1. Manure-derived Antibiotic Resistant Bacteria: Occurrence and Transport.

For several decades, antibiotics have been commonly used in animal farms at therapeutic levels to treat diseases and at sub-therapeutic levels for growth promotion and prophylactic purposes. The widespread use of antibiotics in animal farm environments has resulted in high levels of antibiotic resistant bacteria in animal waste. The primary objectives of this research include: 1) To isolate E. coli and Enterococci from dairy manure and to determine their resistance to the major antibiotics. 2) To examine the transport behavior of antibiotic resistant bacteria.

Our results showed that high percentages of the E. coli isolates were resistant to cephalothin, ampicillin, tetracycline and erythromycin. For Enterococcus, in addition to the aforementioned 4 antibiotics, they were also resistant to gentamicin and ciprofloxacin. Our investigations into the transport behavior of antibiotic resistant E. coli suggested that tetracycline resistance could significantly increase the mobility of manure-derived E. coli. The outer membrane proteins (OMPs) of the E. coli strains were extracted and profiled using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The SDS-PAGE results led to the hypothesis that the OMP TolC, which is part of the AcrAB-TolC drug efflux pump, was at least partly responsible for the increased mobility of tetracycline resistant E. coli (This hypothesis has been confirmed by my group using TolC negative mutant).

To further test the influence of tetracycline resistance on the transport of E. coli within aquifer materials, my group isolated tetracycline resistant and tetracycline susceptible E. coli from Lake Michigan. Consistent to our previous findings, we observed that 1) tetracycline resistance could significantly increase the mobility of E. coli; and 2) the OMP TolC was related to the increased mobility of tetracycline resistant E. coli.

2. Influence of Environmental Stresses (e.g., Desiccation and Starvation) on Bacterial Transport within the Groundwater System.

Fecal bacteria released into the environment experience a variety of environmental stresses. For instance, when traveling through the unsaturated layer of soil, bacterial cells commonly experience desiccation stresses (i.e., bacterial cells are directly exposed to air) before entering the underlying groundwater system. As the natural environment is usually nutrient poor, starvation represent another common environmental stress for bacterial cells. Such environmental stresses could reduce cell viability and alter cell surface properties, which in turn may influence bacterial transport behavior within the groundwater system. My group has investigated the impact of desiccation under various relative humidity (RH) conditions on the survival and transport of E. coli. Our results showed that desiccation significantly reduced E. coli viability and desiccation under high RH conditions increased both cell hydrophobicity and mobility. For cell starvation, we found that starvation led to significant increase in E. coli mobility and viability only if both pH and ionic strength of the electrolyte solution used for starvation were different from those of the growth media.

3. Colloid Straining within Saturated Porous Media.

Over the past several years, I have performed a series of experiments to examine colloid straining within saturated porous media under conditions that are unfavorable for colloid deposition (i.e., strong repulsive interaction between colloid particle and sand surface). These studies have led to a uniform relationship that can be used to describe colloid straining kinetics. Particularly, I have found that for non-spherical particles, the straining kinetics was controlled by the minor size of the particles. For polydisperse colloid suspensions (i.e., mixture of colloids of different sizes), as the straining of the larger particles reduced the size of pore openings, the straining of the smaller particles was enhanced. In contrast, because the smaller particles occupied the spaces that were otherwise available for the straining of the larger particles, the straining kinetics of the larger particles was reduced. For heterogeneous porous media (i.e., mixture of sands of different sizes), we found that the number-averaged sand size should be used to describe straining kinetics.

My group is now working on colloid straining under conditions that are favorable for colloid deposition. It is hypothesized that because deposited colloids will reduce the size of pore openings, colloid straining will be enhanced by colloid deposition. A mathematical model is being developed to simulate the kinetics of colloid straining under favorable conditions.

4. Relationship between Temporal Variations in Precipitation and Groundwater Recharge.

This is Alice Egan’s M.S. thesis research. The rate of groundwater recharge has profound implications for the sustainable use of groundwater resources. My group has examined the relationship between groundwater recharge (based on base flow extraction) and annual precipitation for many watersheds within the state of Wisconsin. Although annual groundwater recharge rate was found to be closely related to annual precipitation, it was frequently observed that groundwater recharge rate could vary significantly (e.g., by a factor of 3) when annual precipitation was roughly constant. Our investigation into the potential effects of air temperature suggested that air temperatures variations were unlikely to be the primary factor behind the observed annual recharge patterns. We then developed the hypothesis that the depth/frequency of precipitation was a major cause of the variations in annual groundwater recharge. To test this hypothesis, we are using the soil-water-balance (SWB) model ( to systematically investigate the relationship between precipitation patterns and variations in groundwater recharge.

5. Effects of Cell Outer Membrane Proteins on the Transport of E. coli within the Groundwater System.

Through the study of the transport of tetracycline resistant E. coli within saturated sand packs, my group hypothesized that the outer membrane protein (OMP) TolC could enhance the mobility of E. coli. Additionally, other researchers have hypothesized that the OMP Ag43 could enhance the deposition (i.e., reduce the mobility) of E. coli. To test these hypotheses, I have worked with Prof. Sonia Bardy (UWM, Department of Biological Sciences) to create E. coli mutants that lack OMP TolC or OMP Ag43. Column transport experiments were then performed to examine the transport behavior the wild type E. coli strain, the TolC negative mutant and the Ag43 negative mutant under a range of water chemistry conditions. Our findings confirmed both hypotheses. Cell surface properties were determined and the XDLVO theory was applied to explain the observed trend in E. coli transport behavior.

6. Can Enterococcal Surface Protein (esp) and Bacteroides spp. Be Used as Microbial Source Tracking Tools in the Groundwater System?

The fast and reliable detection of groundwater microbial contamination and the identification of the contamination sources are of critical importance to the protection of public health. It was recently reported that Enterococcus faecium specific esp gene (espfm) was more prevalent in sewage and septic system samples than livestock, wild animal and bird samples and it could potentially be used as a molecular marker to identify human sources of fecal pollution. Additionally, it was shown that host specific Bacteroides genetic markers (e.g., the 16S rRNA HF183 marker) can be used to determine the source of fecal pollution. The effectiveness of these emerging tools as an index of human fecal pollution for the groundwater system, however, will depend on the transport behavior of E. faecium strains with and without esp as well as Bacteroides.

My group performed column transport experiments to determine the mobility of E. faecium E1162 (isolated from human sources) and its esp negative mutant E1162Δesp. Our results suggested that E1162Δesp had significantly higher mobility than E1162. Therefore, E. faecium strains from non-human sources can travel over longer distances along the groundwater flow direction than E. faecium strains from human sources. If used for the groundwater system, the espfm gene technique may underestimate the contribution from human fecal pollution. In contrast, our results showed that Bacteroides fragilis displayed significantly higher mobility than the common fecal contamination indicator, E. coli, suggesting that Bacteroides can be a promising microbial source tracking tool for the groundwater system. It is also noteworthy that the results from the E. faecium study provide another example that cell surface protein could have significant impact on bacterial transport within the groundwater system.