Transcript S. marcescens - York College of Pennsylvania

Vesicle-Mediated Transfer of Antibiotic Resistance Between
Klebsiella pneumoniae and Serratia marcescens
Ondraya Espenshade
Department of Biological Sciences, York College of Pennsylvania
Taken from http://medinfo.ufl.edu/year2/mmid\
bms5300/bugs/klebpne.html
Figure 1. Taken from Z. Li, A. Clarke and T. Beveridge. 1998.
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.
REVIEW OF LITERATURE
RESEARCH METHODS AND DESIGN
• MVs can be used as predatory response of Gramnegative to Gram-positive or other Gram-negative
bacteria under low nutrient conditions (Figure 1 and 2;
Zusheng et al. 1998).
K. pneumoniae cells will be transformed with
• 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).
• 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).
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).
• 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.
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.
Figure:
(pcDNA3.1
plasmid)
Klebsiella
to Serratia
Serratia to
Klebsiella
Klebsiella
Serratia
450 bp
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. Nontransformed K. pneumoniae and S. marcescens cells will be used as
negative controls.
EXPECTED TRANSFORMATION EFFICIENCY OF
S. MARCESCENS AND K. PNEUMONIAE
K. Pneumoniae
K. pneumoniae
to S. marcescens
S. marcescens
to K. pneumoniae
1x106 CFU
1x103 CFU
1x105 CFU
S. marcescens
1x107 CFU
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.
• 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 hospitalacquired infections.
MVs will be visualized using electron microscopy
(EM) and isolated. DNase and Proteinase K will be
added to remove any extracellular DNA or
bacteriophages.
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.
INTRODUCTION
Positive Control
gentamicin resistance plasmid (pcDNA3.1 plasmid)
using heat shock and CaCl2 method then placed under
gentamicin selection pressure.
PCR will be performed using specific primers
amplifying the pcDNA3.1 plasmid and the presence of
gentamicin resistance in vesicles will be determined.
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).
Negative Controls
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.
Table 1. Colony Forming Units were calculated to determine vesiclemediated transformation efficiency of S. marcescens and K. pneumoniae.
S. marcescens is expected to have a higher transformation efficiency than
K. pneumoniae.
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.