Microbiology Exam 4 (Staphs and Micrococcus)
Describe the macroscopic appearance of staph aureus on BAP
The colonies are gray, white or pale yellow. They are 50% beta 50% gamma. they are medium sized colonies and after 24 hours they are opaque
Clumping factor
bound coagulase
Determine the pathogenesis of staphylococcus aureus
infection sis developed in conjunction with virulence of the strain, size of the inoculum, the immune status of the host
Protein A
interferes with attachment of host phagocytes to microbe
TSST-1
is the toxin associated with toxic shock syndrome
VISA
Vancomycin intermediate staph. aureus
Beta Lactamase
makes organism resistant to B-lactam drugs
Principle/Reagent of the mannitol fermentation test
reagent used is mannitol and 7.5% NaCl solution. This test is used to determine the species of staph. The high salt concentration is tolerated by staphs but not micrococcus. S.AUREUS AND S.SAPROPHYTICUS WILL FERMENT MANNITOL AND S. EPIDERMIDIS DOESNT FERMENT MANNITOL. MICROCOCCUS LUTEUS DOESNT GROW ON THE MEDIA.
Heterotroph
require an organic source of carbon for survival
Autotroph
require carbon dioxide can synthesize organic molecules from inorganic sources
Ammonification
A microbial metabolic activity that releases organic nitrogen in the form of ammonia. Microbes cover ammonia into nitrite/nitrates through nitrification. Fermentation involves the reduction of inorganic molecules other than oxygen. enteric contain and enzyme nitrate reductase and can reduce nitrate to nitrite in denitrification nitrite can be reduced to nitrogen gas.
Hospital acquired
A nosocomial infection, the cause of sepsis, pneumonia, surgical wound infections, S. aureus isolates should be tested on a muller hinton media with ixicilli or be tested for PBP2a ( a test for penicillin binding protein)
Principle/Reagent of the beta-lactamase tests
A rapid test to determine if an organism is sensitive to B-lacram drugs. The procedure uses a B-lactamase disk that is dampened with sterile water, a colony of the organism is smashed on the disk. A color change indicates the organism is resistant to B-lactam drugs. S AUREUS
enterotoxin
A-E, H and I are associated with food poisoning
lipase
Break down fatty acids on the host skin tissue
Identify the laboratory test that best determines if an organism is a staphylococcus vs. streptococcus
Catalase test
Name the most reliable test for staphylococcus aureus
Coagulase test
Simmons citrate media
Contains sodium citrate as the only source of carbon containing compound in the media. Bacteria that possess citratepermease can transport these molecules into the cell and convert them to pyruvate. When the test is positive, the bromthymol blue turn the media royal blue. Enterobactor aerognes: Positive E.Coli: Negative
Malonate media
Contains sodium malonate, yeast extract, and a small amount of glucose to promote growth of organisms otherwise are slow to respond. If the organism utilizes malonate, the media will be alkalinized and changes to a deep blue color (Positive test) Enterobacter aerogenes: Positive E.coli: Negative
Leukocidin
Destroys host WBC's
Nitrate reduction test
Enterococcus faecalis: Not a nitrate reducer P. aeruginosa: Is a nitrate reducer (no3 to Nitrogen gas) E.Coli:Nitrate reducer (No3 to No2) If it turns pink when you put the reagents in, its a nitrate reducer (Nitrate to Nitrite) if it doesn't turn pink right away then put zinc in and if it does turn pink its neg and not a nitrate reducer. if its colorless after zinc its a nitrate reducer no3 to nitrogen gas
Match significant toxins and enzymes with the conditions they are associated with
Enterotoxins A-E, H and I are associated with food poisoning. TSST-1 caused toxic shock syndrome, Misc hemolysis are both alpha and beta hemolysins have lethal effects of a variety of cell types, coagulase and catalase enzymes- having catalase enzymes may destroy hydrogen peroxide substances found in human cells used to kill microbes and coagulase converts fibrinogen to fibrin
Identify virulence factors of the organisms discussed in this unit
Enzymes- Lipases, hyaluronidase, staphylokinase, coagulase, beta lactamase Toxins- Leukocidin, enterotoxins, epidermolytic toxins, cytolytic toxins, Protein A
outline the principles of the catalase test and the coagulase test
Free coagulase present in an organism separates staphylococcus aureus from other species of staph. some bacteria synthesize the enzyme catalase. When 3% H2O2 is hydrolyzed, gaseous oxygen is released in the form of bubbled
Identify with staphylococcus aureus is considered normal flora vs. when it is considered a pathogen
Frequently its part of the normal flora but an infection sis developed in conjunction with virulence of the strain, size of the inoculum, the immune status of the host
list the biochemical, fermentative and enzymatic tests used to identify and differentiate micrococcus spp. from different species of staphylococcus
Glucose fermentation, Microdase test, lysostaphin test, Furazolidone, bacitracin
Identify characteristics that are common among micrococcaceae and staphylococcaceae
Gram positive coccis, Aaerobic or facultative, nonfastidious, nonmotile, catalase positve, will reduce nitrates to nitrites
Clinical conditions associated with staphylococcus aureus
Infections of hair or skin (boil, furuncle, carbuncle), impetigo, scalded skin syndrome, pneumonia, osteomyelitis, metastatic infection, enterocolitis (food poising)
outline why each toxin and enzyme is specifically virulent
Lipases-Break down fatty acids on the host skin tissue Hyluronidase- dissolves host connective tissue Staphylokinase- dissolves host fibrin coagulase- clots plasma beta-lactamase- makes organism resistant to B-lactam drugs Leukocidin- destroys host WBC's Enterotoxin- Causes food poisoning Cytolytic toxin- destroys cellular material of the host Protein A- interferes with attachment of host phagocytes to microbe
Test results for all the staph and micrococcus tests. KEY POINTS
MICROCOCCUS: Microdase +, lysostaphin -, Glucose fermentation -, won't tolerate the 7.5% NaCl in MSA STAPHYLOCOCCUS: Microdase -, lysostaphin +, Glucose fermentation + Staphylococcus aureus: Coagulase +, Mannitol fermentation +, grows easily in the presence of 7.5% NaCl staphylococcus saprophyticus is resistant to novobiocin staphylococcus Epidermidis is sensitive to novobiocin
Community acquired
MRSA Up to 41% is community acquired, primarily the cause of infections, not associated with any one risk factor as once thought, avoid sharing of razors, towels
MRSA
Methicillin resistant staph. aureus.
Outline the progression of methicillin intermediate and resistant strains of staph aureus and vancomycin intermediate and resistant strains
Methicillin- S aureus used to be sensitive to penicillin. now 80% of non-mesa strains are resistant to penicillin. Methicillin was developed to combat strains of penicillin resistant S.aureus. Many strains of S. aureus now are resistant to methicillin, these are MRSA varieties. Vancomycin can be used to treat MRSA strains, but this is the last resort. Vancomycin intermediate s. aureus- vancomycin was once treated for MRSA. VISA was first noted in japan in 1996. VRSA was first seen in 2002 in michigan, pennsylvania, and new york. Laboratories should screen for VRSA by putting suspected strains on brain heart infusion agar with the addition of vancomycin. Growth after 24 hours incubation should cause one to suspect VRSA
Identify Micrococcaceae and staphylococcaceae organisms that are considered normal flora in/on different body sites, identify those that are pathogens.
Micrococcus Luteus is normal skin flora. Stomatococcus mucilaginosus is normal flora of respiratory flora. Staphylococcus aureus id by far the most important human pathogen. Staphylococcus Saprophyticus is leading cause of UTI. Staphylococcus Epidermidis is associated with nosocomial infections and endocarditis.
Hydrolysis test
Organisms that have the enzyme urease break down urea into ammonia and carbon dioxide. Urea slants have a phenol red pH indicator pH changes from yellow to pink if nh3 in produced a pH of >6.8 causes the media to turn bright pink
principle/Reagent of the coagulase test
Reagant: rabbit plasma This is the best test to identify staphylococcus Aureus. Latex agglutination test can also work. The coagulase enzyme present in staph Aureus react with the "reacting factor" in the reagent solution and fibrinogen to form a fibrin clot. free coagulase0a thrombin-like substance. STAPH AUREUS IS POSITIVE FOR THIS TEST (CLUMPING OCCURS) latex agglutination coagulase procedure use latex beads coated with fibrinogen to detect the clumping factor. bound coagulase is attached to the bacterial cell walls unlike free coagulase in the culture filtrates.
Principle/Reagent of the bacitracin test
Reagent Bacitracin ("A" disks) disk susceptibility procedure using .04 units of bacitracin disks. Staph and micrococcus species can be differentiated by using this method. STAPHS ARE RESISTANT AND WILL GROW RIGHT UP NEXT TO DISK. MICROCOCCUS SENSITIVE TO THE DISK 10MM OR >
principle of the glucose fermentation test
Reagent is 10% glucose and pH indicator bromthymol blue This test is used to differentiate between staphylococcus from micrococcus. STAPHYLOCOCCUS FERMENT GLUCOSE. MICROCOCCUS DOES NOT FERMENT GLUCOSE THE MEDIA REMAINS GREEN OR BLUE.
Principle of the catalase test
Reagent is 3% H2O2 Determines the presence of catalase enzyme. If bubbling occurs when the reagent is combines with the organism that is a positive test. ALL STAPHYLOCOCCUS AND MICROCOCCUS ARE CATALASE POSITIVE.
Principle/Reagent of the furaolidone sensitivity test
Reagent is furazolidone disk susceptibility using 100ug furazolidone disks. This is useful in the differentiation of micrococcus and staph species. STAPHS ARE SENSITIVE 15MM OR > MICROCOCCUS ARE RESISTANT WITH ZONE SIZE 6MM-9MM
Principle/Reagent of the novobiocin sensitivity test
Reagent used is novobiocin This test identifies staphylococcus saprophyticus. Method requires the use of a 5ug novobiocin disk. S. SAPROPHYTICUS IS RESISTANT WITH A ZONE SIZE OF 6MM-12MM
principle/Reagent of the lysostaphin test
Reagent: lysostaphin An endopeptidase present on the staphylococcus staphylolyticus organism that cleaves the cell walls of other organisms. cloudy organism suspensions are negative (No lysis) Clear organisms suspensions are positive (cells have lysed) STAPHYLOCOCCUS ORGANISMS ARE SUSCEPTIBLE TO THIS LYSOSTAPHIN ACTIVITY AND WILL BE LYSED. (POS) MICROCOCCUS WILL NOT LYSE (NEG)
principle of the nitrate reduction test
Reagents are a-naphtylamine, sulfanilic acid, and zinc. Determines the ability of an organism to reduce nitrate to nitrite or nitrogen gas. Used to separate staph and micrococcus from other aerobic gram positive cocci. STAPHYLOCOCCUS AND MICROCOCCUS WILL REDUCE NITRATES. Enterococcus faecalis: Not a nitrate reducer P. aeruginosa: Is a nitrate reducer (no3 to Nitrogen gas) E.Coli:Nitrate reducer (No3 to No2) If it turns pink when you put the reagents in, its a nitrate reducer (Nitrate to Nitrite) if it doesn't turn pink right away then put zinc in and if it does turn pink its neg and not a nitrate reducer. if its colorless after zinc its a nitrate reducer no3 to nitrogen gas
Cellular respiration properties
Step Viridians: capnophilic P. aeruginoas: Aerobic B. fragilis: Anaerobic Campylobacter: microaerophilic
VRSA
Vancomycin resistant staph aureus
aerotolerant anaerobes
can grow in the presence of oxygen but will thrive better without. fermentation
Faculatative anaerobe
can grow with or without oxygen, but grow better with oxygen.
Cytolytic Toxin
destroys cellular material of the host
Hemolysin
destroys red blood cells
Principle/reagent of methicillin resistance test
determine if we have an organism resistant to methicillin drugs.
Hyaluronidase
dissolves host connective tissue
Staphylokinase
dissolves host fibrin
Nitrogen cycle
many types of nitrogen based conversions are performed by microorganisms, and specific bacterial species are essential in the steps of nitrogen fixation where nitrogen is brought back into the cycle. the processes performed by nitrogen fixing bacterial species are essential that life as we know would not exist without bacterial intervention. Bacteria themselves require nitrogen to synthesize amino acids, DNA, RNP, and ATP
Outline key reactions for the differentiation of micrococcus species and staphylococcus species
microdase test, lysostaphin test, glucose fermentation, furazolidone test, bacitracin test
Olbigate aerobe
organism that grow only with oxygen
Microaerophiles
organism that prefer low oxygen 2-10% saturation during aerobic respiration
Obligate anaerobes
organisms that grow without oxygen
Principle/reagent of the microdase test
reagent is 6% tetramethyl-p-phenylenediamine dihydrochlordide. In the presence of oxygen, the oxidase will react with the reagent a form a dark purple color. this means the test is positive. MICROCOCCUS ARE POSITIVE AND STAPHS ARE NEGATIVE.
Lysostaphin
staphylococcus organism are susceptible to lysostaphin and will be lysed
epidermolytic toxin
toxic shock syndrome
