Tetracycline Resistance in Aerobic and Anaerobic Bacteria

Recovered from Landfill Leachate

Neil Kirkpatrick, Jenny Will, and James McGaughey

Biological Sciences Department, Eastern Illinois University, Charleston, IL

Introduction and Purpose

Solid waste landfills are inhabited by various microorganisms including bacteria. Leachate accumulating within these landfills has the potential to contaminate surrounding groundwater and surface waters with potentially pathogenic bacteria, some of which may exhibit resistance to currently prescribed antibiotics. Tetracycline, an antibiotic which has found widespread use in a variety of applications, may be useful as a model substance for investigating development of bacterial resistance.

The purposes of this experiment were to estimate the density of aerobic and anaerobic bacteria in landfill leachate and surrounding groundwater and to evaluate their resistance to tetracycline.

	Landfill

Methods

Leachate from a closed landfill and adjacent groundwater samples were obtained from a regional landfill site. All samples were extracted via a well/pump collection system integrated with the landfill and the surrounding ground. Samples were diluted 10^-1 to 10^-3 using phosphate buffered saline. Ten milliliter portions were vortexed and filtered through 0.22mm membrane filters. Replicate filters were transferred onto pads with 2mL of Plate Count broth. One half of the plates contained 20mg mL^-1 tetracycline. The other half contained Plate Count broth without tetracycline. Of these plates, one half were incubated aerobically and the other half in a BBL GasPak Anaerobic System for 48 hours at 35^oC . Statistical analyses were performed. A Mann-Whitney test was used to determine the probability of significant differences between tetracycline resistant and tetracycline sensitive populations.

	Groundwater sample (left); Leachate sample (right)
	BBL Gaspak Anaerobic System
	Manifold filtration system

Results

	Figure 1: Mean heterotrophic count of bacteria from groundwater.
	Figure 2: Mean heterotrophic count of bacteria from leachate.
	Figure 3: Heterotrophic bacteria resistant to tetracycline.

Populations of aerobic heterotrophs from groundwater ranged from 2,000-4,000mL^-1; anaerobic populations ranged from 1,000-1,200mL^-1.  Populations of aerobic heterotrophs from leachate ranged from 400 – 38,000mL^-1; anaerobic populations ranged from 1,000 – 9,100mL^-1.  33% of aerobic and 12% of anaerobic populations from groundwater were resistant to tetracycline.  1% of aerobic and 10% of anaerobic populations from leachate were resistant to tetracycline.  Mann-Whitney tests indicated a significant difference for tetracycline resistance among aerobic and anaerobic bacteria recovered from groundwater and aerobic bacteria recovered from leachate.  Mann-Whitney tests indicated no significant difference for tetracycline resistance among aerobic heterotrophic bacteria recovered from groundwater.

Summary

Populations of aerobic and anaerobic bacteria recovered from landfill leachate and groundwater displayed a wide range of values.  Resistance to tetracycline was highest among bacteria isolated from groundwater and lowest among bacteria isolated from leachate samples.  Anaerobic bacteria seemed to be more resistant to tetracycline than aerobic bacteria.  Tetracycline appeared to maintain its effectiveness in reducing populations of heterotrophic bacteria with the exception of the aerobic groundwater populations.

References

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Acknowledgements

Funding and support from the College of Sciences; Department of Biological Sciences and the Undergraduate Research Council, Eastern Illinois University.  Dr. Scott Meiners for his help with data analysis and statistical calculations.  Matt Gresk for his help and support with the poster design.