Methicillin Resistant Staphylococcus Aureus

Barbara Jennings-Spring Seminar in Molecular Biology 360Smith College

What Is MRSA? MRSA is Methicillin Resistant Staphylococcus aureus

Is a bacteria that is resistant to a synthetic penicillin- methicillin.

MRSA causes a variety of disseminated, lethal infections in humans.

Has the ability to easily transfer resistant genes to other species directly and indirectly

Overuse of antibiotics imposes selective pressures which mediates the acquisition of resistance

Objective To gain a broader understanding of the resistance mechanisms and virulence factors involved with MRSA and how this disease impacts on a physical and global level

Research

History of MRSAThe basic Biology of Staphylococcus aureusMolecular Basis For Virulence factors And ResistanceClinical Presentation Of DiseaseDetection Of pathogenBiotechnology Treatments Public Health StrategiesPolitical And Social Consequences

A Timeline Of Antibiotic Resistance 1941 Penicillin1943 Streptomycin1945 Cephalosporins 1950 Tetracycline1952 Erythromycin1956 Vancomycin

1960 Methicillin1962 Lincomycin1962 Quinolones

1970 Penems

1980 Monobactams2010 Could this be the end of an antibiotic era???

History Of S aureus Resistance

The Basic Characteristics Of S aureusGram positiveNon-motileSphericalGrows in chainsResembles clumps of grapesGolden colorHemolytic pattern on blood agarProduces coagulase and catalase enzymes

img/staph_em.jpg www.aic.cuhk.edu.hk/ web8/mrsa.htm

Mechanism Of Resistancehttp://www.jci.org/cgi/content/full/114/12/1693/F1http://www.jci.org/cgi/content/full/114/12/1693/F1

Horizontal Gene Transfer-Another Mechanism For Resistancehttp://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs

Summary of Virulence Determinants Of Staphylococcus aureushttp://textbookofbacteriology.net/staph.htmlhttp://textbookofbacteriology.net/staph.html

Virulence Factors: Avoiding Host Defenses

Cell Wall

Cytoplasmic membrane- Osmotic barrier prevents disequilibrium of ionic content. Preventing cell osmotic instability and susceptibility to lysis

Polysaccharide capsule-slime layer; adhesin. Inhibits phagocytosis

Petidoglycan-Allows bacteria to attach hosts cell membranes

Protein A- Immunological disguise.

Invasive enzymesCoagulase Complex-Seals off infection, preventing phagocytic engulfment

Protease, lipase, & DNase provide nourishment for MRSA bacterium

FAME-(Fatty acid modifying enzyme) modifies the anti-bacterial lipids side chain-inactivating antibiotic action

Staphylokinase-Fibrinolysisn aids the in spreading factor

Hyaluronidase- Destroys connective tissue

Damage To The Host: Extracellular Products Leukocidins-Kills White blood cells (WBCS)

Alpha, Beta, Delta toxins-These damaging toxins bid to to cell wall surface, forms a pore, and cellular machinery of host cell leak out

Source Of MRSA InfectionsSome infections are caused by own epithelial flora-self contaminationNasal carriage most commonHospitals*Dirty hands, towels, and daycareAirborne?????Community

Predisposing Factors Of SusceptibilityIntegument injuryBurns and traumaForeign objectsA history of chronic InfectionsHormonal changes and stressImmunocompromised

Clinical Manifestations Of MRSA A localized, superficial abscess or

Invasion of lymphatics, blood, and major organs

Superficial Infections

Scalded Skin Syndrome: Classic Toxic Shock

www.aafp.org/afp/ 20000815/804.html

S. aureus Impetigowww.med.sc.edu:85/ fox/staph-impetigo.jpg

Systemic S aureus In the Lower spine.

Systemic Menstrual Toxic Shock By MRSAMost major organs fail with disseminated MRSA (TSS-1)www.web.net/terrafemme/ cashnightmare.htm

How Accurate Can Your Diagnosis Of MRSA Be?http://jcm.asm.org/cgi/content/full/38/6/2378

TABLE 1. 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 18to 24h ofincubation

Identification method

% of isolates showing positive reactions

Accuracy of medium in discriminating S.aureus and CoNS

S. aureus (n=114)

CoNS (n=22)

Sensitivity (%)

Specificity (%)

CHROMagar Staph aureus

100

0a

100

100

DNase

98.0

4.6

98.0

95.4

MSA

98.0

36.5

98.0

63.5

Coagulaseb

100

0

100

100

a S. chromogenes produced a natural carotenoid (orange or red) pigment and gave a slightly pink color. The isolate was identified by API20 Staph.

b All coagulase testing was confirmed by the standard tube method.

Biotechnology: Current Drug Treatments For MRSA MRSA Drugs of Choice: Linezolid-Protein synthesis inhibitor Daptomycin-Causes membrane depolarization in bacteria-so no membrane transportVancomycin-Acts by interfering with the construction of cell wall. Still works well with other antibioticsAlternatives: Synercid, RifampinThird-Line agents: TMP-SMX (Sulfa)

Biotechnology: Drugs In DevelopmentOritavancin-Binds to normal cell wall precursorsTigecyclin-Works on efflux pumpsDalbavancin- Bacteriacidal

Biotechnology: A Novel Vaccination For S Aureus

Development of StaphVAX, a polysaccharide conjugate vaccine against S. aureus infections

The results of the phase 3 clinical trials of the vaccine (Staph VAX) will be presented 2006 according to the NIH.

Public Health Response and CDC Technical help for healthcare professionals

National program of surveillance

Evidence-based educational campaigns

National resource library

Researching S. aureus toxins

More info? Go to www.cdc.goc (CDC,2005)

PreventionDraining infections must be kept covered

Talk to your physician about wound management techniques

Wash hands frequently with soap and water

Avoid sharing personal items

Wipe objects down with alcohol.

Advise health care workers to wash their hands before touching you or your hospital equipment

The Real Cost Of Infectious Diseases

Rising Rates Of Resistant Bacterial Infections=Rising Budget

SummaryMultiple MRSA isolates are circulating in your local hospital and communityMRSA has many mechanisms resistance and virulence factorsMecA gene is the gene responsible for methicillin resistanceMany of the MRSA isolates are encoded with the Sccmec mobile element in themMRSA must be isolated and treated aggressively to prevent secondary infections and spread

Thats All Folks!! Any Questions????Staph cells attaching photo courtesy of Dr. Sharon Peacock- University of Oxford

References

1 Mitchell, David.MRSA.whats New. Inoculum. Volume 8, number 2 (1999) 1-12. 2 textbookofbacteriology.net/resantimicrobial.html 3 healthsciences.columbia.edu/ dept/ps/2007/mid/2006/transcript_02_mid22.pdf 4 http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs 5. Foster, Timothy. The staphylococcus aureus superbug.J. clin IvestigationVolume number114 (2004) 1693-1696. 6. www.channing.harvard.edu/4a.htm 7. ww.ncbi.nlm.nih.gov. 8. www.aafp.org/afp/ 20000815/804.html 9. Journal of Clinical Microbiology, June 2000, p. 2378-2380, Vol. 38, No. 6 0095-1137/04.00+010. www.FDA.com (FDA archives)11.www.postgradmed.com/issues/2001/10_01/hoel.htm 12. www.cdc.gov/ncidod/hip/aresist/mrsa_CDCactions.htm13. www.medscape.com14 http://www.nabi.com/images/factsheets/fsStaphVAX.pdf

In the early 1970s physicians were very confident about treating bacterial infections. But ,their confidence was shaken by the emergence of resistance to multiple antibiotics by Staphylococcus aureus. MRSA stands for Methicillin Resistant Staphylococcus aureus (S aureus). These bacteria colonize the skin in approximately 40% of healthy people. Frequently S aureus is cultured from nasal passages in approximately 30% of the healthy population. The evolution of the increasing microbial resistance is multifactorial, but the chronic overuse of antibiotics geographically has imposed selective pressures on many pathogens including S aureus. These selective pressures mediate the acquisition of resistance and foreign DNA[4] Multiple resistance to antibiotics developed quickly after WW ll. In the beginning this was not of great concern because there were many pharmaceutical companies developing newer antibiotics to combat the problem of resistance stemming from their overuse during the war. But, by the late 1950s, concern arose from the fact that there was only one drug left called vancomycin. Other concerns about vancomycin stems from the fact that it has very unpleasant side effects and is potentially toxic to the hosts cells (hearing loss and nephrotoxicity). Serendipitously, the 1960 breakthrough of methicillin occurred (beta-lactamase stable penicillin) and so concern faded, but only for a short time. MRSA first appeared in 1961 in the United Kingdom after one year from the first introduction to clinical practice. By the late 1970s and 80s MRSA emerged in Australia. Today, it remains one of the most significant nosocomial pathogens. Something to note from this timeline is that the development of new antibiotics has decreased significantly, especially in the past 20 years. This is directly due to the cost involved to develop new antibiotics. [1,2]. Staphylococci are gram positive, non-motile, and perfectly spherical, measuring approximately 1 micrometer. S aureus grow in chains and resemble bunches of grapes. The 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, S aureus strains grow out a hemolytic pattern. S aureus also display golden color of colonies when grown on agar aerobically[3]. This schematic diagram from Journal of clinical investigation illustrates the mechanism of resistance in MRSA. Methicillin resistance requires the presence of the chromosomally localized mecA gene. The mec A gene is the gene responsible for methicillin resistance and is part of a mobile genetic element found in many MRSA strains called SCCmec. To date, There are at least five different SCCmec elements. These elements integrate at the same time site in the chromosome by a mechanism involving site-specific recombination and excision from the chromosome at attBscc, that is a part of an open reading frame of unknown function near the origin of replication. The genetic mechanism responsible for the transfer of these mobile elements are uncertain. However, what we do know is that mecA gene in MRSA is responsible for the synthesis of penicillin binding protein2A. The MecA gene expression alters PBP2A in S aureus resulting in a loss of target affinity. The mecA gene encodes a new b-lactam-insensitive to penicillin[5]The development and acquisition of resistance in MRSA also requires the presence of other mechanisms. Horizontal gene transfer is a mechanism by which plasmids (resistant genes contained in small packets) found inside the cytoplasm of the bacteria have the ability to transfer resistance genes between the same and different species. The three different mechanisms involved in resistance are: 1) Conjugation is where there is cell-to-cell contact, in other words, this is bacterial sex. Some scientists speculate that this is the main mechanism by which resistant gene transfer occurs. 2) Transformation is where bacteria from the external environment is acquired. 3) Transduction involves bacteriophages transfering DNA between two closely related bacteria[4]. Now that we understand some of the mechanisms involved with MRSA acquiring antibiotic resistance, I would like to turn some attention to the many intrinsic virulence factors of S. aureusFigure taken from Textbook of Bacteriology. MRSA expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) 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]. The cell wall consists of a cytoplasmic membrane that is responsible for osmotic regulation of ion content. The cell wall 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 of infection by leaking of bacterial machinery[7]. The peptidoglycan allows the bacteria to attach hosts 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 others bind to receptor 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. The molecules unique to gram positive cocci are teichoic acids and glycopeptides cell wall fragements that bind to hosts 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,]. 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 WBCs 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 a 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 abscess formation. Staphylokinase also known as fibinolysin dissolves fibrin clots by promoting the coversion of plasminogen to the fibrolytic enzyme plasmin (Phage conversion). Hyalurondase (a joint 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 ].All of these described above are membrane damaging toxins. Leukocydins act on polymorhonuclear leukocytes. The accumulation of pus at the infected site is caused by the dead-lysed wbcs. The Type 3 a-b toxins interfere with host cell function. The alpha toxin characterized as the most potent. The (a-toxin) expression involves hemolysin. Alpha toxin can be 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. Beta toxin hydrolyzes membrane lipids. Beta D-hemolysin has detergent properties. G toxin-mechanism unknown according to the literature, but a good guess is cytotoxicity.[4].According to recent statistics 60% of the population carry S aureus on their skin with no subsequent consequences because of healthy immune systems S. aureus is also carried in the nasal passages in 40% of the population. Nasal carriage of S aureus raises the risk of a subsequent infection. MRSA is one of the most common nosocomial infections. Recently, cases of skin infections caused by MRSA have been identified in the community.Infection by staphylococci is usually from a combination of bacterial virulence factors and diminution in host defenses. Skin injury from surgery, trauma burns can lead to a serious MRSA infection, especially if the patient has a history of chronic bacterial infections treated with multiple rounds of antibiotics overtime. Direct inoculation of S aureus bacteria into the blood by indwelling catheters is a very important route to note. Also metal plates in joints, sutures, and skin staples may set the stage for the organism to survive. Hormonal changes and stress may lower host resistance and increase the risk of an opportunistic infection, especially in people with AIDS, diabetes, kidney dialysis/transplant, and people receiving chemotherapy.

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 may not respond to multi-antibiotic combinations

Carbuncles are large, painful lesions that many times produce fever, high white blood cell counts, and they invariably have ineffective drainage sites.

This is nonmenstrual Toxic Shock Syndrome and Scalded Skin Syndrome .This syndrome can present with hypotension (low-blood pressure), erythema (redness), fever, and multisystem dysfunction. Most cases of nonmenstrual toxic shock syndrome occur in newborns and in the postoperative setting.The lesion starts out as a superficial pustule that ruptures and forms a characteristic yellow-honey to a brown-red crust as seen above. Improving facial hygiene would prevent this type of infection that is very common in children. This is a 44 y/o IV Drug Abuser With Back Pain And S aureus Osteomyelitis Of Lumbar Spine. Staph osteomyelitis and discitis involving L5, with extension across the L4-5 disc to erode L4 and extension into S1(sacrum). The L5 vertebral body is destroyed. The obliteration of the disk space is seen by the red arrows. The signal of the scan has been lost in the lumbar portion due to the destruction of the bone and surrounding tissue.

Major organs failure with disseminated MRSA Toxic Shock Syndrome (TSS-1). This is an Unfortunate systemic invasion of a young girl with TSS related organ failure and systemic invasion. Super absorbent tampons with minor abrasions to the vaginal mucosa have been implicated in many cases of TSS-1TABLE from Journal of Clinical Microbiology, June 2000 shows comparison of the levels of accuracy of reactions of S.aureus isolates on CHROMagar S aureus, DNase, and MSA and of coagulase testing of CoNS after 18to 24h ofincubation. As shown above, it appears that the traditional coagulase method for detecting S aureus is as specific as the chromgar with both of them having 100% specificity[8]Antibiotics generally work in 5 ways: 1) Inhibition of nucleic acids- Chloroquine, Rifampin; 2) Inhibition of protein synthesis- tetracyclines, linezolid, and chloraphenicol; 3) Action on cell wall by interfering with proteins that construct cell wall-Penicillin; vancomycin; 4) Action on cell membrane-Polymyxcin; 5) Interference with enzyme mechanism- sulfa drugs. The use of Vancomycin is parenteral only. Toxic effects must be monitored. Linezolid 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 is a lipopeptide antimicrobial that causes membrane depolarization in bacteria which ceases membrane transport in the bacteria. Newly approved for treating skin and wound ulcers of diabetics. Highly bacteriacidal, although no oral therapy is available[10]Oritavancin binds to normal cell-wall precursors, as does vancomycin, but it also inhibits an earlier step (transglycosylation) in cell-wall synthesis and can be given once daily. Tigecyclin is taken orally and appears to have broad antimicrobial activity. It has activity against organisms that are resistant because of their efflux pumps. Tigecyclin also has activity against organisms that have undergone ribosomal modification. Dalbavancin is currently under clinical trials. It has long half-life so it can be given once per week. According to the Journal of antimicrobial Chemotherapy (2005) Dalbavancin is bactericidal against methicillin-susceptible and resistant Staphylococcus aureus, in both the presence and absence of human blood serum. Resistance was not observed in any isolate tested. After serial passage, bacterial populations were more homogeneous in their susceptibility to dalbavancin than to vancomycin or teicoplanin. Conclusion: Dalbavancin is bactericidal for staphylococci. Resistance to this semi-synthetic glycopeptide is not readily developed in vitro. Journal of Antimicrobial Chemotherapy The vaccine was created at The National institutes Of health and is one of several vaccines in development, however, no other product has reached the advance clinical trials like Staph VAX [11]. The S aureus vaccination contains the same polysaccharides found in the outer coating of the most dangerous stains of S aureus-types 5,8. These are the virulent strains that are responsible for 85% of S aureus infections. The outercoating serves as a predacious mechanism that allows the bacteria to evade host defenses (immune system). The Staph vax contains the purified S aureus polysaccharide linked to a large carrier protein molecule. This combination appears to be recognizable as a foreign invasion to the human immune system. This triggers the body to synthesize antibodies to the S aureus polysaccharides in response to vaccination[14]CDC provides technical help and referrals to state and local health departments, doctors, nurses, and other professionals. A national program of surveillance is available for serious infections with MRSA. Also, the CDC has launched evidence-based educational campaign to prevent antimicrobial resistance. A national resource library is being built to identify genetic patterns or relationships. Finally, they are researching the role of staph toxins to provide answers for hospitals and researchers *For more info go to www.cdc.goc

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

Even though annual deaths from infectious disease have decreased over the past decade, a world-wide impact from infectious diseases and antibiotic resistance (MRSA) remains substantial. As shown in this figure, infectious diseases remain the third leading cause of death in the United States and the second cause of death worldwide. Infectious diseases also lead to compromised health and disability and contribute to the most of the disability adjusted life years( DALYs) each year throughout the world.[13] According to recent statistics, over the past decade the budget of the National Institute for Allergy and Infectious Disease (NIAID) has quadrupled; spending on emerging infections diseases has increased from less than $50 million in 1994 to greater than $ 1.7 billion projected for 2005, partly due to the boost in biodefense. However, NIAD supported intramural and extramural investigators have contributed exponentially to the global effort to identify and combat infectious agents like MRSA.