Transcript MRSA - Mark Bryson

Identification of Staphylococcus
aureus and Determination of Its
Methicillin Resistance by MatrixAssisted Laser Desorption/Ionization
Time-of Flight(MALDI-TOF) Mass
Spectrometry
Zongmin, Du, Ruifu Yang, Zhaobiao Guo, Yajun
Song, Jin Wang
Institute of Microbiology and Epidemeology.
Beijing, China
By: Mark Bryson
Overview
Staphylococcus Aureus
 Bacteria
 Antibiotics/Penicillin
 β-Lactam Antibiotics
 Antibiotic Resistance
 MRSA
 Paper
 MALDI-TOF MS
 Future
 Conclusions
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Staphylococcus Aureus
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Common causes of bacterial infections.
1 of 32 species in the staphylococcus
genus.
First conclusively described by German
Physician Anton Rosenbach in 1884.
Non-motile bacterium that grows in
clusters like grapes.
Etymology
 “Staphyle” is Greek for bunch of grapes.
 “Cocci “- spherical bacteria.
 “Aureus” is Latin for gold.
• Added because it grows in large yellow
colonies
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1 micrometer in diameter.
Gram positive bacteria.
Gram Positive vs.
Gram Negative
Staphylococcus Aureus
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Lives on mucous membranes of skin.
Nasal membranes provide an ideal habitat.
Extremely durable. Can exist for years in a dormant state.
So resilient due to its large cell wall compared to other
bacterium.
Second leading cause of hospital acquired pneumonia
Resistant forms and normal forms typically attack people with
weakened immune systems
Results of Infection
• Causes superficial skin lesions, infection of hair follicle, sty (infection of
the eyelid)
• Boils - deep pus filled abscesses.
• Swimmer’s ear, middle ear infections, and urinary tract infections
Bacteria
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Common Features of Bacteria
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Free living prokaryotes - No nucleus
Surrounded by a Cell Wall
Plasma Membrane – permeable membrane.
Cytoplasm – contents of the cell
Ribosomes – build proteins
Three Types of Bacterial Shapes
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Coccus(Spherical)
Spiral
Rods
Weigelt. John A. MRSA New York : Informa Healthcare, c2008.
Specific Diseases Caused
by Staphylococcus Aureus
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Scalded Skin Syndrome (SSS)
 Rash
 Extremely Sensitive
 Skin looks like it is burned
Toxic Shock Syndrome (TSS)
 Produce a Toxin(TSST-1)
 Fever, rash, nausea, breathing difficulties.
Virulence factors that help
SA
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1. Attachment
 Binding of bacterial proteins on the host and invader.
 Like a ping pong ball covered with Velcro
2. Evasion of Host Defense
 Host produces fatty acids and lipids that attack bacteria
– SA produces lipases(destroy fatty acids)
 Proteases – enzymes released by SA that cut proteins
into small pieces.
 SA produces proteins that destroy antibodies
• Protein A, hides bacteria from antibody.
– Binds with wrong orientation
– Causes free antibodies to clump.
Virulence factors that help
SA
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Superantigens
– Cause over stimulation
– TSST-1 Toxic Shock Syndrome Toxin
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Cause the production of a large amount of T-cells
Large secretion causes the release of a protein called Interleukin-2(IL2)
Large concentrations of (IL-2) lead to fever, nausea, and vomiting.
– Can also cause cascading immune responses
– Damage cells that line blood vessels, respiratory distress and
multiple organ failure.
3. Tissue Invasion
– Bind and create holes in cell membrane
Antibiotics
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Antibiotics - Drugs that kill bacteria
Use of Antibiotics in the 20th Century was one of the
biggest success stories.
Natural flora = non disease causing bacteria
Antibiotics kill not only the bacteria but also the
natural flora.
All antibiotics lose their ability to kill bacteria
 The drugs don’t change but the populations of
bacteria that cause infections have changed in
subtle and important ways.
Penicillin
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In 1928 Sir Alexander Fleming spread
SA on agar plates. He accidentally left
one outside the lab.
A mold grew on the outside plate that
prevented growth of SA.
Caused by penicillin.
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Structure
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Beta Lactam
Thiazolidine Ring
Acyl Side Chain
Penicillin
Penicillin
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Before WWII most wartime death was caused by
bacterial infection. Wound infections.
Penicillin became available in 1940‘s and it was mass
produced because it revolutionized treatment. It was
hailed as a “miracle drug”
It lowered the death rate by over 80%
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How penicillin works.
 It disrupts the formation of the cell wall.
 Cell Wall = Amino Acids and sugars
 Prevents large building blocks from being
incorporated into the cell wall.
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Penicillin Binding Protiens (PBP) - are a group of
proteins that are characterized by their affinity for and
binding of penicillin. They are a normal constituent of
many bacteria. All beta lactam antibiotics bind to PBP
to have their effect of preventing cell wall construction
by the bacterium.
Beta-Lactam Antibiotics
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Class of Antibiotics that contain a β-lactam ring.
β-lactam: Four membered ring
Cyclic amide structure
Common Antibiotics
 Cephalosporins
 Penicillins
 Carbapenems
Beta Lactam Structure
Penicillin
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Methicilln
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Derivative of penicillin
Methicillin introduced in 1959
MRSA appeared 2 years later
Resistant SA have a PBP that does
not bind Methicillin.
Methicillin
Resistance
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“It is not difficult to make microbes resistant to penicillin by
exposing them to concentrations not sufficient to kill them and
the same has occasionally happened in the body. The time may
come when Penicillin can be bought in the shops. There is the
danger that the ignorant man may easily under dose himself by
exposing microbes to non lethal quantities of the drug and make
them resistant.”
-- Alexander Fleming
December 11th,1945
Nobel Laureate Speech
Mechanisms of Resistance to
Drugs
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Stop Drugs from binding
 Non-porous cell walls
 New PBP’s
 Random mutation of new PBP
Modify Drugs
 More than 250 B-Lactamases
Modify Target
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Production of more target. Need more antibiotic.
Bacterial Resistance
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Production of β-Lactamases
 Break apart the lactam ring of penicillin.
 Hydrolyze the amide bond
 Typically not a single enzyme but many closely
related β-lactamases.
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Led to a search of penicillin derivatives not
susceptible to β- lactamases.
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Methicillin is not affected by β-Lactamases
 Methicillin posses an acyl side chain that
prevents hydrolysis.
Counteracting Resistance
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Combination of Drugs
• Augmentin
• Approved in 1984
• Amoxicillin - β-Lactam antiobiotic
• Clavulanic Acid - prevents βlactamases from working
• Acts via competitive inhibition
and binds to the enzyme
Amoxicillin
Clavulanic Acid
MRSA
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MRSA - Methicillin-resistant Staphylococcus
aureus - a strain of Staphylococcus aureus that is
resistant to antibiotics. Specifically, beta-lactams
antibiotics, which includes penicillin's and its
derivatives.
MSSA - Methicillin-sensitivie(suceptible)
Staphylococcus aureus – the remainder of
Staphylococcus aureus that are not resistant to
antibiotic treatment.
The first documented outbreak of MRSA in the
United States occurred at Boston City Hospital in
1968.
Two theories on the development of MRSA
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1. MRSA strains developed resistance from one
resistant clone of SA.
2. Acquired resistance from a source outside SA.
Electron micrograph depicting a group of
MRSA bacteria (CDC/Janice Carr)
MRSA
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Four Antibiotics Approved by the FDA for MRSA.
 Vancomycin
 Linezolid
 Daptomycin
 Tigecycline.
Vancomycin
Linezolid
Daptomycin
Tigecycline
MRSA Statistics
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2007
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Approximately 32% (89.4 million persons) and 0.8% (2.3 million persons) of
the U.S. population is colonized with S. aureus and MRSA respectively.
Hospital
• The proportion of healthcare-associated staphylococcal infections that
are due to MRSA has been increasing:
• 2% of S. aureus infections in U.S. intensive-care units were MRSA
in 1974
• 22% in 1995,
• 64% in 2004.
• There are an estimated 292,000 hospitalizations with a diagnosis of S.
aureus infection annually in U.S. hospitals. Of these approximately
126,000 hospitalizations are related to MRSA.
Hospital vs. Community acquired MRSA (2005)
• 85% of all invasive MRSA infections were associated with healthcare
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2/3 outside Hospital
1/3 in Hospital.
Purpose of Study
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Evaluate the performance of MALDI-TOF MS in detecting
clinical isolates of S. Aureus and its methicillin resistance based
on a database search and software analysis.
Analyzed 76 Clinical Isolates of S. aureus
Methicilin resistance in S. aureus based on the spectral
differences between MRSA and MSSA.
Most commonly used methods that rely on assessment of
various bacterial metabolite activities are usually complicated
and time-consuming.
Hope to find a fast method that can be used to quickly diagnose
MRSA.
MALDI-TOF
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Matrix-Assisted Laser Desorption/Ionization – Time of Flight
Alanine
(MALDI - TOF)
Franz Hillenkamp and Michael Karas at the University of Munster in
Germany introduced MALDI as a new ionization technique in 1988.
 They found that the amino acid alanine could be ionized more
easily if it was mixed with the amino acid tryptophan and irradiated
with a pulsed 266 nm laser.
 The tryptophan was absorbing the laser energy and helping to
ionize the non-absorbing alanine.
Technique:
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Analytes are co-crystallized in an organic matrix.
Matrix
• Matrix is comprised of small molecules that are able to absorb light at a
characteristic wavelength.
• Protects the biomolecule from being destroyed by direct laser beam
• Facilitate vaporization and ionization
• Acidic
Tryptophan
MALDI-TOF
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Matrix is deposited onto the MALDI
Target.
 Target is typically metal
Solvent is Evaporated.
Crystallized surface is desorbed by
nano-second laser pulses.
Matrix and Analyte molecules are
transferred into the gas phase.
Analytes are ionized without
significant fragmentation.
Coupled in series with a Time-of
Flight(TOF) Mass Spectrometer.
• Measures time from ion
source to detector.
MALDI-TOF
Experiment
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5-Chloro-2-mercaptobenzothiazole
In a previous study, spectra obtained from Intact Cell Mass
Spectrometry(ICMS) of MSSA and MRSA were readily discriminated
in previous studies.
Suggested that MALDI-TOF MS has potential for MRSA
identification.
α-cyano-4-hydrocinnamic acid
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Materials
 MALDI Matrixes
• 5-Chloro-2-mercaptobenzothiazole(CMBT)
• α-cyano-4-hydrocinnamic acid(α-cyano)
• Triflouroacetic acid(TFA)
• 18-crown-6 ether.
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Saturated solutions of CMBT and α-cyano dissolved in
Triflouroacetic Acid
• Acetonitrile/methanol/water (1,1,1 v/v/v)
• 0.1% formic acid and 0.01M 18-crown-6 ether.
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Seven Peptides for instrument calibration. Externally calibrated
using a mixture of the peptides.
Calibrants were dissolved in 0.1% TFA/Water Solutions and mixed
with an equal volume of α-cyano matrix solution
18-Crown-6-Ether
Instrument
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MALDI-TOF MS equipped with a nitrogen laser
light (337 nm).
Mass Range: m/z 500-10,000 Da.
Accelerating voltage of 15 kV.
The instrument was supported by a software
system which had spectral profiles of numerous
strains of bacteria. Ability to compare databases
and add to the database.
Results: MRSA and MSSA
Spectra
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A = S. aureus methicillin-suceptible strain
B = S. aureus methicillin-resistant strain.
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Differ in m/z 500-3500 Da.
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Results
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Most peaks were in the m/z 800-3500 Da.
Sample Prep: 20 min per isolate.
Reproducibility
 12 replicates of each isolate
 Repetitious analysis of 3 isolates at 10-day intervals
Results
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Isolates 43,44, and 65 were analyzed three times at a 10-day interval under
controlled experiment conditions.
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No spectral differences between the three batches were observed.
Overall Profiles were steady and reproducible.
Results
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(Polymerase Chain Reaction) PCR Tests
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Nuc-Gene exists only in S. aureus. Used for specific detection of S. aureus.
• A nuc-based PCR test was used as a reference method for MALDI-TOF MS
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Mec-A gene is the genetic determinant of methicillin resistance in Staphylocci.
• Mec-based PCR assay was performed to confirm methicillin resistance in the
isolates.
• Mec-A gene is the gene that encodes an altered PBP
Results
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S. aurues
 74% of the isolates were identified as S. aureus by MALDITOF MS
 Nuc-based PCR test demonstrated that all 76 isolates in the
study were S. aureus.
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Methicillin Resistance
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33 out of the 76 strains were positive for Mec-A gene.
All were classified into MRSA group by MALDI-TOF MS.
7 Strains that were negative for MecA gene were classified
into MRSA group by MALDI-TOF MS.
Low Accuracy due to:
 Incomplete Database
 Sheep blood was used on agar plates rather than horse
blood.
Results
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Cluster Analysis was performed with the aid
of software to separate groups into MRSA
and MSSA.
Future
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Vaccines
Bacteriophages – viruses that infect bacteria
• Before the use of antibiotics phage treatment was used
• Advantages
• Kill both resistant and sensitive strains of bacteria
• Inexpensive
• Disadvantage
• If not purified they can make the patient sicker
• Have to pass FDA Standards
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Phage Enzymes
• Vincent A. Fischetti, Ph.D. Rockefeller University, New York
• After viral infection phages produce an enzyme that creates holes in the cell wall.
• Adv
• Highly specific
• Kill resistant and drug susceptible bacteria
• Does not involve infecting the patient with entire phage virus.
• Disadvantages
• Unstable
Conclusions
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The study demonstrated that MSSA and MRSA differed in the
spectral profiles in the m/z 500-3500 under the experimental
conditions.
MALDI-TOF MS of clinical isolates is a fast, simple and specific
method.
Highly Reproducible
Further Improvement in method is necessary.
 Different results between PCR and MALDI test.
 Condition differences may have caused the discrepancy in
categorization of the isolates in this study.
Bibliography
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Barrett FF, McGhee RF, Finland M: Methicillin-resistant Staphylococcus aureus at Boston city hospital. New
England Journal of Medicine 1968;279:448
Ciba Foundation. Antibiotic Resistance: Origins, Evolution, Selection, and Spread. England: John Wiley & Sons.
1997.
Cuevas, Carlos F. Amáblez. Antimicrobial Resistance in Bacteria. United Kingdom: Bioscience. 2007
Deurenberg RH, and Stobberingh EE. The Evolution of Staphylococcus Aureus. Infec Genet Evol. 29 Jul 2008. 12
Sep 2008.
Freeman-Cook, Lisa. Staphylococcus aureus infections. Philadelphia: Chelsea House Publishers, c2006.
Klevens RM et al. Clinical Infectious Diseases 2006;42:389-91)
Klevens, R. Monina, et al. Invasive Methicillin-Resistant Staphylococcus aureus Infections in the United States.
Journal of the American Medical Association. Chicago. 18 Oct 2007. 1 Oct 2008.
Kuehnert MJ et al. Emerging Infectious Diseases. 2005;11:868-72.)
Kuehnert MJ et al. Journal of Infectious Diseases. 2006;193:172-9.) Staphylococcal Disease Burden
Wax, Richard et Al. Bacterial Resistance to Antimicrobials, 2nd Edition. Boca Raton: Taylor and Francis Group.
2008.
Weigelt. John A. MRSA New York : Informa Healthcare, c2008.
Zongmin, Du, Yang, Ruifu, Song, Yajun, and Jin Wang. Identification of Staphylococcus and Determination of It’s
Methicillin Resistance by Matrix Assisted Laser Deportion Ionization Time of Flight Mass Spectrometry. Journal of
Analytical Chemistry, 2 Aug 2002. 12 Sep 2008.
MRSA in Healthcare Settings. Center for Disease Control. 3 Oct 2003. 19 Oct 2008
http://www.cdc.gov/ncidod/dhqp/ar_MRSA_spotlight_2006.html.
Thanks
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Professor Powers
Dr. Lantz
Shin Wound
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Photo taken August 2007
5 Stitches on the left shin
Antibiotic Treatment
 Initially on Keflex. (Slow effect on the infection)
Keflex
• Beta Lactam Antibiotic
• Cephalosporin
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Switched to Trimethoprim after cultures results and
determination of MRSA infection.
• Antibiotic prescribed mainly for urinary tract infections
• Prescribed for viral infections as well.
Trimethoprim
MRSA in the Press
GVSU Lanthorn:
October 29th, 2007
MRSA in the Press
Grand Rapids
Press: Fall 2007
MRSA in the Press
Grand Rapids Press:
Fall 2008
GVSU Research
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http://www.gvsu.edu/s3/index.cfm?id=65B7671E-EA91B8B2-1884433B7163A3A3
Final Note
The future of our country is in your hands. Be sure to Vote Tomorrow!!!!
Questions???
Human Defenses
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Antibodies are small proteins produced by the immune
system that coat or bind to foreign invaders. Therefore,
easier to recognize.
Complement binds to antibodies and bacteria.
Killed by white blood cells by phagycytosis.
After 4-7 days the white blood cells
move the bacteria to lymph nodes.
Human Defenses
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The skin produces oils that inhibit bacterial growth.
Saliva contains lysozyme, an enzyme that kills bacteria.
Bacteria inhaled are trapped in mucous and cilia and moved
toward the back of the mouth so that the bacteria are
swallowed.
The GI track is killer to bacteria.
Determination of
Resistance
- Disk Diffusion Assay – Used to determine resistance of
antibiotics
1. Small disks are soaked in different
antibiotics and then placed on a plate
containing bacteria.
2. If antibiotic kills the bacteria then there is no
growth around the small disks. If they are
resistant then uninhibited growth can occur.