Current Research Projects and Funds

My laboratory conducts Environmental Microbiology research. Currently, four graduate and three undergraduate students are involved in the projects described below helped by an excellent lab manager, Mrs. Glenda Kohlhagen.

Project 1. Circulation of Antibiotic Resistance Genes in Animal Feeding Operations and Adjacent to Them Environments (supported by grants from the National Pork Board and National Sea Grant College Program.)

Microbial resistance to antibiotics is not new but the number of resistant microorganisms is increasing.  Animal feeding operations (AFO) are generally viewed as important sources of environmentally-observed antibiotic resistance genes (ARG) whereas their relative contribution to ARG migration and accumulation in the environment is not yet clear.  Comparative profiles of ARG in swine (SFO) and poultry (PFO) operations, ARG concentrations, and their fate in the environment are addressed in this project.  Due to their abundance and diversity, tetracycline resistance genes (tet) are used as model ARG.  In 2005-2006, we surveyed the profiles and abundances of tet genes in microbial communities of a SFO and PFO, and in the adjacent to them the Altamaha- and Satilla River (Georgia, USA) watersheds.  Neither AFO had a record of antibiotic usage within 3 years preceding the study.  Eleven tet genes - tetA(P), (E), (K), (L), (M), (O), (Q), (T), (S), (X) and (W) - encoding all three known tetracycline resistance mechanisms were targeted in this study. The community-based profiles of these genes have been monitored in animal feces, waste lagoons, manure-treated and non-treated soils, outbound streams, and lakes. The PFO has revealed the presence of tet(K), (L), (M), (O), (Q), (T), (X) and (W) genes; whereas tetA(P), (M), (O), (Q), (X) and (W) have been observed in the SFO.  Concentration of tet(M) increased as animal wastes passed through the lagoons. Between the first and the fourth PFO waste treatment lagoons, its concentration augmented from 0.04 to 1.3 copies per one 16S rRNA operon, thus demonstrating approximately 30-fold enrichment.  The presence of tet(M) genes with the amplicon sequences highly similar to these observed in waste lagoons was observed in manure-treated soils and adjacent to both the SFO and PFO creeks and lakes.  Tet(O) was detected in soils bordering the swine- but not poultry AFO at concentrations below 0.1 copy/16S rRNA operon.

 Three more varied upon the animal profiles, numbers, and antibiotic usages SFO (including the previously sampled operation) are included in 2007 survey. Based on the array of 16 tet genes, this study revealed conservativeness of ARG profiles in the SFO, and confirmed a high distinction of these profiles from those observed in the PFO.  No correlation between the ARG profiles and antibiotic dosage has been observed yet.  The enrichment and potential for environmental discharge was observed for three among twelve tet genes detected in the SFO feces and lagoons. Our study clearly demonstrates (i) high presence of tet genes in AFOs even in the absence of direct tetracycline selective pressure, (ii) diversity between SFO and PFO tet gene profiles, and (iii) selective enrichment of tet genes in waste lagoons and their discharge to the environment.

Future studies will address relationships between the discharge of tet genes from AFO and the abundances of integrons classes 1 through 3 in these AFO and adjacent to them environments, and between the molecular structure of tet genes, their mobility, and persistence in the environment. 

Project 2.  Tetracycline Resistance and Integron Genes Related to Animal Waste Contamination of Riverine and Estuarine Environments Including Oyster Beds (supported by grants from the National Sea Grant College Program and Georgia Department of Natural Resources.)

Animal and human wastes impact water quality and shellfish harvestability in 19 of 59 Georgia sub-watersheds. In 2003-2005, sediments of the Altamaha, Satilla, and Savanna River estuaries were surveyed for the presence of microorganisms resistant to environmentally relevant (1-5 mg L^-1) tetracycline concentrations.  Three fractions of microorganisms were isolated with respect to their association to sediments: fraction 1 (F1, planktonic), fraction 2 (F2, loosely associated to sediments); and fraction 3 (F3, strongly associated to sediments). In all three estuaries, the highest abundance of tetracycline resistant (TR) microorganisms was observed in F3.  Microbial fractions were surveyed for the presence of ten tet genes. Tet(M), tet(Q), and tet(T) were found responsible for the TR in the Altamaha and Savannah River estuaries.  Only tet(T) was detected in the Satilla River microbial community. These results indicate that in estuaries, TR microorganisms are primarily associated with sediment particles and that the TR gene profiles vary between the estuaries possibly indicating different origins of TR microorganisms in their watersheds.

In 2007-2008, we plan to devise and test a tool for tracking point-source fecal contamination in oyster beds based on tet genes, and to devise and test a supplementary to coliforms indicator for non-point source fecal contamination in oyster beds based on int (integron) genes.  Deboy Sound (Altamaha River estuary, anthropogenically impacted) is chosen for this study.  Water, sediment, and oysters will be collected every 4 months.  DNA will be extracted, and 16 tet and 3 int genes will be targeted and quantified.  Data will be normalized to 16S rDNA copies. Resulting data will be compared to coliform counts.  If our theory is correct, we will observe mid to high numbers of target genes in Deboy Sound that may or may not correlate to coliform counts. Coastal managers and RiverKeepers will be involved with the project through Georgia DNR and Georgia River Network. 

Project 3.  Efficacy of Biocides towards Particle-Associated and Freely-Dispersed Clay Microorganisms (supported by grants from IMERYS.)

Biofouling during the storage and oversea transportation present a problem for commercial kaolin products.  We hypothesized that biofouling happens because some microorganisms strongly associated with kaolin particles are insensitive to biocides because they are sheltered from the biocides by these particles.  During the summer of 2004, we started experiments targeting this hypothesis.  In our experiments, the biocides were applied at commercial dosage either (i) directly to the clay slurries with consequent elution of freely-dispersed (FD), loosely- (LA), and strongly particle - associated (SA) microbial communities, or (ii) directly to the eluted from clay FD-, LA-, and SA microbial communities.  No biocide effect was observed for LA and SA microorganisms in the first experiment whereas all the FD microorganisms were killed by the biocides at their commercially used dosages.  No difference in the resistance of LA, SA, and FD to biocides was observed in the second experiment, all were sensitive.  The data obtained indicate that the resistance of particle-associated microorganisms to biocides is governed by their attachment to and protection from clay particles rather than by their higher resistance to biocides per se.   

Our current study evaluates the effects that biocides pose on particle-associated and freely-dispersed microorganisms, and how to differentiate these effects for different physiological groups of microorganisms including sulfate- and iron-reducers.  Our future study will address the detachment of particle-associated microorganisms from kaolin particles that should increase their sensitivity to biocides.