Progress 10/01/11 to 09/30/16
Outputs
Target Audience:Target audience ranged from local to international. Students presented at several meetings including ones at Montana State University and Internationally (Italy). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provides training opportunitites for students. It allows undergraduate students to engage in hands-on laboratory research. It also provides opportunities for graduate students to particpatein studies that focus on agriculture. How have the results been disseminated to communities of interest?The results have been disseminated to the public in seminars locally, nationally, and internationally. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
To better study mechanisms involved in the progression of mastitis, in collaboration with Dr. Eric Wilson at Brigham Young University we have begun to establish a physiologically relevant mouse model of Staphylococcus aureus (S. aureus) mastitis. Unlike our preliminary studies in E. coli establishing the mouse model using S. aureus required many studies to determine the infectious dose. We are now well positioned to look at how different strains of S. aureus isolated from mastitic cows impact the outcome of mammary gland infection. We have used a S. aureusstrain that has the saeR/S two-component sensory system knocked-out to begin to identify pathogen factors important for establishment of a mammary gland infection. Gaining knowledgeof what virulence factors are used by S. aureus to cause disease will guide vaccine designs. Ournewly developed murine mastitis model will be used to test the hypothesis that virulence factors regulated by the SaeR/S two-component gene regulatory system of S. aureus contribute to mastitis by dysregulating the initial inflammatory response to the pathogen challenge. We have characterized samples from the equine nares and have found that numerous other Staphylococcus species reside as normal microbiota in the equine nares.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
c. Guerra, F. E., Addison, C.B., de Jong, N.W.M., Azzolino ,J., Pallister, K.B.,, van Strijp, J., and J. M. Voyich (2016) Staphylococcus aureus SaeR/S-regulated Factors Reduce Human Neutrophil Reactive Oxygen Species Production. J. Leuk. Biol. 100: 1005-1010
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target audience ranged from local to international. Students presented at several meetings including ones at Montana State University and Internationally (Italy). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provides training opportunitites for students. It allows undergraduate students to engage in hands-on laboratory research. It also provides opportunities for graduate students to particpatein studies that focus on agriculture. How have the results been disseminated to communities of interest?The results have been disseminated to the public in seminars locally, nationally, and internationally. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Using S. aureus strains isolated from mastitic cattle we generated new data in regard to bovine immunity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. We described the cloning and function of bovine CCL28 and documented the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms including S. aureus isolated from mastitic cows. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Zurek OW, Pallister KB, Voyich JM. Staphylococcus aureus inhibits neutrophil-derived IL-8 to promote cell J Infect Dis. 2015:212
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Pallister KB, Mason S, Nygaard TK, Liu B, Griffith S, Jones J, et al. (2015) Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria. PLoS ONE 10(9): e0138084. doi:10.1371/journal.pone.0138084
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Students presented the findings at public seminars at Montana State University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has once again provided opportunities for undergraduates to work on the project. It has supported biotech capstone projects. How have the results been disseminated to communities of interest? The results have been disseminated locally at Montana State University. What do you plan to do during the next reporting period to accomplish the goals? We will continue to determine the characteristics and incidence of Staphylococcus in the anterior nares of horses. We have also begun to identify the proper anatomical niche of this microorganism in horses but conducting studies looking at the presence of Staphylococcus in the guttural pouch. These accomplishments as outlined in the previous section are directly related to the goal of determining the incidence of S. aureus in horses and its zoonotic potential.
Impacts
What was accomplished under these goals?
The focus of the current study was to characterize S. aureus virulence factors as well as antibiotic resistance from equine isolates and compare these results to human isolates. We investigated 26 individual horses three separate times for the presence of S. aureus. To do this nasal swabs were taken from both left and right nostrils of the subjects and S. aureus was isolated from the microbiota and confirmed using RAPIDEC Staph® (Biomerieux) . Our data suggest 7 individual horses were colonized with S. aureus. However, in the past year we sent strains for 16s sequencing and have determined that the equine population has several different Staphylococcus species including: S. vitulinus, S. equorum, S. saprophyticus, S. delphini, S. haemolyticus, S. lugdenensis, S. warneri, and S. simiae. Using PCR we investigated the presence of toxin and antibiotic resistance genes including: mecA, sbi, tsst-1, lukS-PV, lukF-PV, hla, hlga, and blaZ. PCR results indicate all isolates have blaZ (gene that confers penicillin resistance), a high prevalence of alpha toxin (hla), and the sbi gene that encodes an immunomodulatory protein important in complement evasion. We have also begun to use RT-PCR to confirm PCR results and to determine if the virulence genes are actually expressed. Finally by measuring hemolytic activity we are determining the virulence capacity of these non-aureus strains of Staphylococcus. Collectively, these studies indicate that S. aureus is not the common inhabitant of equine nasal passages and demonstrate that other staphylococcal species are common inhabitants. We are in the process of determining if these other isolates have virulence capacity and whether or not these isolates contain antibiotic resistance.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Staphylococcus aureus (S. aureus) is a Gram-positive, non-spore forming, and non-motile bacterium that is part of human and animal normal microbiota. However, S. aureus can invade its host and cause a variety of diseases such as toxic-shock syndrome, sepsis, and necrotizing pneumonia, as well as skin and soft tissue infections. S. aureus is the paradigm for drug resistance and also contains an abundance of virulence factors that are directly cytotoxic to host cells including hemolysins and cytolytic toxins. As virulence factors of S. aureus are well characterized in human populations, they are not well understood in equine populations. The focus of the current study was to characterize S. aureus virulence factors as well as antibiotic resistance from equine isolates and compare these results to human isolates. We investigated 26 individual horses two separate times for the presence of S. aureus. To do this nasal swabs were taken from both left and right nostrils of the subjects and S. aureus was isolated from the microbiota and confirmed using RAPIDEC Staph® (Biomerieux) . Our data suggest 7 individual horses were colonized with S. aureus. Using PCR we investigated the presence of toxin and antibiotic resistance genes including: mecA, sbi, tsst-1, lukS-PV, lukF-PV, hla, hlga, and blaZ. PCR results indicate all isolates have blaZ (gene that confers penicillin resistance), a high prevalence of alpha toxin (hla), and the sbi gene that encodes an immunomodulatory protein important in complement evasion. Several isolates appear to contain mecA, the gene responsible for methicillin-resistance, but through MIC analysis, only two samples show enough resistance to be considered MRSA. Through measuring hemolytic activity it was found that several strains did not have hemolytic activity which was surprising as several strains expressed hla. Collectively, these studies indicate that S. aureus is a common inhabitant of equine nasal passages and that these isolates contain antibiotic resistance and virulence factors common with strains colonized on humans. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Data for the project was collected by two pre-veterinary students as part of their capstone projects. It provided hands-on field and laboratory experience for these students. How have the results been disseminated to communities of interest? The results were presented at the American Association of Microbiology in Denver, CO (May 2013) and at 3 different public forums at Montana State University. Three public seminars were given on the outcomes of these experiments. What do you plan to do during the next reporting period to accomplish the goals? We are in the process of determining if the guttural pouch of horses contains Staphylococcus aureus and have started to confirm via 16S analysis the presence of Staphylococcus aureus in both the nares and guttural pouch. We will also continue to analyze virulence gene expression in the isolates and to determine relative capacity to cause disease. Finally, we intend to try to link presence of S. aureus in the nares or guttural pouch with incidence of infection.
Impacts
What was accomplished under these goals?
We have started to determine the characteristics and incidence of Staphylococcus in the anterior nares of horses. We have also begun to identify the proper anatomical niche of this microorganism in horses. These accomplishments as outlined in the previous section are directly related to the goal of determining the incidence of S. aureus in horses and its zoonotic potential.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
1. Long DR, Mead J, Hendricks JM, Hardy ME, Voyich JM, 18?-Glycyrrhetinic Acid Inhibits MRSA Survival and Attenuates Virulence Gene Expression.Antimicrob Agents Chemother. 2013. 57: 241-247.
2. Olson ME, Nygaard TK, Ackermann L, Watkins RL, Zurek OW, Pallister KB, Griffith S, Kiedrowski MR, Flack CE, Kavanaugh JS, Kreiswirth BN, Horswill AR, Voyich JM. Staphylococcus aureus nuclease is a SaeR/S-dependent virulence factor. 2013. Infect. Immun. 81: 1316- 24.
3. Watkins, R.L, Zurek, O.W., Pallister, K.B., and J. M. Voyich. The SaeR/S two-component system induces interferon-gamma production in neutrophils during invasive Staphylococcus aureus infection. 2013. Microbes and Infection 15(10-11):749-54.
4. Zurek, O., T.K. Nygaard, R. L. Watkins, K. B. Pallister, V. J. Torres, A. R. Horswill, and J. M. Voyich. The Role of Innate Immunity in Promoting SaeR/S-mediated Virulence in Staphylococcus aureus 2014. J. Innate Immun. **Editors Choice.
5. Nygaard TK, Pallister KB, Zurek OW, and J.M.Voyich. The impact of ?-toxin on host cell plasma membrane permeability and cytokine expression during human blood infection by CA-MRSA USA300.J Leukoc Biol. 2014 in press
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Staphylococcus aureus (S. aureus) is a Gram-positive, non-spore forming and non-motile bacterium that is part of human and animal normal microbiota. However, S. aureus can invade its host and cause a variety of diseases such as toxic-shock syndrome, sepsis, and necrotizing pneumonia, as well as skin and soft tissue infections. S. aureus is the paradigm for drug resistance and also contains an abundance of virulence factors that are directly cytotoxic to host cells including hemolysins and cytolytic toxins. As virulence factors of S. aureus are well characterized in human populations, they are not well understood in equine populations. The focus of the current study was to characterize S. aureus virulence factors as well as antibiotic resistance from equine isolates and compare these results to human isolates. We investigated 30 individual horses two separate times for the presence of S. aureus. To do this nasal swabs were taken from both left and right nostrils of the subjects and S. aureus was isolated from the microbiota and confirmed using RAPIDEC Staph (Biomerieux) . In related studies we are investigating the clonal isolates of S. aureus from nasal microbiota of healthy individuals. In total, 413 healthy volunteers from the community performed their own nasal swabs on both nostrils. Presence of S. aureus in nasal microbiota was determined by plating on mannitol-salt agar containing 7.5% sodium chloride. Coagulase positive (putative S. aureus) isolates were first selected based on mannitol fermentation and confirmed by API Staph detection system (Biomerieux) to ensure samples were S. aureus and not Staphylococcus intermedius or Staphylococcus haemolyticus. Using spa typing we determined the predominant clones from 96 of the S. aureus strains isolated from nasal microbiota and have determined prevalence of methicillin-resistance based on presence of the mecA cassette conferring methicillin-resistance. PARTICIPANTS: Barry Kreiswirth, Ph.D. Director Tuberculosis Center Public Health Research Institute, Newark, NJ, Helps with molecular epidemiology analysis of nasal isolates. Kyler Pallister, Montana State University, research technician performs experiments and trains students. The following students worked on this project: Meet Patel, Hannah Bigelow, Katie Rowe, Eli Sward. All students attend Montana State University. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our data suggest 13 individual horses were colonized with S. aureus. Using PCR we investigated the presence of toxin and antibiotic resistance genes including: mecA, sbi, tsst-1, lukS-PV, lukF-PV, hla, and blaZ . Preliminary PCR results indicate all isolates have blaZ (gene that confers penicillin resistance), a high prevalence of alpha toxin (hla), and the sbi gene that encodes an immunomulatory protein important in complement evasion. Surprisingly, several isolates appear to contain mecA, the gene responsible for methicillin-resistance, and the lukS-PV and lukF-PV genes encoding the two-component toxin PVL. TSST-1, the gene encoding the toxic-shock syndrome toxin-1 was present in one isolate. Collectively, these studies indicate that S. aureus is a common inhabitant of equine nasal passages and that these isolates contain antibiotic resistance and virulence factors common with human strains. One of the goals of our study is to determine if nasal isolates have adapted to the human nares and to determine how closely the isolates are related. To date we have sequenced 3 USA300 nasal isolates. Sequence data demonstrates three of the nasal isolates, NC324 (23824), NC61 (23811) and NC28 (23878) are from the ST8 multi-locus sequence type (clonal cluster 8) and are clonally related. We are now investigating the sequences of the virulence regulators such as agr and saeR/S. We have demonstrated that the sequence of saeR/S in strain NC61 varies from the predicted sequence of saeR/S in USA300 isolates and looks similar to saeR/S from USA200. This is an interesting finding that we confirmed by sequencing the saePQRS genes. We also have started to investigate variations in alpha-toxin expression and hemolytic activity in isolates from nasal microbiota compared to isolates from infection. To test the hypothesis that differential expression of virulence factors modulates pathogenic potential of nasal isolates, we evaluated expression of alpha-toxin using TaqMan real-time RT-PCR after exposure to human blood in a USA300 nasal isolate and in a well characterized USA300 isolate called LAC originally isolated from a patient with a skin and soft-tissue infection. Surprisingly, the USA300 nasal (23864) isolate had a more robust expression of alpha-toxin at 30 minutes post exposure to human blood than the USA300 infection isolate, LAC (compare a ~215 fold increase in 23864 to a 95 fold-increase in LAC). We also investigated hemolytic capacities among nasal and infection isolates by dropping equal amounts of cells on sheep blood agar plates and have developed a high throughput screen to compare hemolytic activities in the isolates. Differences in hemolytic activity were evident and with our newly developed assay are quantifiable suggesting either genetic differences in hemolytic toxins (potentially absence of some hemolytic toxins) or differential expression of hemolytic toxins (potentially mutations in S. aureus regulatory systems).
Publications
- No publications reported this period
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