor sporulation or to premature death of cells such that they have insufficient time to sporulate. The timing and the extent of the presence of the heatsensitive forms will indicate which explanation is correct. If it is a sporulation defect, future studies can be employed to determine at what stage of sporulation the process becomes arrested. It may be at a very early stage such that the cells do not enter the sporulation process, or it may be blocked at a later stage. The Bacillus sporulation process is well characterized, and so sporulation gene expression data derived from an RNA-seq approach could be utilized (17). Arrest at specific stages of sporulation would result in a failure of those genes whose involvement is downstream of the block to be expressed. Gene expression patterns can thus indicate where the defect lies. However, this analysis is beyond the scope of this initial pilot project.If growth and/or sporulation is inefficient under these soil and incubation conditions, we will test soils with increased alkalinity, increased plant organic material, and increased calcium levels, parameters which were reported in the older scientific literature to improve B. anthracis replication in soil (3).For objective 2: To determine if vegetative bacterial cells can sporulate in soil and how efficient is this process. The sporulation process does not occur in an infected animal until the postmortem process is underway and the bloodstream bacteria in the carcass are exposed to air (presumably oxygen). It is not known if bacterial cells only sporulate within the carcass and are then released into the soil or if bacterial cells released from the carcass can enter the soil and subsequently sporulate. The objective 1 studies are concerned with sporulation arising from growth of the organisms in soil. In this objective, we will assess the capacity of bacteria grown in a more nutrient rich environment to sporulate when expelled into a soil environment. My hypothesis is that the bacterial cells released into the soil from the infected animal will sporulate more efficiently than cells actually growing in the soil environment. This may be due to the energy stores present in bacteria growing in the bloodstream being substantially different from bacteria growing in a more nutrient unbalanced environment such as soil. For this objective, Sterne bacterial cells rather than spores will be inoculated into the soil-bearing tubes. The bacteria will be serially passaged in brain heart infusion broth (Difco) in midlogarithmic growth phase to eliminate spores from the sample. Absence of spores will be verified by plating heat-treated samples of the cells. The samples will be incubated and processed as above and examined for the emergence of heat-resistant spores. Cells will be added to the soil at inoculum ranges of 104 to 106 viable cells and the samples collected over a three month time frame (at least initially). The experiment will be repeated with the sampling interval determined based on the initial experiment. If spores are produced, the experiment will be repeated with the BclA-GFP and BclA-mCh fusion strains and emergence of the spores imaged by fluorescent microscopy.Anticipated results and alternative approaches. The environment of the bloodstream of an infected animal is profoundly different from the environment a bacterial cell encounters in soil. It is expected, therefore, that the chemical composition of the cytoplasm of cells from an infected animal and its physiologic state will differ markedly from that of bacteria replicating in soil. Levels of energy stores (ATP, GTP) would differ along with the composition of amino acid and nucleoside pools. Thus one would expect that energy demanding processes, like sporulation, would differ quantitatively between bacteria from the two environments. The bacteria in this experiment will be cultured in a very complex and rich growth medium (brain heart infusion broth) and may better reflect the biochemistry and physiology of an organism emerging from an infected animal. Prior studies of environmental replication of B. anthracis have used spore inoculation of soil. Therefore, the results of this experiment will provide new information regarding the ecology of B. anthracis interactions with the environment.For objective 3: To determine if bovine blood contamination of soil, as would be the case following death of a cow with anthrax, promotes growth and sporulation of B. anthracis in soil. It is possible that bacterial cells may not sporulate in soil in the objective 2 experiments, yet do so in the real world situation of a dead infected animal. This might be due to the bacteria released from the animals being mixed with blood and other bodily fluids from the dying or dead animal. To test whether animal bodily fluids are needed for sporulation to occur in soil, or increase the efficiency of sporulation in soil, I will examine the contributions of blood to sporulation by B. anthracis in soil. The Sterne bacterial cells will be cultured as above and prior to their addition to the soil will be pelleted by centrifugation and resuspended with citrated bovine blood (Carolina Biological Supply Co.). The same bacterial inoculum ranges will be used and the blood will be used undiluted and diluted 1:2, 1:4, 1:8 and 1:16 with sterile normal saline. Cells will be resuspended in saline only as a control. Appearance of heat-resistant spores will be assessed as described above.Anticipated results and alternative approaches. I predict that the addition of the blood will improve sporulation efficiency of cells encountering the soil environment. This would be an evolutionary advantage for an organism that kills its host as vegetative bacteria but must emerge as spores in soil to effectively infect future hosts. The dilution series will provide an indication of the strength or abundance of the signaling species in the blood. However, if increased sporulation efficiencies are not realized, the experiments will be repeated with blood which has incubated at 37ºC for a week prior to mixing with the bacterial cells. It is possible that chemical cues stimulating sporulation appear only during postmortem degradation of the blood.