By: Mark Bryson

1. Identification of Staphylococcus aureus and Determination of Its Methicillin Resistance by Matrix-Assisted Laser Desorption/Ionization Time-of Flight(MALDI-TOF) Mass SpectrometryZongmin, Du, Ruifu Yang, Zhaobiao Guo, Yajun Song, Jin WangInstitute of Microbiology and Epidemeology. Beijing, China By: Mark Bryson

2. Overview Staphylococcus Aureus Bacteria Antibiotics/Penicillin β-Lactam Antibiotics Antibiotic Resistance MRSA Paper MALDI-TOF MS Future Conclusions

3. Staphylococcus Aureus • 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 • 1 micrometer in diameter. • Gram positive bacteria.

4. Gram Positive vs. Gram Negative

5. Staphylococcus Aureus • 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

6. Bacteria • Common Features of Bacteria • 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 • Coccus(Spherical) • Spiral • Rods Weigelt. John A. MRSA New York : Informa Healthcare, c2008.

7. Specific Diseases Caused by Staphylococcus Aureus • 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.

8. Virulence factors that help SA •  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.

9. Virulence factors that help SA • Superantigens • Cause over stimulation • TSST-1 Toxic Shock Syndrome Toxin • Cause the production of a large amount of T-cells • Large secretion causes the release of a protein called Interleukin-2(IL-2) • 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

10. Antibiotics • 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.

11. Penicillin Penicillin • 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. • Structure • Beta Lactam • Thiazolidine Ring • Acyl Side Chain

12. Penicillin • 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% • 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. • 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.

13. Beta-Lactam Antibiotics Class of Antibiotics that contain a β-lactam ring. β-lactam: Four membered ring Cyclic amide structure Common Antibiotics Cephalosporins Penicillins Carbapenems Methicilln Derivative of penicillin Methicillin introduced in 1959 MRSA appeared 2 years later Resistant SA have a PBP that does not bind Methicillin. Beta Lactam Structure Penicillin Methicillin

14. Resistance • “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

15. Mechanisms of Resistance to Drugs • 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 • Production of more target. Need more antibiotic.

16. Bacterial Resistance • 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. • Led to a search of penicillin derivatives not susceptible to β- lactamases. • Methicillin is not affected by β-Lactamases • Methicillin posses an acyl side chain that prevents hydrolysis.

17. Counteracting Resistance Amoxicillin Clavulanic Acid • Combination of Drugs • Augmentin • Approved in 1984 • Amoxicillin - β-Lactam antiobiotic • Clavulanic Acid - prevents β-lactamases from working • Acts via competitive inhibition and binds to the enzyme

18. MRSA Electron micrograph depicting a group of MRSA bacteria (CDC/Janice Carr) • 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 • 1. MRSA strains developed resistance from one resistant clone of SA. • 2. Acquired resistance from a source outside SA.

19. MRSA • Four Antibiotics Approved by the FDA for MRSA. • Vancomycin • Linezolid • Daptomycin • Tigecycline. Vancomycin Linezolid Daptomycin Tigecycline

20. MRSA Statistics • 2007 • 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 • 2/3 outside Hospital • 1/3 in Hospital.

21. Purpose of Study 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.

22. MALDI-TOF Alanine Tryptophan • Matrix-Assisted Laser Desorption/Ionization – Time of Flight (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: • 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

23. MALDI-TOF • 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.

24. MALDI-TOF

25. 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. Materials MALDI Matrixes 5-Chloro-2-mercaptobenzothiazole(CMBT) α-cyano-4-hydrocinnamic acid(α-cyano) Triflouroacetic acid(TFA) 18-crown-6 ether. Saturated solutions of CMBT and α-cyano dissolved in Acetonitrile/methanol/water (1,1,1 v/v/v) 0.1% formic acid and 0.01M 18-crown-6 ether. 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 Experiment 5-Chloro-2-mercaptobenzothiazole α-cyano-4-hydrocinnamic acid Triflouroacetic Acid 18-Crown-6-Ether

26. Instrument 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.

27. Results: MRSA and MSSA Spectra A = S. aureus methicillin-suceptible strain B = S. aureus methicillin-resistant strain. Differ in m/z 500-3500 Da.

28. 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

29. Results • Isolates 43,44, and 65 were analyzed three times at a 10-day interval under controlled experiment conditions. • No spectral differences between the three batches were observed. Overall Profiles were steady and reproducible.

30. Results (Polymerase Chain Reaction) PCR Tests 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 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

31. Results • S. aurues • 74% of the isolates were identified as S. aureus by MALDI-TOF MS • Nuc-based PCR test demonstrated that all 76 isolates in the study were S. aureus. • Methicillin Resistance • 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.

32. Results Cluster Analysis was performed with the aid of software to separate groups into MRSA and MSSA.

33. Future • 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 • 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

34. Conclusions 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.

35. Bibliography 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.

36. Thanks • Professor Powers • Dr. Lantz

37. Shin Wound • Photo taken August 2007 • 5 Stitches on the left shin • Antibiotic Treatment • Initially on Keflex. (Slow effect on the infection) • Beta Lactam Antibiotic • Cephalosporin • Switched to Trimethoprim after cultures results and determination of MRSA infection. • Antibiotic prescribed mainly for urinary tract infections • Prescribed for viral infections as well. Keflex Trimethoprim

38. MRSA in the Press GVSU Lanthorn: October 29th, 2007

39. MRSA in the Press Grand Rapids Press: Fall 2007

40. MRSA in the Press Grand Rapids Press: Fall 2008

41. GVSU Research http://www.gvsu.edu/s3/index.cfm?id=65B7671E-EA91-B8B2-1884433B7163A3A3

42. Final Note The future of our country is in your hands. Be sure to Vote Tomorrow!!!!

43. Questions???

44. Human Defenses 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.

45. Human Defenses 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.

46. 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.