1. Vesicle-Mediated Transfer of Antibiotic Resistance Between Klebsiella pneumoniae and Serratia marcescens Ondraya Espenshade Department of Biological Sciences, York College of Pennsylvania PROJECT SUMMARY Klebsiella pneumoniae and Serratia marcescens are among the most opportunistic pathogens and frequently encountered gram-negative organisms in nosocomial infections. Recent work has shown that gram-negative bacteria release membrane vesicles (MV), which contain proteins, lipopolysaccharides, phospholipids, RNA and DNA, from their surfaces during growth. MVs have been shown to transfer antibiotic resistance between different generas, such as E. coli and Salmonella. Because K. pneumoniae and S. marcescens both produce MVs and are usually resistant to the same treatments, the goal of our study is to determine if MVs represents an alternative means of genetic transfer between these bacteria. Bacteria will be transformed with a gentamicin resistance plasmid and vesicle production confirmed through EM. Vesicles will be isolated and combined with sensitive bacteria. We predict that resistant bacteria will be observed and the presence of the plasmid confirmed by PCR analysis. Resistance will be observed in both species confirming MV-mediated recombination, which may explain recent rises in antibiotic resistant bacterial infections. INTRODUCTION Several paths of bacteria antibiotic resistance have been characterized such as vertical gene transfer, horizontal gene transfer, conjugation, transformation, and transduction. Potential new path of resistance has been suggested involving membrane vesicles (MVs). Klebsiella pneumoniae and Serratia marcescens are gram-negative bacilli found in soil, water and are part of the normal intestinal flora in humans. K. pneumoniae is the second most frequently isolated colon-related bacterium in clinical laboratories and accounts for a large percentage of hospital- acquired infections. S. marcescens occurs predominantly in hospitalized patients that have undergone previous antibiotic treatment causing urinary tract infections in catheterized patients and septicemia. It can spread in hospital wards and transmitted by inhalation therapy equipment. Figure 2. Large arrows = lysed E.coli K-12 cells; Small arrows = MVs of P. aeruginosa. Predatory response of P. aeruginosa to E. coli K-12 cells. Bar = 1 mm. (Taken from Zusheng et al. 1998). Figure 3. PCR analysis of vesicles from E. coli O157:H7. Left lane, vesicles containing gfp; Right lane, purified pGFP. Confirms DNA present in vesicles. (Taken from Yaron et al. 2000). REVIEW OF LITERATURE MVs can be used as predatory response of Gram- negative to Gram-positive or other Gram-negative bacteria under low nutrient conditions (Figure 1 and 2; Zusheng et al. 1998). DNA shown in vesicles with PCR analysis using GFP primers (Figure 3; Yaron et al. 2000). DNA transfer confirmed using Vero cell cytotoxicity using E. coli and Salmonella supporting transfer of DNA between genera of bacteria (Yaron et al. 2000). OBJECTIVES To conduct a controlled experiment to determine if DNA is present in the MVs of K. pneumoniae and S. marcescens. To determine if DNA present in MVs can be transferred between K. pneumoniae and S. marcescens. To determine if DNA transferred between K. pneumoniae and S. marcescens can be expressed, indicating MVs represent an alternative means of genetic transfer between these bacteria. EXPECTED RESULTS Vesicles from both genera will be visualized using electron microscopy (Figure 1). PCR using specific primers will show gentamicin resistance plasmid presence in vesicles of both K. pneumoniae and S. marcescens. Gentamicin selection pressure of transformed cultures will confirm transfer and transcription of the gentamicin resistance plasmid. PCR of these cells will show presence of plasmid at 450 BP (Figure 4). Both sensitive cultures will result in transformed, gentamicin- resistant cultures confirming MV-mediated genetic transfer between generas. RESEARCH METHODS AND DESIGN K. pneumoniae cells will be transformed with gentamicin resistance plasmid (pcDNA3.1 plasmid) using heat shock and CaCl 2 method then placed under gentamicin selection pressure. MVs will be visualized using electron microscopy (EM) and isolated. DNase and Proteinase K will be added to remove any extracellular DNA or bacteriophages. PCR will be performed using specific primers amplifying the pcDNA3.1 plasmid and the presence of gentamicin resistance in vesicles will be determined. MVs from the transformed K. pneumoniae cells will be combined with S. marcescens sensitive cells in gentamicin selective broth culture containing DNase and Proteinase K. Cells that grow will be purified and PCR will be performed amplifying the pcDNA3.1 plasmid to further confirm if present in cells. Transformation Efficiency will be calculated then the procedure will be repeated using MVs of S. marcescens and K. pneumoniae cells. LITERATURE CITED Yaron, Sima, Glynis L. Kolling, Lee Simon, and Karl R. Matthews. 2000. Applied and Environmental Microbiology. 66:4414-4420. Zusheng, Li, Anthony J. Clarke and Terry J. Beveridge. 1998. Journal of Bacteriology. 180:5478-5483. Klebsiella pneumoniae. Available from: http://www.medinfo.ufl.edu/year2/mmid/bms5300/bugs/klebpn e.html Serratia marcescens. Available from: http://medinfo.ufl.edu/year2/mmid/bms5300/bugs/klebpne.ht ml Acknowledgements: Special thanks to Dr. Kaltreider for his endless guidance and support and thanks to Gary Milkovich for his continuous insight and help. EXPECTED TRANSFORMATION EFFICIENCY OF S. MARCESCENS AND K. PNEUMONIAE K. Pneumoniae K. pneumoniae S. marcescens S. marcescens to S. marcescens to K. pneumoniae 1x10 6 CFU 1x10 3 CFU 1x10 5 CFU 1x10 7 CFU Table 1. Colony Forming Units were calculated to determine vesicle- mediated transformation efficiency of S. marcescens and K. pneumoniae. S. marcescens is expected to have a higher transformation efficiency than K. pneumoniae. Figure: 450 bp Positive Control (pcDNA3.1 plasmid) Klebsiella to Serratia Serratia to Klebsiella KlebsiellaSerratia Negative Controls Figure 4. MV-mediated transformation. Positive control will be the pcDNA3.1 plasmid. It is expected there will be bands for the K. pneumoniae MVs added to the S. marcescens cells and vice-versa. Non- transformed K. pneumoniae and S. marcescens cells will be used as negative controls. Figure 1. Taken from Z. Li, A. Clarke and T. Beveridge. 1998. Taken from http://medinfo.ufl.edu/year2/mmid\ bms5300/bugs/klebpne.html