Research:

Research Interests:

  • Microbiology of rural groundwater supplies.
  • Detection and significance of antibiotic resistant bacteria in surface and ground water.
  • Microbiology of wetlands used to treat domestic wastes.
  • Detection of opportunistic pathogens (Acinetobacter spp., Aeromonas spp., and slow-growing pigmented heterotrophic bacteria) in rural drinking water supplies.
  • Development of highly selective, yet sensitive, culture methods for detection of microorganisms of public health significance (pathogens and indicator organisms) in water and wastewater.
  • Effect of antecedent growth conditions on the sensitivity of waterborne pathogens and indicator organisms to disinfectant stress.
  • Detection of small-sized (filterable) bacteria in aquatic environments.
  • Evaluation of Bifidobacterium spp. as alternative indicators of fecal pollution of water.
  • Detection and distribution of Arcobacter spp.and Enterobacter sakazakii in the environment.
  • Effect of environmental stress (e.g., starvation, acid, temperature) on the morphology and physiology of sublethally-injured surviving bacteria

Current Graduate Adivsees :

  • Emily A.Kenney (M.S. Candidate) -Thesis Research : Detection and Survival of Enterobacter sakazakii in Aqueous Environments.

  • Matthew G.McCarroll (M.S.Candidate) -Thesis Research: Application of the BIOLOG Microplate System to monitor the Physiological Response of Environmentally Stressed Bacteria.

Research Activities & Publications:

Short Summaries/Abstracts of Selected Research Projects:

  • Thunberg, R.L., A.J. Sexstone, J.P. Calabrese, and G.K. Bissonnette. Effects of Antecedent Fermentative and Respiratory Growth on the Detection of Chloramine-Stressed Escherichia coli and Salmonella typhimurium. Antecedent growth conditions have been shown to influence the subsequent response of a particular organism to stress. In vitro laboratory studies were performed to assess the effects of antecedent growth conditions on the recovery of Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 following chloramine disinfection. Six- and 18-h cultures of each organism were grown under aerobic, fermentative, and nitrate-reducing conditions prior to disinfection. At predetermined time intervals during a 10-min exposure to chloramine, survivors were surface-plated on nonselective recovery media to determine C.t values. It was observed that nitrate-reducing growth predisposed the test organisms towards an increased sensitivity to chloramine stress over cells grown under fermentative or aerobic conditions (p<0.01). Our findings suggest that conclusions drawn from experiments utilizing cultures of enteric bacteria grown under aerobiosis may not accurately reflect the capabilities of environmental isolates, which may grow under the anaerobic nitrate-reducing environment of the intestinal tract man and animals. Thus, extrapolation of the results of in vitro studies to those conditions occurring in situ or in vivo must be done with caution as environmental conditions rarely reflect those created in the laboratory.

  • Hench, K.R., A.J. Sexstone, J. Coleman, K. Garbutt, J. Skousen, and G.K. Bissonnette. Removal of Pathogens and Indicator Organisms from Wastewater Using Small-Scale Constructed Wetlands. Constructed wetlands are being promoted as low-cost, low-maintenance systems for treatment of domestic wastewater in areas where standard septic tanks fail. In order to evaluate the efficacy of constructed wetlands for small communities located in rural areas, four small-scale wetland mesocosms containing two plant treatments (no plants/control, mixture/combination of Typha, Scirpus, and Juncus species) were planted into pea gravel. Each mesocosm received 5 gal/day of primary-treated domestic sewage. Mesocosms were monitored on a monthly basis over a two-year period for total suspended solids (TSS), 5-day biochemical oxygen demand (BOD), total Kjeldahl nitrogen (TKN), dissolved oxygen (DO), and temperature. Fecal coliforms, enterococci, Salmonella, Shigella, Yersinia, and coliphage densities were also determined. Significant differences between influent and effluent water quality were observed in TSS, BOD, and TKN. Increased DO and reduction in fecal coliforms, enterococci, Salmonella, Shigella, Yersinia, and coliphage populations were also observed. Greatest microbial reductions were observed in the planted mesocosms, with removal efficiencies more apparent during the initial compared to the second year of operation. Less effective removal during the second year may have been a result of accumulation of suspended solids by plant roots, leading to decreased inactivation of microbes in planted mesocosms.

  • Lillis, T.O., and G.K. Bissonnette. Detection and Characterization of Filterable Heterotrophic Bacteria from Rural Groundwater Supplies. The chemical and physical environment of ground water may contribute to the existence of a subpopulation of small-sized bacteria (filterable bacteria) that fails to be trapped on conventional 0.45 micrometer-pore-size membrane filters during routine bacteriological water quality analyses. Individual groundwater supplies in a rural setting were examined by a double membrane filtration procedure to determine the presence of heterotrophic plate count (HPC) bacteria capable of escaping detection on conventional pore size (0.45 micrometer) membrane filters but retained on 0.22 micrometer-pore-size filters. Maximum recovery of filterable HPC bacteria occurred on low-nutrient concentration R2A agar incubated for seven days at 28°C. Similarly, identical cultural conditions gave enhanced detection of the general HPC population on 0.45 micrometer membrane filters. A 17-month survey of ten well water supplies conducted with the cultural conditions described above resulted in detection of filterable bacteria in six of the groundwater sources. The proportion of filterable bacteria in any single sample never exceeded 10% of the total HPC population. A majority of the colonies appearing on the smaller porosity membrane filters were pigmented. Subsequent identification of randomly selected isolates obtained from these filters indicated that some of these filterable bacteria might be opportunistic pathogens.

  • McElwain, R.D., and G.K. Bissonnette. Survival and Recovery of Arcobacter spp. from Aquatic Environments. In vitro studies have been initiated to examine the survival of Arcobacter butzleri, a recently recognized pathogen associated with humans and domestic animals, in groundwater environments. Arcobacter butzleri proved to be relatively resistant to exposure to the nutrient-depleted environment of groundwater at 15°C as demonstrated by D-values of approximately 14 days (time required to reduce the population by 90%). In comparison, identical experiments conducted with the standard water quality indicator organism, Escherichia coli, resulted in D-values of 20 days. In addition, we observed that incorporation of blood to recovery media permitted efficient detection of survivors under aerobic incubation conditions, thereby providing an alternative to more costly and complex microaerophilic incubation. We are extending our studies to include other stress temperatures (5 and 25°C) and to develop a selective recovery medium that incorporates both specificity and selectivity.

 

  • Kenney, E.A., and G.K. Bissonnette. Detection and Survival of Enterobacter sakazakii in Aqueous Environments. The goals of this M.S. research project are: (1) to optimize a culture-based method for the detection and isolation of E. sakazakii in environmental samples; (2) to investigate the possible environmental reservoirs of E. sakazakii (such as wastewater and surface water); and (3) to evaluate the ability of E. sakazakii to survive in theses aqueous environments under varying conditions. In order to detect and isolate E. sakazakii from microcosm and natural samples, a differential and selective medium based on glucosidase activity will be initially investigated.

 

  • McCarroll, M.G., and G.K. Bissonnette. Application of the BIOLOG Microplate System to Monitor the Physiological Response of Environmentally Stressed Bacteria. The overall goal of this M.S. research project is to evaluate the ability of the BIOLOG system as an assay for the physiological response of bacteria exposed to environmental stress. The test organisms Escherichia coli, Enterobacter sakazakii, and Salmonella typhimurium will be exposed to such stresses as heat, acid, and starvation. The potential benefit of this study is the possible addition of a new method to aid in the monitoring of the physiological response of bacteria following environmental stress.

 

Publications (since 1990):

  • Calabrese, J.P., and G.K. Bissonnette. 1990. Improved detection of acid mine water-stressed coliform bacteria on media containing catalase and sodium pyruvate. Canad. J. Microbiol. 36:544-550.
  • Calabrese, J.P., and G.K. Bissonnette. 1990. Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria. Appl. Environ. Microbiol. 56: 3558-3564.
  • O'Neill, C.E., and G.K. Bissonnette. 1991. Antecedent oxygen growth conditions and recovery of heat-stressed Escherichia coli. J. Food Protect. 54: 90-93.
  • Shirey, J.J., and G.K. Bissonnette. 1991. Detection and identification of groundwater bacteria capable of escaping entrapment on 0.45 micrometer-pore-size membrane filters. Appl. Environ. Microbiol. 57: 2251-2254.
  • Shirey, J.J., and G.K. Bissonnette. 1992. Sheen formation and growth response of groundwater bacteria to reduced oxygen concentrations during incubation of M-endo medium. Canad. J. Microbiol. 38: 261-266.
  • Calabrese, V.G.M., R.E. Elsavage, G.K. Bissonnette, and A.J. Sexstone. 1993. Mobility and enhanced biodegradation of a dilute waste oil emulsion during land treatment. J. Indust. Microbiol. 12: 13-20.
  • McKeon, D.M., J.P. Calabrese, and G.K. Bissonnette. 1995. Antibiotic resistant gram-negative bacteria from rural groundwater supplies. Water Res. 29: 1902-1908.
  • Snyder, J.W., C.N. Mains, R.E. Anderson, and G.K. Bissonnette. 1995. Effect of point-of-use, activated carbon filters on the bacteriological quality of rural groundwater supplies. Appl. Environ. Microbiol. 61: 4291-4295.
  • Shirey, J.J., and G.K. Bissonnette. 1997. Recovery of coliform bacteria from rural groundwater supplies using reduced oxygen concentrations during incubation. Canad. J. Microbiol. 43: 583-588.
  • Sexstone, A.J., J.G. Skousen, J.P. Calabrese, D.K. Bhumbla, J.B. Cliff, and G.K. Bissonnette. 1999. Iron removal from acid mine drainage by wetlands. Proc. 16th Natl. Meet. Amer. Soc. Surface Mining and Reclamation, pp. 609-620.
  • Sexstone, A.J., M. Aiton, G.K. Bissonnette, K. Fleming, K. Kineer, K. Hench, T. Bozicevich, B. Cooley, and E. Wiant. 2000. A survey of home aeration units operating in six West Virginia counties. EPA Small Flows Quaterly 1: 38-46.
  • Coleman, J., K. Hench, K. Garbutt, A.J. Sexstone, G. K. Bissonnette, and J. Skousen. 2001. Treatment of domestic wastewater by three plant species in constructed wetlands. Water, Air, and Soil Pollut. 128:283-295.
  • Nayak, R., P.B. Kenney, and G.K. Bissonnette. 2001. Inhibition and reversal of Salmonella typhimurium attachment to poultry skin using zinc chloride. J. Food Protect. 64:456-461.
  • Lillis, T.O. and G.K. Bissonnette. 2001. Detection and characterization of filterable heterotrophic bacteria from rural groundwater supplies. Lett. Appl. Microbiol. 32:268-272.
  • Thunberg, R.L., A.J. Sexstone, J.P. Calabrese, and G.K. Bissonnette. 2001. Effects of antecedent fermentative and respiratory growth on the detection of chloramine-stressed Escherichia coli and Salmonella typhiurium. Canad. J. Microbiol.47:777-781
  • Coleman, J., K. Hench, K. Garbutt, A. Sexstone, G. K. Bissonnette, and J. Skousen. 2001. Treatment of domestic wastewater by three plant species in constructed wetlands. Water, Air, and Soil Pollut. 128:283-295.
  • Hench, K.R., G.K. Bissonnette, A.J. Sexstone, J.G. Coleman, K. Garbutt, and J.G.Skousen. 2003. Fate of physical, chemical, and microbiological contaminants in domestic wastewater following treatment by small constructed wetlands. Water Res. 37:921-927.
  • Hench, K.R., A.J. Sexstone, and G.K. Bissonnette. 2004. Heterotrophic community-level physiological profiles of domestic wastewater following treatment by small constructed subsurface flow wetlands. Water Environ. Res. 76:468-473.

 

Funded Research Projects (P.I. and Co-P.I. grants ):

  • Standard Plate Count Bacteria and Opportunistic Pathogens in Rural Groundwater Supplies - Water Research Institute/U.S. Geological Survey.
  • Microbial Degradation of Oil Waste Emulsions - Kaiser Chemical and Aluminum Company .
  • Modification of Coliform Methodology to Assess the Bacterial Quality of Rural Groundwater Supplies- Water Research Institute/U.S. Geological Survey .
  • Removal of Iron and Manganese from Acid Mine Drainage by Cattail Wetlands: Chemical and Microbiological Processes - WVU Energy and Water Research Center/National Mine Land Reclamation Center .
  • Bacterial Quality of POU Filters for Treatment of Rural Groundwater Supplies - USDA/CSRS .
  • In Situ Removal of Metals and Acidity from Pretreated Mine Drainage Using Typha Wetlands - WVU Energy and Water Research Center/National Mine Land Reclamation Center.
  • Efficacy of Coliform Presence-Absence Testing and the Coliphage Assay for Monitoring the Microbiological Quality of Individual Rural Groundwater Supplies - Water Research Institute/U.S. Geological Survey .
  • Wetland Plant Selection for Wastewater Treatment - EPA/WVU Cooperative Extension Service .
  • Determination of Fecal Coliform Bacteria, Biological Oxygen Demand, Total Suspended Solids, and Nitrate in Effluents from Home Aeration Units of Varying Age and Maintenance - Environmental Services Training Division, WVU National; Research Center for Coal and Energy.
  • Potomac Headwaters Sampling Project - West Virginia Department of Agriculture and Water Research Institute .
  • Evaluation of Biocidal Polymers for the Treatment of Domestic Water Supplies - Water Research Institute/U.S. Geological Survey.