Progress 10/01/14 to 09/30/19
Outputs
Target Audience:Understanding the mechanisms by which bacteria survive desiccation will be of interest to several audiences. Dry surfaces, equipment, and dried samples could harbor bacteria in a desiccated state and initially be undetected, but the bacteria could regain viability given suitable rehydration conditions. The food safety industry surveys processing plants and food products to detect contamination. Projects that engineer microbes for various applications, such as the development of biosensors, may need to consider how bacterial recovery from desiccation will affect an application in which live bacteria are subjected to drying conditions. In addition, clinical laboratories will be concerned with the impact of desiccation on bacterial detection and survival, particularly with regard to bacterial pathogens present in samples analyzed by these laboratories. Survival from desiccation stress may be important for additional concerns such as bioterrorism. Changes/Problems:Due to limitations in budget and personnel, we were not able to carry out additional experiments to address the changes in amounts of proteins, DNA and RNA present in cells during desiccation and rehydration. Genes that are involved in desiccation tolerance were not identified. When additional funds become available, more work will be done to address the mechanisms of survival during desiccation. What opportunities for training and professional development has the project provided?Two part-time technicians have worked on this project over the past three years. Both of these individuals had earned their B.S. degrees in science and were interested in advancing their research skills. They were trained in methods used to grow bacterial cultures, desiccate and rehydrate cell samples, follow culture growth and assess culture concentration by optical density, and measure colony forming units by dilution plating. They were trained in sterile technique, working in a BSL2 laboratory, maintaining equipment and keeping a laboratory notebook. Their experiences in this project significantly enhanced their training and lead to full time employment in their fields of interest. One of the technicians took a job in the marine biology field, and the other technician was hired by a local biotechnology company. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Past experiments focused on studying the effects of desiccation on survival of Escherichia coli K-12, which is a commonly used, avirulent laboratory strain. With this organism, we found that only about 0.001% of the original culture sample, which had been desiccated and rehydrated in buffer, could form colonies on solid growth medium during the first 12 hours of rehydration. Gradually, the number of colony forming units (CFUs) increased during the next 36 hours to about 15% of the original CFUs of the culture prior to desiccation. This increase in CFUs reflected an increase in the ability of the cells to divide and form colonies since the actual number of cells measured by microscopy and the optical density of the culture samples did not increase significantly during this time. An additional series of experiments were carried out with a strain of E. coli that had a mutation in the asd gene that codes for the synthesis of diaminopimelic acid (DAP), which is required for cell wall synthesis. Without addition of DAP to the medium, cells can't divide. When submitting this strain to desiccation and rehydration in buffer without DAP, we observed a similar recovery over time when the cells were plated on solid growth medium with added DAP. Since the cells could not divide during rehydration without DAP, the recovery in the number CFU's observed on solid medium with added DAP suggested the implementation of repair mechanisms. To support this conclusion, the addition of chloramphenicol to the rehydration buffer significantly decreased recovery indicating that active protein synthesis was required. These studies were extended to one avirulent strain of Salmonella enterica Typhimurium. Preliminary results indicated that the number of CFUs after desiccation and during rehydration increased two-fold to sixty-fold depending on the stage of culture growth prior to desiccation without change to the optimal density of the culture. As with E. coli, we concluded that this increase in CFUs during rehydration was due to an increase in the ability of the cells to divide and form colonies. Therefore, this study demonstrated that both E. coli and S. Typhimurium were able to survive desiccation conditions when rehydrated in a non-growth supporting buffer and regained the ability to form colonies when plated on solid growth medium.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:In addition to our work with E. coli, we will continue to evaluate desiccation survival by various strains of S. enterica Typhimurium. During this past year, my technician took another position and left the lab, but I have identified a student who is interested in working on this project; his training will begin during the month of January. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The survival of of S. enterica Typhimurium, particularly those strains altered in cell wall structures, will be evaluated during recovery from desiccation for comparison to the parent strain and to our previous results with E. coli. Relative humidity, temperature, and time of desiccation are some of the variables that will be addressed. Culture density and cell number will be quantified during rehydration to monitor significant changes. As time permits, we also will quantify the amount of DNA, RNA and protein present in these cells before desiccation and during rehydration in order to address mechanisms of desiccation tolerance.
Impacts
What was accomplished under these goals?
Past experiments focused on studying the effects of desiccation on survival of Escherichia coli K-12, which is a commonly used, avirulent laboratory strain. With this organism, we found that only about 0.001% of the original culture sample, which had been desiccated and rehydrated in buffer, could form colonies on solid growth medium during the first 12 hours of rehydration. Gradually, the number of colony forming units (CFUs) increased during the next 36 hours to about 15% of the original CFUs of the culture prior to desiccation. This increase in CFUs reflected an increase in the ability of the cells to divide and form colonies since the actual number of cells measured by microscopy and the optical density of the culture samples did not increase significantly during this time. These studies were extended to one avirulent strain of S. enterica Typhimurium. Preliminary results indicated that the number of CFUs after desiccation increased two-fold to sixty-fold depending on the stage of culture growth prior to desiccation without change to the optimal density of the culture. As with E. coli, we conclude that this increase in CFUs during rehydration was due to an increase in the ability of the cells to divide and form colonies. To address the mechanism of desiccation tolerance, we have worked to isolate mutants of S. Typhimurium that lack components of cell wall structures, which may be critical to cell integrity during times of severe dryness. Using various genetic approaches, we have succeeded in making deletions in genes that code for structures such as flagella and O-antigen. These mutants will be tested for desiccation tolerance and compared to the parent strain to assess the importance of these structures for survival and recovery from desiccation.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The advances made with understanding the mechanisms by which bacteria survive desiccation will be of interest to several audiences. The food safety industry surveys processing plants and food products to detect contamination. Dry surfaces, equipment, and dried food products could harbor bacteria in a desiccated state, be initially undetected, but viable given suitable rehydration conditions. Projects that engineer microbes for various applications, such as the development of biosensors, may need to consider how bacterial recovery from desiccation will affect an application in which live bacteria are subjected to drying conditions. In addition, clinical laboratories will be concerned with the impact of desiccation on bacterial detection and survival, particularly with regard to bacterial pathogens present in samples analyzed by these laboratories. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The part-time technician hired to work on this project was trained in methods used to grow bacterial cultures, desiccate and rehydrate cell samples, measure cell concentration by optical density, and measure colony forming units by dilution plating. In addition, this person was trained in keeping a laboratory notebook, calculating CFU/ml and determining the percent recovery during rehydration. Sterile technique, working in a BSL2 laboratory, and maintaining equipment were also involved in the training. The technician was advised to take a microbiology course and positioned himself to secure a full-time job in the field of marine biology. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will continue to evaluate the recovery of S. enterica Typhimurium from desiccation stress. To extend our current experiments, we will vary the length of desiccation from three days to weeks to assess the extent to which the cells can survive prolonged dehydration. In addition, the density of the culture (either at mid-log or stationary phases) will be varied to determine if culture density affects the ability of the cells to enter a viable but nonculturable state. Optical density of the culture samples will be measured during rehydration to monitor significant changes in culture density, and microscopy will be used to quantify the number of cells present during rehydration. As time and funds permit, we will quantify the amount of DNA, RNA and protein present in these cells before desiccation and during rehydration in order to eventually address mechanisms of desiccation recovery.
Impacts
What was accomplished under these goals?
Our past experiments focused on studying the effects of desiccation on survival of Escherichia coli K-12, which is a commonly used, avirulent laboratory strain. With this organism, we found that only about 0.001% of the original culture sample, which had been desiccated and rehydrated in buffer, could form colonies on solid growth medium during the first 12 hours of rehydration. Gradually, the number of colony forming units increased during the next 36 hours to about 15% of the original colony forming units of the culture prior to desiccation. This increase in colony forming units reflected an increase in the ability of the cells to divide and form colonies since the actual number of cells measured by microscopy and the optical density of the culture samples did not increase during this time. During this past year, we certified the lab as BioSafety Level 2 and have extended our studies to an avirulent strain of Salmonella enterica Typhimurium. Culture growth was standardized. Two major variables were tested that included comparing mid-log to stationary phase cultures and exposure to continuous light versus dark conditions during desiccation and rehydration. The stationary phase cultures, desiccated and rehydrated in buffer under either light or dark conditions, showed an approximate 2-fold increase in colony forming units (CFU), from 1.4% to about 3% of the original desiccated CFU over three days of rehydration. In contrast, mid-log phase cultures showed an average 60-fold increase in CFU, from 0.05% to 3% of the original desiccated CFU, during three days of rehydration regardless of the light or dark incubation conditions. During rehydration of all cell samples, there was no significant change in optical density of the samples indicating no significant increase in actual cell number but rather an increase in the ability of the cells to form colonies. These preliminary results suggested that light versus dark exposure during desiccation and rehydration did not significantly affect survival, whereas culture growth phase or culture density may influence the number of CFU detected immediately following rehydration and the subsequent increase in CFU over time.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The advances made with understanding the mechanisms by which bacteria survive desiccation will be of interest to several audiences. The food safety industry surveys processing plants and food products to detect contamination. Dry surfaces, equipment, and dried food products could harbor bacteria in a desiccated state, be initially undetected, but viable given suitable rehydration conditions. Projects that engineer microbes for various applications, such as the development of biosensors, may need to consider how bacterial recovery from desiccation will affect an application in which live bacteria are subjected to drying conditions. In addition, clinical laboratories will be concerned with the impact of desiccation on bacterial detection and survival, particularly with regard to bacterial pathogens present in samples analyzed by these laboratories. Changes/Problems:During this past year, we merged my lab with a colleague's lab in order to attain BSL2 certification. This change will allow us to broaden our studies by evaluating desiccation survival with other organisms such as S. enterica Typhimurium for comparison to our work with E. coli. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will evaluate the recovery of S. enterica Typhimurium from desiccation stress using various conditions of relative humidity, temperature and time of desiccation as well as various conditions of rehydration, similar to what we've accomplished with E. coli K12. Optical density of the culture samples will be measured during rehydration to monitor significant changes in culture density, and microscopy will be used to quantify the number of cells present during rehydration. If time permits, we will quantify the amount of DNA, RNA and protein present in these cells before desiccation and during rehydration in order to eventually address mechanisms of desiccation recovery.
Impacts
What was accomplished under these goals?
Our past experiments focused on studying the effects of desiccation on survival of Escherichia coli K-12, which is a commonly used, avirulent laboratory strain. With this organism, we found that only about 0.001% of the original culture sample, which had been desiccated and rehydrated in buffer, could form colonies on solid growth medium during the first 12 hours of rehydration. Gradually, the number of colony forming units increased during the next 36 hours to about 15% of the original colony forming units of the culture prior to desiccation. This increase in colony forming units reflected an increase in the ability of the cells to divide and form colonies since the actual number of cells measured by microscopy and the optical density of the culture samples did not increase during this time. In our effort to broaden this study to other organisms, the laboratory had to become certified as a BioSafety Level 2 (BSL2) lab. We merged my lab with the lab of a colleague, installed the proper equipment and successfully attained BSL2 certification. Preliminary work has been initiated with Salmonella enterica Typhimurium. We have several avirulent strains and have worked to establish growth conditions for these organisms in preparation for desiccation/rehydration experiments. These organisms will be subjected to various desiccation and rehydration conditions for comparison to our previous results with E. coli K12.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The advances made with understanding the mechanisms by which bacteria survive desiccation will be of interest to several audiences. The food safety industry assays food products to detect contamination, and dried products in particular could harbor bacteria in a desiccated state. Projects that engineer microbes for various applications may need to consider how bacterial recovery from desiccation will affect a specific application in which bacteria are subjected to drying conditions. In addition, clinical laboratories will be concerned with the impact of desiccation on bacterial survival and detection, particularly with regard to bacterial pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will continue to evaluate the recovery of E. coli B from desiccation stress using various conditions of relative humidity, temperature and time of desiccation as well as various conditions of rehydration. In addition, we will begin to quantify the amount of DNA, RNA and protein present in E. coli K-12 and E. coli B before desiccation and during rehydration. An additional interest is to study desiccation survival using a pathogenic organism such as Salmonella enterica for comparison to E. coli; a BSL2 laboratory will be required for these experiments.
Impacts
What was accomplished under these goals?
Our past experiments focused on studying the effects of desiccation on survival of Escherichia coli K-12, which is a commonly used, avirulent laboratory strain. With this organism, we found that only about 0.001% of the original culture sample, which had been desiccated and rehydrated in buffer, could form colonies on solid growth medium during the first 12 hours of rehydration. Gradually, the number of colony forming units increased during the next 36 hours to about 15% of the original colony forming units of the culture prior to desiccation. This increase in colony forming units reflected an increase in the ability of the cells to divide and form colonies since the actual number of cells measured by microscopy and the optical density of the culture samples did not increase during this time. Preliminary work has been initiated with E. coli B to broaden these studies to additional organisms and determine if there is a similar response during recovery from desiccation. The initial experiments showed that E. coli B had a several order of magnitude increase in colony forming units during rehydration, similar to the results with E. coli K-12. These experiments will be repeated using various desiccation and rehydration conditions. Optical density of the culture samples will be measured during rehydration to monitor significant changes, and microscopy will be used to quantify the number of cells present during rehydration.
Publications
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