Gram + = Gram Positive; Staphylococcus Aureus (SA); S. Epidermidis (SE); Virulence Factor (VF)

FUN2: 11:00-12:00Scribe: Maggie Law

Tuesday, November 4, 2008Proof: Kallie Law

Dr. WaitesGram Positive BacteriaPage1 of 8

Gram + = gram positive; Staphylococcus aureus (SA); S. epidermidis (SE); Virulence factor (VF)

Gram Positive Bacteria

Objectives [S1]:
Introduction to several group of important groups of microorganisms that cause human disease; talk about their habitats, types of infections they cause, specific virulence factors, laboratory characteristics, how you identity people with the disease, and insights into their importance in the overall contribution to human disease.
Two step approach: lectures and labs where you will be able to see the microorganisms and look at the case studies with different diagnostic tests and how they can be identified in a laboratory.
[S2]Go over the characteristics, epidemiology, virulence factors, associated diseases, and laboratory detections of clinically important gram + positive cocci and gram + positive bacilli.
Will discuss staphylococcus, streptococcus, enterococcus, listeria, corynebacterium, and bacillus.
Other gram + bacteria that are important but do not need to know all of them.
Staphylococcus [S3]
General information
Very important human pathogen---most important in the genus Micrococcaceae.
Two other genera in the family—stomatococcus & micrococcus
Not important causes of human disease
Undergoing taxonomic changes; organisms found in mouth and skin, but rarely cause human diseases except in extraordinary circumstances
Diagram of Staphylococcus aureus (SA) on the slide—looks like a bunch of grapes; bacteria are really colorless; must be colored in order to see them in clinical specimens—reason for the gram stain.
Gram staining is used to characterize and color bacteria
SA is a gram + that appears and grows in groups or clusters; name means “grape-like.”
Habitat [S4]
SA
is the most important pathogen in the staphylococcus group—most important human pathogens overall
Very successful pathogen due to the way it can exploit the niches in the human body; some bacteria can cause disease in normally healthy people; other bacteria must wait until defenses are down to cause disease—called “opportunists.”
Reason for success it because it can find a way to live in body without causing disease; found in the nares of 50-75% of healthy people
Adapted micro ecological habitat so it can exist in human without causing disease, yet successful pathogen because if the given the right circumstances or if it is found in the right part of the body, it can cause devastating diseases.
Important pathogen in healthcare setting due to nosocomial infections—infection acquired in a healthcare setting; many people get infections from being at the hospital due to the fact that hospitals harbor bacteria, there are lots of antibiotics present that selective for the most resistant bacteria and the most successful bacteria can move around and live on inanimate surfaces like bedside toilets, bed rails, etc. and from person to person from people that are colonized with the organisms that can cause disease.
Very important nosocomial organism because it can live on inanimate surfaces for long periods of time
S. epidermidis & other groups of staphylococci—not as important human pathogens
Staphylococcus epidermidis is common on the skin; on the skin of everyone in the room; as long as it stays on the skin, it usually doesn’t cause problems; much less of a virulent organism than SA
SE is also in your conjunctiva even though it does not usually cause disease there.
S. saprophyticus
Mainly known to cause urinary tract infections in young women; not usually cause infections in men because they are not as common in men due to the fact that women have a short urethra and it is easier for the bacteria to get into the urinary tract from the outside.
Lab Characteristics of all staphylococci and differentiating characteristics that will allow you to separate them—if you suspect a patient has a staphylococcal disease, you must collect a diagnostic specimen so you can find the staphylococcus and then determine what type it is so you can determine if it is likely to cause disease, the type of management that you would provide for the patient, etc. [S5]
Picture of what staphylococcus looks like on a gram stain—dark purple cocci in clusters, typically in small or large groups—can tell it is a staphylococcus but not what particular type it is—look at the gram stain and be pretty sure that it is a staphylococcus though some bacteria can look similar
When looking at gram stains and bacteria in general, a gram + cocci such as a staphylococcus or a streptococcus are about 1 micron in size (a RBC is about 6-9 microns in size), so about 1/6th the size of RBC for comparison
Grow aerobically—not particular about what they grow on—very hardy organisms
Are beta-hemolytic—means that on a blood agar plate (soy agar with 5% sheep blood to grow bacteria—important because most common bacteria will grow on agar; the blood cells provide enrichment and nutrients that the bacteria need) you COMPLETELY lyse the RBC and you can see through the plate because the RBC are lysed by the hemolysin that is present.
One of SA virulence factors (VF) is that it has a hemolysin that lyses the RBC; allows the RBC to open and get the things out of the cells that it needs to grow; this is a characteristic that you can see and it is a great trait at differentiating bacteria—whether or not they are hemolytic
Beta hemolytic – complete lysis of the RBC
Produces an enzyme called coagulase—enzyme that will convert fibrinogen into fibrin and produce a clot; staphylococcus secretes coagulase into its environment as well as produces a bound coagulase called clumping factor on the surface of the cells.
Important VF because it allows staphylococcus to protect itself by coating itself with fibrin in order to protect it from antibodies, complement, WBC, and other things that are part of the body’s immune system that would be trying to fight it—way of protection.
Trait that will differentiate SA from other staphylococci that we refer to collectively as the coagulase negative staphylococcus.
Catalase enzyme is another one produced that will help differentiate a staphylococcus from a streptococcus; enzyme that converts hydrogen peroxide to water and oxygen;
Produces bubbles
VF that allow it to protect itself from peroxide—can use peroxides as an antiseptic if you have an infection you could put peroxide on the infection or wound
Can it will help kill bacteria because bacteria that LACK catalase to break down the peroxides will be killed because the hydrogen peroxide will be toxic
Fermentation of mannitol
One way to selectively grow SA in clinical specimens is to grow specimen on mannitol salt agar; contains high concentration of about 7.5% NaCl that will keep many bacteria except staphylococcus from growing
Also has mannitol in there as the sole carbohydrate source with a pH indicator—a SA will produce acid from mannitol; the plate will change from red to yellow(depicted on the image on the slide)
Mannitol fermentation (changing the plate to yellow) is another characteristic of SA.
Catalase Test[S6]—this test is a way to differentiate staphylococcus from streptococcus
Catalase test will separate staph from strep
1st test to do if you have a gram + coccus and are not sure if it’s in the strep or staph group
See bubbles when you add hydrogen peroxide to some of the colonies—MEANS IT’S A POSITIVE TEST AND ITS NOT A STREPTOCOCCUS and it is probably a staphylococcus
Coagulase Test [S7]
1st way—measure the production of coagulase by taking rabbit plasma and putting some bacteria into the tube and wait to see if clotting occurs
If the plasma clots, then it’s a positive test for coagulase
SA will usually be positive fairly quickly; however, if the clot is not produced right way, it must be incubated for at least 24 hours before you can conclusively say it is not an SA.
Microbiologists like to do things quickly so they have a more rapid coagulase test where you can actually do the test faster by looking for clumping of latex particles that have the fibrinogen coated on the latex particles
Cell Wall [S8]—many of the bacteria have various VF and antigens that can be expressed on the bacteria’s surface
Some VF are secreted into the environment; others are expressed on the cell’s surface
Some strains will have a capsule or a polysaccharide slime layer; capsule is important to help protect the organism from phagocytosis
SA is an extracellular pathogen that produces disease if it can escape white cells; if white cells phagocytise it, the organism will be killed so it cannot go out and do it’s business as a bacteria
capsule helps prevent its opsonizaiton
The slime layer that is seen in some strains of SA and other strains of staphylococci help it attach to things—one of the best ways to get inside the blood stream of a patient that has an IV cathedar in their vein is to attach to plastic—some staphylococci can easily attach to inanimate surfaces that allows them to hang on to keep from being washed off or removed; important way to have organisms attach to things
Peptidoglycan layer—peptidoglycan is important in the cell wall structure of gram + bacteria; acts as an antigen and helps hold the crystal violet in the cell wall during a grain stain
Teichoic acids—important antigenic structures—part of the structures that help bind the organism to the epithelial cells in the host; bacteria have to have a way to attach to host to cause disease; part of the attachment structures in gram + bacteria are related to teichoic acids and the polysaccharides there.
Protein A is another VF of SA—helps it to cause disease because it binds to the Fc fragment of IgG; it binds the Fc fragment of IgG and destroys the antibody, the antibody can’t opsonize the bacteria; another way bacteria has in order to evade the host’s immune response
All of the stuff provides the rigidity of the cell wall to keep the bacteria osmotically stable; on the outside of the cytoplasmic membrane
Clumping factor—bound coagulase mentioned earlier
Antigenic Structures & VF of SA [S9]
In addition to things about binding the fibronectin on the host cell, teiochic acid is part of the staphylococcal cell wall that helps produce the pyogenic effect—hallmark feature of staphylococcal infections are puss-forming abscesses; pyoderma or pyogenic infections of inflammatory cells, primarily neutrophils—typical of staphylococcal infection
Peptidoglycan of the cells wall is partly responsible for the stimulation of neutrophils to come to the site of staphylococcal infection which causes the pyogenic effect
Production of interleukin and opsonic antibody isvalso part of the peptidoglycan cell wall and activation of complement—important in terms of VF
Soluble VF of SA [S10]
Secreted by the cell—ex. coagulase and catalase
Hyaluronidase—hyaluronic acid is part of the glue that holds the tissues together
Keeps the skin and tissues solid—not many bacteria can cause disease if skin is intact because skin is one of the most important forms of protection in bacterial infections
A break in the skin will allow bacteria to get into subcutaneous tissues.
One of the 1st things that a staphylococcus or a streptococcus does once it gets inside the skin is to start to produce hyaluronidase that breaks down the hyaluronic acid and allows the organism to spread through the facial planes to the tissues to spread throughout the body and get into the blood stream
Very important VF
90 plus % produce beta lactamase—enzyme that bacteria produce that breaks down beta lactam antibiotics like penicillin
When penicillin was 1st introduced into clinical practice in the 1940’s, all staphylococci were susceptible to it because it would prevent the cell walls from being formed; in a few years, the bacteria developed the beta lactamase enzyme that would destroy the antibiotic before it could destroy the bacteria; the antibiotics are not active any more—once the beta lactam ring of penicillin is broken open, it can’t do anything to the cell well
MRSA (methicillin resistant SA)—comprise about 60% of all staphylococci that are seen in the hospital; becoming more important as a cause of community acquired infections; are altered penicillin binding proteins
Bacteria synthesize cell walls with the chains of the carbohydrates, NAM & NAG, bound together by amino acids
The cross linking of the cell wall carbohydrates are catalyzed by enzymes, the carboxypeptidases and the endopeptidases that we know collectively as the penicillin binding proteins.
Penicillin works because the beta lactams resemble the substrates for the amino acids of the penicillin binding proteins; bind irreversibly to the penicillin binding proteins and prevent them from cross linking the cell walls
Staphylococci that are the MRSA strains have an altered penicillin binding protein that does not recognize the beta lactam so the cell wall cross linking takes place—important VF because it entirely wipes out a whole class of antibiotics and gets these bacteria
Makes them much for difficult to treat and then they spread from person to person and cause lots of morbidity and mortality in the hospital
Now seeing these in the community acquired variety of staphylococcal infections so doctors are now learning about this and realize about the altered penicillin binding proteins
recognize that they have to prescribe different antibiotics.
Other important enzymes are the fibrinolysins, the lipases, and the nucleases—also things that help destroy components of the host that help cause the inflammatory process and necrosis of tissue that you get in association with the pathologic lesions
Very diverse organism because it can do all kinds of different things—among the many different VF that SA has, not every strain produces every one of them; some are only produced by some strains and not by others.
[S11] Leukocidins—VF that are produced by some strain of staphylococcus that kills WBC; the Paton-Valentine leukocidin is one of the characteristics of the community acquired MRSA infections
If you kill the WBC then the WBC cannot kill you; so the bacteria see an advantage in doing this
Some of the other enzymes release lysomal enzymes which damage tissue and further causes the necrosis and abscesses that you see
Scalded skin syndrome—produced by the exfoliating toxin of SA that interrupts the intercellular junctions of the skin and causes the skin to fall off and exfoliate
Toxic shock syndrome—occurs as a super antigen—stimulates T cells to release cytokines, causes significant endothelial damage and causes a rash and the overall appearance of the toxic shock syndrome was especially associated with tampon use by women.
These are all exotoxins—all exotoxins that are produced by the staphylococcus
Some strains of staphylococcus also produce enterotoxins—can cause food poisoning because it interacts with GI neuroreceptors and causes a lot of nausea and vomiting
Example of food poising that is an entoxication because you can actually ingest the performed toxin that the staphylococcus has produced by foods such as potato salad
Ingest the toxin already and the toxin causes the disease in your body even if you don’t ingest the bacteria
[S12] SA Diseases
Lots of SA infections start out as skin diseases
Image—impetigo on man’s face—honey crusted lesions—referred to as furuncles which are infections around a hair follicle that actually spread and coalesce; carbuncles produced this way
Typically of SA disease—impetigo could also be caused by streptococci but in association with this, you can also get cellulitis, which is a spreading inflammation of the skin (don’t have collection of pus, but is a more diffuse inflammations)
Can progress to bactermia in which bacteria spread throughout the blood stream—in the worst cases of bacteremia you can get the bacteria on heart valves and have a very bad endocarditis
SA can cause severe endocarditis and destroy a heart valve and patient has to undergo surgery to have valve replaced
Not usual, but can cause meningitis—more common to see it as an epidural abscess around the spinal cord
Occasionally SA can cause brain abscesses.
S. Aureus Diseases [S13]
Child with scalded skin syndrome; person with toxic shock syndrome rash
Examples of toxin-mediated diseases
We see pneumonia that occurs in hospitalized patients; don’t think of S. aureus as a cause of community acquired pneumonia
People in hospital, especially with respiratory failures - MRSA can be a significant for ventilator associated pneumonia in hospitalized patients
S. Aureus – single most common cause of osteomylitis (bone infections); can get into joints and cause septic arthritis
Can sometimes get into the urinary tract cause infections; not a common cause of these
Treatment of S. Aureus [S14]
Not going to talk about treatment – get that in pharmacology
Reemphasize that MRSA has caused the requirement that we have to use lots of Vancomycin in the hospital; seeing this in the community as well
Causing concern for possibility Vancomycin resistant S.