Conjugative DNA transfer, antibiotic resistance and MDR bacteria

With thanks to Steve MatsonWho first created this lecture

Antibiotics – a medical miracle

The discovery of antibiotics

changed the medical landscape

http://www.nature.com/nature/journal/v406/n6797

Life expectancy increased by 8 years between 1944 and 1972

Bacterial infection as cause of death plummeted

www.gro-scotland.gov.uk

Deaths in Scotland due to infectious disease per 100,0000

Life expectancy increased by 8 years between 1944 and 1972

Bacterial infection as cause of death plummeted

www.gro-scotland.gov.ukDeaths in Scotland due to TB per 100,0000

The antibiotic resistance problem

Drug resistant bacteria are very wide spread occurring throughout the world

The antibiotic resistance problem

Drug resistance happens quicklyOne 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

MRSA Staph aureus 19,000 deaths

HIV 17.011 deaths

Stats from CDC

The antibiotic resistance problem

85% of the cases of MRSA Staph were acquired in hospitals or other health care settings

MRSA Staph aureus 19,000 deaths

HIV 17.011 deaths

How antibiotics work

evolution.berkeley.edu

How do drug resistant bugs arise?

evolution.berkeley.edu

How do drug resistant bugs arise?

evolution.berkeley.edu

How do drug resistant bugs arise?

evolution.berkeley.edu

How do drug resistant bugs arise?

How did that 1st drug 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 rateFar 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 rateFar 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 drugIf this were the result of point mutations in

the chromosome the rate would be even slower

Vancomycin resistant

The four waves of antibiotic resistance in Staph. aureus

There are many ways of becoming drug resistant

Plasmids are a key to combiningthem 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

Two questions

1– how are plasmids rapidly transferred in a bacterial population?

2 – how do plasmids encode resistance to multiple drugs?

To understand the rapid increase in multiple drug resistant strains of bacteria there are two questions we must answer.

Bacterial conjugation Driven by conjugative plasmids; 1st

example called the fertility factor or F found in some but not all E. coli one of several different types of

conjugative plasmid Mating only between cell with F

(F+) and cell without F (F–) Transfer of information is one-way

from donor to recipient Cells must be in close cell-cell

contact for DNA transfer to occur

F Plasmid

• A 100 kbp plasmid (single copy) with ~ 100 genes– Replicates inside host cell using host machinery for replication– Partitions to daughter cells in a manner similar to chromosome

William Hayes

F Plasmid

• Contains genes encoding synthesis of pillin which is assembled into pili that allow cell contact• F+ cells have pili and F- cells lack pilli• F+ inhibited from making contact with other F+ cells

F Plasmid

• F+ cells conjugate with F– cells– F+ donates single-stranded copy of F to F– cell (rolling circle)– F+ retains copy of plasmid, F- cell converted to F+ by replication of ssDNA donated to the F- cell– Allows F plasmid to rapidly spread through a bacterial population

Bacterial Conjugation

Bacterial conjugation is the primary mechanism used to spread antibiotic resistance among bacterial populations

There will be several million infections involving antibiotic resistant bacteria this year

This is now a very significant health problem

Pumping ssDNA

Pumping ssDNA

Tra I (H) = helicase

Tra Y (R)= nicks donor DNA at oriTand remains covalently linked during transfer

Tra D = links TraY to Type 4 secretion machine

This machine can be a drug target

Look among existing drugsfor small moleculesthat inhibit the Relaxase

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 incompatibility 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

A variety of colorful names have been used to describe these genetic elements Controlling elements Jumping genes Roving genes Mobile genetic elements Transposons

Definition: Transposable genetic elements (transposons) are DNA segments that can insert themselves at one or more sites in a genome. They are ubiquitous among organisms and play an important role in genome evolution.

Remarkably, almost 50% of our chromosomes consist of transposable elements We are still unsure of the normal genetic role,

if any, of these elements

Composite versus simple Tns

Composite Tns contain a variety of genes between two IS elements Transposase is encoded by one of the elements Individual IS elements cannot move

Simple Tn contains short IRs at each end Encode their own transposase and other genes

Transposons carry drug resistance genes onto plasmids called R plasmids

The plasmid can then be transferred to another bacterium by conjugation

How does transposition occur?

Transposition is catalyzed by an enzyme, transposase, encoded by the transposon

The ends of the transposon are critical for transposition

Our genome is filled with transposons and their “fossils” Human genome is typical in terms of abundance and

distribution of mobile elements How do we survive? Elements inserted into introns Vast majority of elements cannot move

There are instances of mutations caused by mobile elements

R plasmids can become increasingly complex through natural selection

http://www.fbs.leeds.ac.uk/staff/profile.php?tag=ONeill_AJ

Integrases can move DNA flanked by direct repeatsFrom plasmids to chromosome and back

CDC

Research into this area iskey to combating TB andother bacterial infections!