Goals and Directions
Elucidation of the mechanisms of persister cell formation in pathogenic bacteria.
Persister cells from ESKAPE pathogens (excepting P. aeruginosa as I have already done this) will be isolated and the RNA will be extracted for RNA sequencing as described above. We will begin with S. aureus and K. pneumoniae and continue to the other species once these have been completed. We will compare the expression profile of persister cells with that of biofilm/stationary cultures in order to identify ORFs differentially expressed in persister cells. I have completed much of the groundwork for the analysis P. aeruginosa persister cell formation and therefore am confident in the ability of myself and my students to achieve further understanding of various pathogens. Mutants of ORFs implicated in persister cell formation will be either screened (from existing transposon libraries, such as that of P. aeruginosa or S. aureus) or generated to determine the contribution of the ORFs to persister cell formation in these species. For species where there is not an available transposon library, we will begin making mutants in ORFs categorized as regulators of transcription or homologs to already identified ORFs in persister cell formation.
Students on this project: Saika, Erik, and Christian
Identification of ORFs necessary for antibiotic resistance and potential vaccine targets in K. pneumoniae and P. aeruginosa.
We will begin with targets already identified from the RNA sequencing analysis of K. pneumoniae biofilms. Students will purify recombinant proteins which will then be evaluated as potential vaccine targets in conjunction with collaborators who have access to mouse models of disease. We will also generate mutants in genes of interest which we will be able to share with our collaborators or potentially use ourselves with the Caenorhabditis elegant (nematode) model. We are also working in conjunction with Dr. Rawat (Fresno State) to understand how low molecular weight (LMW) thiols contribute to survival of P. aeruginosa under conditions of oxidative stress, such as the stress caused by antibiotic therapy. For this project, I am excited about the opportunity for students to spend some time researching what is known about antibiotic resistance in other bacterial systems and use that knowledge to begin selecting ORFs they would like to study in our pathogens of interest.
Students on this project: Tyler, Amorette, and Justin
Characterization of the microbiome and resistome of the American Crow
Birds are among the most common non-human vertebrates in urban environments, and the continuing sprawl of urban habitats leads to increased possibilities for acquisition of antibiotic resistance. Close association with human habitation also increases risk for transmission of these antibiotic resistant organisms to humans through contact and contamination with fecal material. As American Crows (Corvus branchyrhyncos), members of the Corvidae, are widespread throughout North America and tend to roost in large numbers near human settlements, understanding the microbiome and potential carriage of antimicrobial resistant organisms in this species will provide important information about risks to human health in urban areas. The long-term goal of this study is to characterize the microbiome and antimicrobial resistance found in corvid feces. The central hypothesis is that birds roosting in urban environments will have more pathogenic and antimicrobial resistant organisms in their feces than birds in rural environments. The immediate objective is to identify the microbial composition and antimicrobial resistance profile of feces from urban and rural roosting crows. We are completing this project in collaboration with Dr. Katti (Fresno State) and Dr. Eisen (UC Davis).
Students on this project: Lee and Michael
Occurrence and distribution of Black Swift (Cypseloides niger) in the Souther Sierra Nevada
The black swift (Cypseloides niger) is a rare bird that breeds only on sea cliffs and behind mountain waterfalls. Surveys in 2004 have found a steep decline in the coastal population. Of two historic coastal population centers, one in Santa Cruz and San Mateo Counties has been completely extirpated and a second in Monterey and San Luis Obispo Counties has declined from approximately 50 pairs in 1992 to ten pairs in 2008. The southern Sierra Nevada population has not been systematically surveyed in over 75 years. A survey of this population will determine if declines in the coastal population are mirrored by declines in the southern Sierra population. This will provide data that helps identify potential threats, which will allow for more effective management. This project will consist of three parts. First, we will inventory known nest sites and use GIS to quantify level of stream flow and vertical relief. This data will be used to identify potential nesting locations in the southern Sierra Nevada. Second, we will survey a random selection of as many of the potential nesting sites as possible during the breeding window (22 June to 1 September, with some variation based on elevation). These surveys will use the protocol developed by Levad et al (2008) that collects microhabitat data on the nesting sites in addition to maximizing black swift detection probability. Finally, we will use multivariate analysis on the data to determine to what extent characteristics including vertical relief, rate of flow, vegetation, and shade allow black swifts to use waterfalls as nesting sites.
Students on this project: Alex
Prevalence of Tick-Borne Relapsing Fever in Ornithodoros hermsi collected in the Sierra National Forest, California
Tick-Borne Relapsing Fever (TBRF) is a serious vector-borne disease that is endemic to the mountains of Fresno County. The distribution and prevalence of both the bacterial agent (Borrelia spp.) and the soft-tick vector (Ornithodoros hermsi) are poorly understood in the Sierra National Forest. Our goal is to collect O. hermsi near popular recreational locations and at varying elevations within the Sierra National Forest to better understand the distribution and abundance of these ticks. Ticks will be collected using an adapted carbon dioxide (dry ice) baited trap. A series of traps will be deployed overnight near human-occupied cabins, campgrounds, and other man-made facilities in addition to the forested areas in the immediate vicinity on these locations. Traps are inexpensive and easily deployable for immediate reuse at multiple locations. We will test collected ticks for the presence of B. hermsii (and other relapsing fever causing Borrelia) in both their bodies, signifying an active infection, and separately their salivary glands, which would signify the ability of the tick to transmit the bacteria to a host animal or human. Samples will be tested using a polymerase chain reaction (PCR) amplification of the glpQ gene followed by a restriction fragment length polymorphism (RFLP) assay to determine which relapsing fever Borrelia is present in these ticks, as the glpQ is unique to relapsing fever Borrelia and absent from those causing Lyme disease. The data we collect will be used to better understand the distribution of O. hermsi and prevalence of relapsing fever Borrelia in the Sierra National Forest with the intent of assisting public health officials with possible risk assessments. If successful, these protocols can be readily adapted for use in other counties where Tick-Borne Relapsing Fever is endemic.
Students on this project: Cameron, Manny
Elucidation of the mechanisms of persister cell formation in pathogenic bacteria.
Persister cells from ESKAPE pathogens (excepting P. aeruginosa as I have already done this) will be isolated and the RNA will be extracted for RNA sequencing as described above. We will begin with S. aureus and K. pneumoniae and continue to the other species once these have been completed. We will compare the expression profile of persister cells with that of biofilm/stationary cultures in order to identify ORFs differentially expressed in persister cells. I have completed much of the groundwork for the analysis P. aeruginosa persister cell formation and therefore am confident in the ability of myself and my students to achieve further understanding of various pathogens. Mutants of ORFs implicated in persister cell formation will be either screened (from existing transposon libraries, such as that of P. aeruginosa or S. aureus) or generated to determine the contribution of the ORFs to persister cell formation in these species. For species where there is not an available transposon library, we will begin making mutants in ORFs categorized as regulators of transcription or homologs to already identified ORFs in persister cell formation.
Students on this project: Saika, Erik, and Christian
Identification of ORFs necessary for antibiotic resistance and potential vaccine targets in K. pneumoniae and P. aeruginosa.
We will begin with targets already identified from the RNA sequencing analysis of K. pneumoniae biofilms. Students will purify recombinant proteins which will then be evaluated as potential vaccine targets in conjunction with collaborators who have access to mouse models of disease. We will also generate mutants in genes of interest which we will be able to share with our collaborators or potentially use ourselves with the Caenorhabditis elegant (nematode) model. We are also working in conjunction with Dr. Rawat (Fresno State) to understand how low molecular weight (LMW) thiols contribute to survival of P. aeruginosa under conditions of oxidative stress, such as the stress caused by antibiotic therapy. For this project, I am excited about the opportunity for students to spend some time researching what is known about antibiotic resistance in other bacterial systems and use that knowledge to begin selecting ORFs they would like to study in our pathogens of interest.
Students on this project: Tyler, Amorette, and Justin
Characterization of the microbiome and resistome of the American Crow
Birds are among the most common non-human vertebrates in urban environments, and the continuing sprawl of urban habitats leads to increased possibilities for acquisition of antibiotic resistance. Close association with human habitation also increases risk for transmission of these antibiotic resistant organisms to humans through contact and contamination with fecal material. As American Crows (Corvus branchyrhyncos), members of the Corvidae, are widespread throughout North America and tend to roost in large numbers near human settlements, understanding the microbiome and potential carriage of antimicrobial resistant organisms in this species will provide important information about risks to human health in urban areas. The long-term goal of this study is to characterize the microbiome and antimicrobial resistance found in corvid feces. The central hypothesis is that birds roosting in urban environments will have more pathogenic and antimicrobial resistant organisms in their feces than birds in rural environments. The immediate objective is to identify the microbial composition and antimicrobial resistance profile of feces from urban and rural roosting crows. We are completing this project in collaboration with Dr. Katti (Fresno State) and Dr. Eisen (UC Davis).
Students on this project: Lee and Michael
Occurrence and distribution of Black Swift (Cypseloides niger) in the Souther Sierra Nevada
The black swift (Cypseloides niger) is a rare bird that breeds only on sea cliffs and behind mountain waterfalls. Surveys in 2004 have found a steep decline in the coastal population. Of two historic coastal population centers, one in Santa Cruz and San Mateo Counties has been completely extirpated and a second in Monterey and San Luis Obispo Counties has declined from approximately 50 pairs in 1992 to ten pairs in 2008. The southern Sierra Nevada population has not been systematically surveyed in over 75 years. A survey of this population will determine if declines in the coastal population are mirrored by declines in the southern Sierra population. This will provide data that helps identify potential threats, which will allow for more effective management. This project will consist of three parts. First, we will inventory known nest sites and use GIS to quantify level of stream flow and vertical relief. This data will be used to identify potential nesting locations in the southern Sierra Nevada. Second, we will survey a random selection of as many of the potential nesting sites as possible during the breeding window (22 June to 1 September, with some variation based on elevation). These surveys will use the protocol developed by Levad et al (2008) that collects microhabitat data on the nesting sites in addition to maximizing black swift detection probability. Finally, we will use multivariate analysis on the data to determine to what extent characteristics including vertical relief, rate of flow, vegetation, and shade allow black swifts to use waterfalls as nesting sites.
Students on this project: Alex
Prevalence of Tick-Borne Relapsing Fever in Ornithodoros hermsi collected in the Sierra National Forest, California
Tick-Borne Relapsing Fever (TBRF) is a serious vector-borne disease that is endemic to the mountains of Fresno County. The distribution and prevalence of both the bacterial agent (Borrelia spp.) and the soft-tick vector (Ornithodoros hermsi) are poorly understood in the Sierra National Forest. Our goal is to collect O. hermsi near popular recreational locations and at varying elevations within the Sierra National Forest to better understand the distribution and abundance of these ticks. Ticks will be collected using an adapted carbon dioxide (dry ice) baited trap. A series of traps will be deployed overnight near human-occupied cabins, campgrounds, and other man-made facilities in addition to the forested areas in the immediate vicinity on these locations. Traps are inexpensive and easily deployable for immediate reuse at multiple locations. We will test collected ticks for the presence of B. hermsii (and other relapsing fever causing Borrelia) in both their bodies, signifying an active infection, and separately their salivary glands, which would signify the ability of the tick to transmit the bacteria to a host animal or human. Samples will be tested using a polymerase chain reaction (PCR) amplification of the glpQ gene followed by a restriction fragment length polymorphism (RFLP) assay to determine which relapsing fever Borrelia is present in these ticks, as the glpQ is unique to relapsing fever Borrelia and absent from those causing Lyme disease. The data we collect will be used to better understand the distribution of O. hermsi and prevalence of relapsing fever Borrelia in the Sierra National Forest with the intent of assisting public health officials with possible risk assessments. If successful, these protocols can be readily adapted for use in other counties where Tick-Borne Relapsing Fever is endemic.
Students on this project: Cameron, Manny
