Metagenomic Analysis From Equus caballus Fecal Samples
A current focus of research is to identify novel enzymes within microflora communities that could be used to increase efficiency and lower operating cost of applications across multiple industries. The aim of this study was to construct a metagenomic library of the microflora community of intestinal bacteria from equine fecal samples in order to identify new novel enzymes. Fecal samples were obtained from horses fed a mixture of grain and perennial pasture and hay grasses. Several genomic DNA extraction and purification protocols were evaluated to include commercial kits and pretreatments prior to their use. A modified pretreatment method using bead beating with a lysis buffer containing high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA followed by a purification step using QIAamp columns resulted in the highest yield of DNA. This procedure was scaled up in order to obtain a large volume of purified DNA for downstream applications. Multiple electrophoresis techniques were evaluated for the best band resolution of large sized DNA fragments. Total genomic DNA was electrophoresed in 0.8% agarose with 1% polyvinylpyrrolidone (PVP) which resulted in the majority of the chromosomal DNA fragments being located between 23-33 kb. The DNA from this section of gel was extracted and used for the construction of a library that will be screened for novel enzymes. Currently, there is a need in the biofuel industry for cellulases which can aid in the production of ethanol. To identify clones expressing these enzymes, screening will be conducted on carboxymethylcellulose (CMC) agar plates.
Dept of Biological Sciences, Campbell University, Buies Creek, NC
P172 • Zainab Veerani, Brandi Baggett, Brian Seeblack, Julia E. S. Shearer
Plasmid Content and Identification of Environmental Bacterial Isolates From Northern Georgia
Bacteria are ubiquitous in the environment and frequently share genes by the horizontal gene transfer of mobile genetic elements, such as plasmid conjugation. Most bacteria carry plasmids, which encode adaptive genes for specific environments like virulence genes that aid in causing diseases, antibiotic resistance genes, and genes for alternate carbon sources. Class 1 integrons are usually found on plasmids and are associated with spreading antibiotic resistance genes; Class 1 integrons can be identified by the presence of intI1. We are interested in the plasmids carried by bacteria in our immediate surroundings, such as in the soil and in our food. We obtained soil samples from the GGC campus and isolated 16 bacterial strains. All isolates were identified as Gram-positive and were examined for plasmids. Fourteen out of 16 (87.5%) bacterial strains contained plasmids, 10 of which (71.4%) had plasmid bands that were larger than 30 kb, meaning large enough to possibly be conjugative. Eleven of the soil isolates were identified to the genus level by 16S rDNA PCR and sequencing. Total DNA extracted from the GGC soil samples was negative for intI1 by PCR, suggesting the absence of Class 1 integrons. Evidence of Class 1 integrons was found in food isolates. intI1-positive bacterial strains were isolated from organic store-bought chicken breasts. Two isolates were identified to the genus level by 16S rDNA PCR and sequencing and were examined for plasmid content, both containing plasmid bands >30 kb.
School of Science & Technology, Georgia Gwinnett College, Lawrenceville, GA
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P173 • William C. Bible, Michael S. Glennon, David K. Giles
Scavenging of Fatty Acids Confers Phenotypic Advantages to Pseudomonas aeruginosa
Pseudomonas aeruginosa, a common opportunistic pathogen, is known to cause infections in a variety of compromised human tissues. Bacteria are extremely adept at adapting to their environmental and host niches. An emerging mechanism for microbial survival is the incorporation of exogenous fatty acids to alter the cell’s membrane phospholipid profile. The goal of this research was to examine P. aeruginosa’s ability to utilize exogenous fatty acids in a manner that affects its growth and survival. Initially, potential phospholipid alterations in response to pH, temperature, and salt concentrations were examined. Then, a range of host-derived fatty acids not native to P. aeruginosa were added individually to cultures. Bacterial phospholipids were extracted using the Bligh and Dyer technique and examined using thin layer chromatography (TLC). Whereas minor phospholipid alterations were observed in response to various environmental stresses, growth of P. aeruginosa in the presence of linolenic and arachidonic acid resulted in the assimilation of the fatty acids into membrane phospholipids. High performance liquid chromatography (HPLC) was performed to confirm new phospholipid profiles that had been observed by TLC. The potential survival advantages conferred by this phenomenon were examined by exposing P. aeruginosa to several physiologically relevant stresses such as acid stress, salt stress, and resistance to antimicrobial peptides. Preliminary results indicate that P. aeruginosa is gaining survival advantages due to its ability to incorporate exogenous fatty acids.
Dept of Biological and Environmental Sciences, The University of Tennessee at Chattanooga, Chattanooga, TN
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P174 • Lauren M. Dehnart1, Devanshi Patel1, Kevin M. Andres2, Virginia A. Young1
Isolation and Identification of Bacteria Surviving in the Presence of the Long-Term Antimicrobial Protect ’N Shield
This is a continuation of last year’s study of the efficacy of Protect ’N Shield on collegiate wrestling mats. There are two elements to this study; the first part served to monitor the colony forming units (CFUs) surviving on the wrestling mats throughout the year to validate last year’s study. We continued to sample the mats treated with Protect ’N Shield and the untreated mats both before and after mopping with Maxima 135, in an effort to monitor growth trends throughout the semester to test our previous conclusion that Protect ’N Shield should be applied twice a semester at minimum. The second part was investigative; bacteria surviving in the presence of Protect ’N Shield were isolated, identified, and tested for antimicrobial resistance. We selected colonies that visually resembled Staphylococcus species, and then performed biochemical tests to confirm the identity of the isolates. After identification, we tested for resistance to the cleaning agent Maxima 135 and to antibiotics to determine if the surviving bacteria represent a health concern to the wrestlers. The efficacy of Protect ’N Shield on the wrestling mats, the species identification of isolated colonies, and the results of drug resistance tests on the isolates will be discussed.
1 Dept of Biology, Mercer University, Macon, GA; 2 University Athletics, Ottawa University, Ottawa, KS
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P175 • Stephen E. Hayes, Michelle S. Thomas
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