UVM Theses and Dissertations
Format:
Print
Author:
Herzer, Kristi
Title:
Dept./Program:
Civil and Environmental Engineering
Year:
2013
Degree:
MS
Abstract:
The High Aspect Ratio Vessel (HARV) is a novel in vitro system for culturing bacterial cells. The HARV was designed by NASA to create a low fluid shear environment that is reported to model aspects of microgravity when rotated along the horizontal axis (deemed Low Shear Modeled Microgravity, LSMMG). In previous studies with P. aeruginosa and other opportunistic bacterial pathogens, it has been shown that bacteria grown in under LSMMG in the HARV can have increased stress survival phenotypes.
To initially characterize this low fluid shear bioreactor and the phenotypes it induces, we conducted experiments to assess a strain of P. aeruginosa (PA14) which had not yet been investigated. Samples were taken from the HARV in both gravity orientations, low shear modeled microgravity (LSMMG) and normal gravity (NG), and subjected to various stressors and antibiotics. Bacteria were assessed for survival as a function of post-stressed conditions relative to pre-stressed conditions. It was shown that there was not a significant difference between the gravity orientations of the HARV environment for a majority of conditions tested, such as 3M NaC1, 55°C heat shock, 10% v/v ethanol, meropenem, and ciprofloxacin. Specific time points or concentrations yielded statistically higher survival for LSMMG-grown PAI4, such as 5 and 15 min acid stress, 8[mu]g/mL tobramycin, and 1[mu]g/mL polymyxin B. Due to observations during these experiments and subsequent qualitative oxygen availability assays, the passive oxygenation of the HARV system was called into question.
In vitro bacterial culturing is most commonly performed in a shake flask, but this is not an accurate representation of natural bacterial habitats. In the standard in vitro system, aerobic microbes are allowed adequate access to nutrients and oxygen until stationary phase, but also experience a high level of fluid shear. Conversely, in their natural environments bacteria often thrive in a biofilm state, where the bacteria are attached to a surface and experience an oxygen gradient and negligible fluid shear. Oxygen limitation is one of the hallmarks of antibiotic resistance in a biofilm state since oxygen availability is a crucial component to the efficacy of antibiotics. Since oxygen availability is essential for the effectiveness of many antibiotics, and most bacteria persist in a biofilm state, the standard aerated shake flask is not an adequate model to study bacterial pathogens.
In order to characterize the HARV bioreactor and assess the low fluid shear and low oxygen conditions, the antibiotic survivability of three bacterial pathogens was compared in the conventional shake flask, biofilm, and HARV environments. Conventional MICs were determined for shake flask cultures, and 1x MIC and I000x MIC were used throughout these experiments as benchmarks to assess antibiotic survival. We hypothesized that biofilm cultures would have the highest survival after antibiotic challenge, followed by HARV, with shake flaskgrown cultures having the lowest survival. We observed that H influenzae challenged with 1x MIC levofloxacin for 1 h yielded statistically higher survival in the biofilm than the HARV and the HARV has statistically higher survival than the shake flask. Of the thirty-six conditions tested, ten were found statistically significant, and nine of these demonstrated biofilm-grown cells had a higher percent survival than shake flask. The HARV-grown cultures were determined not statistically different from the biofilm or shake flask-grown cultures for this study, but more experimentation is needed to evaluate this system.
To initially characterize this low fluid shear bioreactor and the phenotypes it induces, we conducted experiments to assess a strain of P. aeruginosa (PA14) which had not yet been investigated. Samples were taken from the HARV in both gravity orientations, low shear modeled microgravity (LSMMG) and normal gravity (NG), and subjected to various stressors and antibiotics. Bacteria were assessed for survival as a function of post-stressed conditions relative to pre-stressed conditions. It was shown that there was not a significant difference between the gravity orientations of the HARV environment for a majority of conditions tested, such as 3M NaC1, 55°C heat shock, 10% v/v ethanol, meropenem, and ciprofloxacin. Specific time points or concentrations yielded statistically higher survival for LSMMG-grown PAI4, such as 5 and 15 min acid stress, 8[mu]g/mL tobramycin, and 1[mu]g/mL polymyxin B. Due to observations during these experiments and subsequent qualitative oxygen availability assays, the passive oxygenation of the HARV system was called into question.
In vitro bacterial culturing is most commonly performed in a shake flask, but this is not an accurate representation of natural bacterial habitats. In the standard in vitro system, aerobic microbes are allowed adequate access to nutrients and oxygen until stationary phase, but also experience a high level of fluid shear. Conversely, in their natural environments bacteria often thrive in a biofilm state, where the bacteria are attached to a surface and experience an oxygen gradient and negligible fluid shear. Oxygen limitation is one of the hallmarks of antibiotic resistance in a biofilm state since oxygen availability is a crucial component to the efficacy of antibiotics. Since oxygen availability is essential for the effectiveness of many antibiotics, and most bacteria persist in a biofilm state, the standard aerated shake flask is not an adequate model to study bacterial pathogens.
In order to characterize the HARV bioreactor and assess the low fluid shear and low oxygen conditions, the antibiotic survivability of three bacterial pathogens was compared in the conventional shake flask, biofilm, and HARV environments. Conventional MICs were determined for shake flask cultures, and 1x MIC and I000x MIC were used throughout these experiments as benchmarks to assess antibiotic survival. We hypothesized that biofilm cultures would have the highest survival after antibiotic challenge, followed by HARV, with shake flaskgrown cultures having the lowest survival. We observed that H influenzae challenged with 1x MIC levofloxacin for 1 h yielded statistically higher survival in the biofilm than the HARV and the HARV has statistically higher survival than the shake flask. Of the thirty-six conditions tested, ten were found statistically significant, and nine of these demonstrated biofilm-grown cells had a higher percent survival than shake flask. The HARV-grown cultures were determined not statistically different from the biofilm or shake flask-grown cultures for this study, but more experimentation is needed to evaluate this system.