Transcript Multidrug resistance

Conjugative DNA
transfer, antibiotic
resistance and MDR
bacteria
With thanks to Steve Matson
Who first created this lecture
Antibiotics – a medical miracle
The discovery of antibiotics
changed the medical landscape
http://www.nature.com/nature/journal/v406/n6797
Bacterial infection as cause of
death plummeted

Life expectancy increased by 8 years
between 1944 and 1972
Deaths in Scotland due to infectious disease per 100,0000
www.gro-scotland.gov.uk
Bacterial infection as cause of
death plummeted

Life expectancy increased by 8 years
between 1944 and 1972
Deaths in Scotland due to TB per 100,0000
www.gro-scotland.gov.uk
The antibiotic resistance
problem

Drug resistant bacteria are very wide
spread occurring throughout the world
The antibiotic resistance
problem

Drug resistance happens quickly

One study observed an increase from
0% to 28% drug resistant E. coli in less
than 5 years
The antibiotic resistance
problem

In 2005 there were more deaths in the US from
Methicillin resistant Staphylococcus aureus
than from AIDS
HIV 17,011 deaths
MRSA Staph aureus 19,000 deaths
Stats from CDC
The antibiotic resistance
problem

85% of the cases of MRSA Staph were acquired in
hospitals or other health care settings
HIV 17.011 deaths
MRSA Staph aureus 19,000 deaths
You now know how
antibiotics work
And how drug resistant
bugs arise
evolution.berkeley.edu
And how drug resistant
bugs arise
evolution.berkeley.edu
And how drug resistant
bugs arise
evolution.berkeley.edu
And how drug resistant
bugs arise
evolution.berkeley.edu
How did that 1st streptomysin
resistant bug arise?

A simple error in DNA
replication that produced a
mutation




Occurs at low frequency
Mutation is on the
chromosome
Mutation affects either
ribosomal protein S12 or 16S
rRNA to produce streptomycin
resistance
Does not explain MDR bugs or
high rate of spread
How do we solve this puzzle?

We know that drug resistance spreads at
an alarming rate

Far too fast to be the result of single
mutations in the chromosome that arise
independently
How do we solve this puzzle?

We know that drug resistance spreads at
an alarming rate


Far too fast to be the result of single
mutations in the chromosome that arise
independently
We also know that bacteria become
resistant to more than a single drug

If this were the result of point mutations in
the chromosome the rate would be even
slower
The four waves of antibiotic resistance in Staph. aureus
Vancomycin resistant
There are many ways of
becoming drug resistant
Plasmids are a key to combining
them together in one bacterium
And plasmids are?
Plasmids are a key to combining
them together in one bacterium
A plasmid is an extra-chromosomal DNA molecule
separate from the chromosomal DNA which is
capable of replicating independently of the
chromosomal DNA. In many cases, it is circular
and double-stranded. Plasmids usually occur
naturally in bacteria, but are sometimes found in
eukaryotic organisms
To understand the rapid increase in multiple drug
resistant strains of bacteria there are two
questions we must answer.
1– how are plasmids rapidly
transferred in a bacterial
population?
 2 – how do plasmids encode
resistance to multiple drugs?

Bacterial conjugation




Driven by conjugative plasmids;
1st example =the fertility factor F
Mating only between cell with F
(F+) and cell without F (F–)
Transfer of information is oneway from donor to recipient
Cells must be in close cell-cell
contact for DNA transfer to occur
F Plasmid
William Hayes
• A 100 kb (single copy) with ~ 100 genes
– Replicates using host machinery
– Partitions to daughter cells
A selfish genetic element!
• Encodes pillin which assembles into pili allowing cell contact
• Only F+ cells have pili
• F+ inhibited from contacting other F+ cells
Here’s how it happens
• F+ donates single-stranded copy of F to F– cell (rolling circle)
• F- cell converted to F+ by replication of ssDNA
• F plasmid rapidly spreads through bacterial population
Bacterial conjugation is the
primary mechanism which
spreads antibiotic resistance
among bacterial populations
Pumping ssDNA
Let’s look at the machine
Tra I (H) = helicase
Tra Y (R)= nicks
donor DNA at oriT
and remains
covalently linked
during transfer
Tra D = links TraY to
Type 4 secretion
Machine = pillus
This machine can be a drug target
Look among existing drugs
for small molecules
that inhibit the Relaxase (R)
1 nM
10 nM
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7
These inhibit DNA transfer!
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7
Plasmid transfer provides a
drug target
Plasmid transfer provides
other drug targets
Plasmids that replicate in similar ways (top, red and blue)
compete for resources, and the losing plasmid
is lost from the bacterial cell.
J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404
Plasmid transfer
provides a drug target
An aminoglycoside that binds the small RNA causing plasmid incompatib
can mimic this natural process, causing elimination
of a drug-resistance plasmid (bottom, green).
J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404
Transposable Genetic
Elements are also key to
antibiotic resistance
What is a transposon?
Transposable Genetic
Elements are also key to
antibiotic resistance
Transposable genetic elements (transposons) =
DNA segments that can insert themselves
at one or more sites in a genome.
Remarkably, almost 50% of our chromosomes
consist of transposable elements
Composite versus simple Tns
Transposons can carry drug
resistance genes onto
“R plasmids”
The plasmid can then be
transferred to another
bacterium by conjugation!
How does transposition occur?
Our genome is filled with
transposons and their “fossils”
R plasmids can become
increasingly complex
through natural selection
Wow!
http://www.fbs.leeds.ac.uk/staff/profile.php?tag=ONeill_AJ
Research into this area is
key to combating TB and
other bacterial infections!
CDC