1. Methicillin Resistant Staphylococcus Aureus
Barbara Jennings-Spring
Seminar in Molecular Biology 360
Smith College

2. What Is MRSA? MRSA Is “Methicillin Resistant Staph Aureus”
*It is a bacteria that is resistant to a synthetic penicillin methicillin. Staph aureus colonizes skin, nasal passages, and many other mucous membranes.
*Also causes a variety of disseminated, lethal infections in humans.
* It has the ability to transfer resistant genes easily to other species directly and indirectly
*Overuse of antibiotics is the one of the major reasons for the evolution of MRSA

3. Overview
To gain a better understanding of the molecular mechanisms involved with (MRSA) and how biotechnology continues to combat this super-bug in hospitals and communities throughout the world.

4. Research The Basic Biology Of Staphylococcus Aureus
History Of MRSA
The Basic Biology Of Staphylococcus Aureus
Molecular Basis For Virulence
Clinical Presentation Of Disease
Detection Of Pathogen
Biotechnology Treatments
Public Health Control Strategies
Political And Social Impediments
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5. History Of Antibiotic Resistance
1941 Penicillin
1943 Streptomycin
1945 Cephalosporins
1950 Tetracycline
1952 Erythromycin
1956 Vancomycin
1960 Methicillin
1962 Lincomycin
1962 Quinolones
1970 Penems
1980 Monobactams
2010 Is this the end of an antibiotic era???
Staphylococcus Aureus (Staph A.) developed resistance quickly after WW ll. In the beginning this was not of great concern because there were many pharmaceutical companies developing new antibiotics. But, by the late 1950’s, concern arose from the fact that there was only one drug left called vancomycin. Other concern about vancomycin was from the fact that it has very unpleasant side effects and is potentially toxic to the host’s cells. Serendipitously, the 1960 breakthrough of methicillin occurred (beta-lactamase stable penicillin) and so concern faded. Beta lactamase confers resistance to penicillin. Not surprisingly, MRSA first appeared in 1961 in the United Kingdom only after one-two years from the first introduction to clinical practice. Then in the late 1970’s and 80’s MRSA emerged in Australia. It remains one of the most significant nosocomial pathogens to date. Something to note from this table is that the development of new antibiotics has decreased significantly, especially in the past 20 years because of the cost as well as the ability of bacterial species to out-maneuver biotechnology in such a short period of time [1,2].

6. The Basic Phenotypic Characteristics Of Methicillin Resistant Staphylococcus Aureus
Gram positive
Non-motile
Spherical
Grows in chains
Resembles clumps of grapes
Golden color
Hemolytic pattern on blood agar
Produces coagulase and catalase enzymes
Staphylococci are gram positive, non-motile, and perfectly spherical, measuring approximately 1 micrometer. Because Staph A grow in chains and resemble bunches of grapes, their unusual name is derived from the greek staphyle, meaning “‘bunch of grapes” when observed microscopically in stained specimens taken from colonies grown on blood agar. Staph a. strains generally grow a hemolytic pattern and also display golden color of colonies when grown on agar aerobically[3].

7. Microevolutionary changes-a single point mutation
The molecular genetics of antimicrobial resistance includes three main pathways:
Microevolutionary changes-a single point mutation
Macroevolutionary change- rearrangements occur
Acquisition of foreign DNA
The rise in MRSA and the pathogenesis of this important pathogen is multifactorial. It would be difficult to pin-point any precise role, however; MRSA strain survive very well because they can express a wide array of potential virulence factors by different mechanisms.
The main topics that will be discussed for the large proportion of the paper will be the molecular genetics of antimicrobial resistance in MRSA and mechanisms of antimicrobial resistance, and the virulence factors. The molecular genetics topic will include point mutations, rearrangements, and acquisition of foreign DNA. Then, I will discuss mechanisms of antimicrobial resistance, describing cell wall constituents, the production of enzymes and toxins that promote tissue invasion, and other mechanisms. Examples of these pathways will follow.

8. What are some examples of how single point mutations occur over time?
Beta lactamases will confer resistance to B- lactams (penicillins, cephalosporins)
If you get a single point mutation (substitution of base pair) that involves the target action of PCN or the cephalosporin drug, you will extend the spectum of action of that B-lactamase enzyme
so that a broad range of the cephalosporin or penicillin family will show resistance instead of one single cephalosporin or penicillin family
Mutations on the rpoB gene (RNA polymerase) alters antibiotic binding site, preventing drug action.
B- lactamases will confer resistance to B lactams to antibiotics (e.g penicillin and cepahalosporins) if you get a point mutation that involves the target of action of B-lactamases, a bacteria can and will extend the spectrum of action so that it confers not just to one cephalosporin but the whole cephalosporin family. RNA Polymerase (enzyme) mutation can alter the shape of the binding site through a conformational change, thus preventing antibiotic. activity Rifampin is a semisynthetic derivative of rifamycin and is used in many types of bacterial infections including MRSA[3].

9. What Are Some Examples Of Macro Evolutionary Changes With Resistance?
Rearrangement of DNA segments-by transposons
Transposon—”Jumping Gene” that contributes to antibiotic resistance
Transposons- Contain genes that are for the coding of antibiotic resistance
A macroevolutionary change is an example of rearrangements of certain parts of the DNA through transposons. Transposons are within the cytoplasm of the cell and are literally called “jumping genes”. They are small pieces of protein containig small DNA that encodes certain enzymes to transpose transposn so that it moves from one DNA location to another. Transposons carry antibiotic resistance genes that insert themselves behind one of thef promoter proteins so that they can become active in the resistance mechanism. At the same time, transposons have the innate ability to literally cut themselves out of the bacterial nucleoid or plasmid to the contribute to antibiotic resistance among a population of bacteria or move to other populations.[3]

10. How is the Acquisition of Foreign DNA from other species accomplished?
Conjugative plasmids: Found inside cytoplasm.
Possess the ability to transfer resistance genes to the same and different species
Horizontal gene transfer is common in bacteria and is accomplished by the process known as Transformation in Staph. aureus.
Conjugate plasmids are found inside the cytoplasm and have the ability to transfer resistance genes between the same and different species. For example: Between gram positive to gram negative species. Plasmids are able to acquire a number of different genes to carry out this function of resistance through a process called conjugation. [6].

11. Examples of horizontal Gene Transfers (HGT)
MRSA uses the mechanism of transformation and plasmid conjugation to accomplish horizontal gene transfer[4]

12. How Staph Aureus acquires resistance to methicillin
This schematic diagram from Journal of clinical investigation illustrates how easy it is for Staph. aureus acquires resistance to methicillin and its ability to express different virulence factors. The bacterium express surface proteins called adhesins and WTA. They also secrete many toxins and enzymes by activation of the chromosomal genes. The mechanism of resistance is actually acquired by the insertion of a horizontally transferred element called SCCmec. There are at least five different SCCmec elements that integrate at the same time site in the chromosome by a mechanism involving site-specific recombination. The mecA gene encodes a new b-lactam-insensitive to penicillin[5]

13. Mechanism of Antibiotic Resistance in MRSA
The principal mechanism for rapid spread of antibiotic-resistance genes through different bacterial populations are the genes that are collected on plasmids because they are independently replicated within and passed between bacterial species. Once inside, they get segregated with by tranposons that actively cut themselves out of one DNA location and literally jump into other locations. They are rather promiscuous about moving their antibiotic-conferring genetic baggage![4] Now that we know a little about exchange of foreign DNA amongst different groups of bacteria, I would like to move on to discussing other components of virulence factors such as: The extracellular enzymes, toxins, cell wall and surface. In detail, I will describe how each constituent contributes to resistance of the super bug “MRSA[6].

14. Important Virulence Factors for MRSA: Cell Wall Structures
Cytoplasmic membrane- osmotic barrier
Consists of thick polysaccharide capsule (slime layer; adhesin). Capsules are just tricks to avoid host defenses
Petidoglycan-Used for osmotic stability so bacterial cell wall does not burst due to hypertonic states.
Protein A- immunological disguise. Inhibits oponization , Binds IG’s, leukocyte, chemoattractant, anticomplementary.
Techoic Acid-Acts as a receptor for bacteriophages. Attachment site for mucosal cell receptors.
The cell wall consists of a cytoplasmic membrane that is responsible for osmotic regulation of ion content. Regulates transportation of ions in and out of cell down their concentration gradient. The cell wall consists of a thick polysaccharide capsule. The capsule inhibits phagocytosis, prevents quick disposal of bacterium by white blood cells. A subsequent loss of capsule typically causes loss infection by leaking of bacterial machinery[7]. The peptidoglycan allows the bacteria to attach host’s cell membrane and resist extreme environments. Protein A binds antibodies IgG1, IgG2, IgG4 fc receptors makes the bacteria resistant to phagocytosis. Protein A Inhibits oponization. Oponization is called enhancement attachment. The complement proteins C3b and C4b are known oponosins because they bind to phagocytes. One portion of the molecule binds to some strategically placed microbial proteins while other binds to receptors protein on the phagocytes. In this way, microbes will be engulfed by phagocytes more effectively, but the protein A inhibits this mechanism, Protein A is also leukocyte chemoattractant and anti-complementary. Techoic acids regulate the cation concentration at the cytoplasm, but they also play a much larger role in blocking pathogen associated molecular patterns. In order to prevent infection, initially, the body must be able to recognize the invading organism. They do this by pathogen associated molecular patterns, as described earlier. The molecules unique to gram positive cocci are teichoic acids and glycopeptides cell wall fragements that bind to host’s pattern recognition receptors on a variety defensive cells (bacteriophages, macrophages, and neutrophils) in the body. This results in cytokine response, sometimes too much which leads to other problems that will be discussed later [3,6,].

15. Invasive enzymes As Other Virulence Factors
Coagulase Complex-Produces enzymes that coagulate blood and seal off infection
Protease, lipase, & DNase provide nourishment for MRSA bacterium
FAME-Important in abscess formation. Also it could change anti-bacterial lipids and prolong survival of MRSA in wound.
Staphylokinase-Plasminogen activator enzyme that lyses fibrin clots
Hyaluronidase-It is the spreading factor; hydrolyzes haluronic acid in synovial joints
MRSA has specific enzymes that allow the bacteria to destroy and penetrate host tissues. Coagulase is an extra cellular protein that binds to prothrombin in the host to from this mass of a complex called staphylthrombin. The Coagulase complex is traditional marker for identifying S. aureus in the lab. Also known as the ‘clumping factor” this coagulates the blood and the result is that WBC’s and other body defenses are unable to reach the site of infection. S. aureus typically remains localized or “walled off’ from defenses, so this produces many scary types of localized infections that will be talked about later. S. aureus also express proteases, lipases, Dioxynuclease (DNase), and fatty acid enzyme known as FAME. According to the textbook of bacteriology(2005), the first three provide nutrients for the MRSA bacteria. Fame has been implicated in the formation of abscesses. Staphylokinase also known as fibinolysin dissolves fibrin clots by promoting the coversion of plasminogen to the fibrolytic enzyme plasmin (Phage conversion). Hyalurondase( ajoint lubricant) is the spreading factor present in intracellular ground substances of connective tissue leading to their destruction. Finally, nucleases act on DNA and RNA )[3,4 ].

16. Virulence Factors Cont: Extracellular Products and Toxins (hemolysins) Of MRSA Invasion
Leukocidins-The name implies their job! Leukocidins kill wbc’s. Membrane damaging toxin
Alpha, beta, delta toxin-binds to cell surface, forms pores leaks.
Superantigens: (type 1 toxin) Toxic shock syndrome toxin (TSST-1)
Staphylococcal Enterotoxin-food poisoning
Exfoliation toxin-scalded skin syndrome
All of these described above are membrane damaging toxins. Leukocydins kill wbc’s and are in MRSA.The accumulation of pus at the infected site is caused by the dead-lysed wbc’s. The Type 3 a-b toxins interfere with host cell function. The alpha toxin (a-toxin) expression involves hemolysin. Is described as a monomer that binds to the membrane of susceptible host cell. The subunits oligomerize to from heptameric rings with a central pore that causes the cellular contents of host cell to leak. B toxin hydrolyzes membrane lipids. Beta D-hemolysin has detergent properties. G toxin-mechanism unknown according to the literature, but a good guess is cytotoxicity. Superantigens can be classified as a sort of hybrid bacterial toxin that interacts with a rather large amount of killer T-4 lymphocytes. The activation of these T-4’s causes an excessive amount of cytokine release, inducing autoimmune phenomena. The clinical manifestations of these toxins will be discussed later[4].

17. Virulence Factors Cont: Mechanism Of Superantigens And The Stimulation Of Cytokine Release
Figure from textbookofbacteriology.net of superantigens and the non-specific stimulation of T cells. Superantigens binding directly to class II major histocompatibility complexes (MHC II) of antigen-presenting cells outside the normal  antigen-binding groove. Up to one in five T cells may be activated. Cytokines are released in large amounts, causing the symptoms of toxic shock[3].

18. Summary of Virulence Determinants Of Staph. Aureus
MRSA expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) Figure taken from textbook of bacteiology showing invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); (3) surface factors that inhibit phagocytic engulfment (capsule, Protein A); (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); (5) immunological disguises (Protein A, coagulase, clotting factor); and (6) membrane-damaging toxins that lyse cell membranes (hemolysins, leukotoxin, leukocidin; (7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G, TSST, ET (8) inherent and acquired resistance to antimicrobial agents[2].

19. Source of MRSA Infections
Some infections are caused by own epithelial flora-self contamination
Nasal carriage most common
Hospitals
*Dirty hands, towels, and daycare
Airborne?????

20. Predisposing Factors Of Host Resistance
Integument injury via surgery
Burns and trauma
Foreign objects like indwelling catheters, metals, sutures, implants
A history of chronic bacterial infections with multiple rounds of antibiotics
Hormonal changes and stress
Immunocompromised (AIDS, Diabetes, Chemo)

21. Clinical Manifestations Of MRSA
The lesion usually starts out as an small cut or break in the skin. The lesions can range from small abrasions to large, gaping abscesses
Even the most benign localized abrasion (from tampon insertion) can become the fuel for a devastating, disseminated MRSA systemic infection that do not respond to multi-antibiotic combinations
Vancomycin is last resort and sometimes that does not work as well when the body is overwhelmed with toxins and sepsis, however, we have some newer, less toxic antibiotics available now that I will discuss later.

22. MRSA Infections Go Everywhere
Integumental and soft tissue
Suppurative arthritis-first causative agent
Osteomyelitis-First causative agent
Bacteremia-First causative agent
Pneumonia
Acute and chronic Endocarditis-#1
Bacterial Meningitis-first causative agent
Integumental and soft tissue folliculitis, boils, post op wound infections, central venous lines entry, mammary abscesses very common from chapped skin and stress of childbirth lowering the immune system defenses.
suppurative arthritis Acute inflammation of the synovial membranes accompanied by purulent effusion into a joint and caused by bacterial infection. Also called purulent synovitis, pyarthrosis.

23. Menstrual Toxic Shock By MRSA
Most major organs fail with disseminated MRSA (TSS-1)
Unfortunate systemic invasion
cashnightmare.htm

24. More MRSA Infections
Toxic shock Syndrome-Super absorbent tampons- #1 causative reason
Urinary tract Infections
Scalded skin syndrome and impetigo from picking pimples
Food poisoning-Enterotoxin A in spoiled or contaminated food.
Urinary tract infections-hemmaglutinin proteins and surface proteins mediate attachment to urinary tract epithelium

25. Carbuncle(Boil)
tahilla.typepad.com/.../ super_bug_ jpg

26. 44 y/o IV drug abuser with back pain and Staph Osteomyelitis of lumbar spine.
Staph osteomyelitis and discitis involving L5, with extension across the L4-5 disc to erode L4 and extension into S1. The L5 vertebral body is destroyed. (33.210, , diskitis)
Case 72

27. Classic Toxic Shock: Scalded Skin Syndrome
Toxic Shock Syndrome and Scalded Skin Syndrome According to Mckinnon et al. ( 2005) ”Staphylococcus aureus is the organism responsible for classic toxic shock syndrome and scalded skin syndrome. Toxic shock syndrome can present with hypotension, erythema, fever and multisystem dysfunction. Most cases of nonmenstrual toxic shock syndrome occur in the postoperative setting” /804.html
/804.html

28. Staph. Aureus Impetiigo
Don’t pick your face!!!!!
fox/staph-impetigo.jpg

29. Getting A Lab Diagnosis For MRSA
FIG .from Journal of Clinical Microbiology, June 2000, p , Vol. 38, No / Comparisons of different agar media showing various reactions of 18 selected Staphylococcus isolates and one CoNS isolate mixed with enterococci. (A) CHROMagar Staph aureus with no antibiotic supplementation showing pink-to-red reactions for all S. aureus isolates. Row 4 spot 1 shows a blue color reaction for CoNS mixed with enterococci. Row 5 spots 2 and 3 show a white color for CoNS. (B) MSA showing yellow positive reactions and pink-to-red negative reactions of isolates. (C) CHROMagar Staph aureus supplemented with oxacillin showing the oxacillin (methicillin) resistance of isolates. The color of MRSA isolates is pink to red. (D) DNase agar showing clearing around positive isolates. As described above,MRSA is detected by the ability to grow on agar medium containing micrograms of oxacillin. The lack of growth of Methicillin suseptible Staph is shown on Agar C
MRSA

30. How Accurate Can Your Diagnosis Of MRSA Be?
TABLE from Journal of Clinical Microbiology, June 2000 shows comparison of the levels of accuracy of reactions of S. aureus isolates on CHROMagar Staph aureus, DNase, and MSA and of coagulase testing of CoNS after 18 to 24 h of incubation. As shown above, it appears that the traditional coagulase method for detecting staph is as specific as the chromgar with both of them having 100% specificity[8]

31. Current Drug Treatments For MRSA
[Methicillin-resistant] –MRSA Drugs of Choice: 
Linezolid(protein synthesis inhibitor), Daptomycin,
Vancomycin
Alternatives:  Synercid, Rifampin
Third-Line agents:  TMP-SMX
Antibiotics generally work in 5 ways: Inhibition of nucleic acids- Chloroquine, Rifampin. Inhibition of protein synthesis- tetracyclines, linezolid, and chloraphenicol. Action on cell membrane-polymoxin,Penicillin; vancomycin Interfernce with enzyme mechanism- sulfa drugs
The use of Vancomycin is parenteral only. Toxic effects must be monitored. Linzezolid is available parenteral and orally. Resistant strains are already developing. But, the dual formulation is the major advantage because outside hospital treatment is much more likely to cure infection. The drug has 100% bioavailability and that enables a physician to get equivalent serum levels both orally and parenterally. Daptomycin newly approved for treating skin and wound ulcers of diabetics. Highly bacteriacidal, although no oral therapy.[10]

32. Drugs In Development Oritavancin-can be given once daily
Tigecyclin-orally broad antimicrobial activity
Dalbavancin- Currently undergoing clinical trials. Has long half-life so it can be given once per week
(FDA, 2005)

33. How Close Is Staph Vaccine?
Pretty close. The results of the phase 3 testing of the vaccine (Staph VAX) will be presented soon according to the NIH.
The vaccine was created at the National institutes Of health and is one of several vaccines in development, however, no other products have reached the advance clinical trials like Staph VAX [11]

34. Public Health Response-What Is Being Done To Combat MRSA?
The CDC provides technical help and referrals to state and local health departments, doctors, nurses, and other professionals
The CDC provides national program of surveillance for serious infections with MRSA.
CDC launched evidence-based educational campaign to prevent antimicrobial resistance
CDC building national resource library to identify genetic patterns or relationships
CDC researching the role of staph toxins-to provide answers for hospitals and researchers
*For more info go to

35. What can you do to prevent MRSA from attacking You?
Keep draining infections of skin, covered with clean dry bandages
Talk to your physician about wound management techniques
Advise family to wash hands frequently with soap and water, count to at least “20”,especially after dressing a gaping wound.
Avoid sharing personal items such as towel, razors, bed linens with people who have sores or have come home from the hospital recently
Wipe objects down with alcohol.
If you are in the hospital please advise you nurse or physician to wash their hands before touching you or your hospital equipment

36. What Are The Social And Political Costs To Us?
Overall, infectious diseases remain the third leading cause of death in the United States each year. Infectious diseases are approximately the second leading cause of death worldwide (Emerging infections, 2005) As shown in the figure. Of the estimated 57 million deaths that occur throughout the world each year, ≈15 million, >25%, are directly caused by infectious diseases. Millions more deaths are due to secondary effects of infections!

37. The Real Cost Of Infectious Diseases Like MRSA
Leading causes of disability-adjusted life years (DALYs) due to infectious and parasitic diseases (2002 estimates). Lower respiratory infections, HIV/AIDS, diarrheal diseases, and malaria are among the infectious diseases that contribute to the most DALYs each year throughout the world.[13]

38. Rising Rates Of Resistant Bacterial Infections=Rising Budget
Figure taken from www. medscape.com is Budget of the National Institute for Allergy and Infectious Disease (NIAID), FY The overall NIAID budget rose from $1.06 billion in FY1994 to $4.4 billion (estimated) in FY2005. Funding for emerging infectious diseases rose from $47.2 million in FY1994 to $1.74 billion in FY2005 (est.)[13].     

39. That’s All Folks!! Any Questions????
Staph cells attaching photo courtesy of Dr. Sharon peacock- University of Oxford

40. References
1 Mitchell, David.MRSA.”what’s New”. Inoculum. Volume 8, number 2 (1999)
2 textbookofbacteriology.net/resantimicrobial.html
3 healthsciences.columbia.edu/ dept/ps/2007/mid/2006/transcript_02_mid22.pdf 4
5. Foster, Timothy. The staphylococcus aureus “superbug”.J. clin Ivestigation
Volume number114 (2004) 6.
7. ww.ncbi.nlm.nih.gov.
/804.html
9. Journal of Clinical Microbiology, June 2000, p , Vol. 38, No /
10. (FDA archives)
13.