Transcript Staph Notes File - Carolinas College of Health Sciences

CATALASE POSITIVE, GRAM POSITIVE COCCI
Genera include:
Staphylococcus
Micrococcus
Rothia
Macrococcus
Planococcus
Alloiococcus
What is Catalase?
CATALASE:
Principle: Catalase is an enzyme that can split hydrogen peroxide (H2O2) into water and oxygen. Organisms which possess the
enzyme catalase decompose the hydrogen peroxide releasing oxygen gas resulting in bubbles = positive test. This test is used to
differentiate Staphylococci from Streptococci.
Reagents: 3% Hydrogen peroxide. Store in refrigerator.
2 H2O2
catalase
2H20 + 02
Procedure:
1. With a sterile loop, pick up the organism to be tested.
2. Gently rub it on a clean microscope slide.
2. Add a “free-falling” drop of the H2O2 reagent directly onto the organism on the slide.
3. Observe for a bubbly reaction.
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* Do not cool your loop in the blood agar media or touch the blood agar when picking up the colony. Blood agar is
catalase positive and false positives may occur.
Interpretation: Bubbling in the peroxidase or immediate effervescence is a positive test.
Staph - Catalase positive
Strep - Catalase negative
STAPHYLOCOCCUS
- gram positive cocci in clusters
- catalase positive
- ferments glucose
- non-motile
- aerobic
On BAP colonies look opaque, circular, smooth, entire colonies “butyrous” (butter-like
consistency) and may appear in color from orange-yellow-dirty white-bright white
COAGULASE TO SPECIATE:
Staph aureus - coagulase positive
Staph epidermidis - coagulase negative
Coagulase
Principle: Coagulase is a prothrombin-like substance when combined with normal plasma
factors forms a thrombin-like substance which activates fibrinogen to form fibrin which is a clot.
This test aids in the differentiation of Staph aureus from other Coag-Negative Staphylococci
(Staph epidermidis).
+
2 Types of Coagulase:
1. Free Coagulase (extracellular)
-
Free Coagulase is measured with the tube method. Bacteria is inoculated
into a rabbit plasma tube and incubated at least 4 hours at 35 C. This is an
extracellular enzyme and requires the presence of a plasma factor known
as CRF (coagulase reacting factor). CRF is similar to thrombin which converts fibrinogen to
fibrin to form a clot.
2. Bound Coagulase:
Bound Coagulase is bound to the cell wall and acts directly on fibrinogen to form a fibrin clot.
This test may be done on a microscope slide.
Slide Coagulase Test:
1. Make a heavy suspension of organism in distilled water
and place 1 drop on a clean microscope slide
2. Add 1 drop of plasma
3. Stir the mixture
4. Observe for clumping within 10 seconds (Positive test)
** False positives may occur if observed longer than 10 seconds or if colonies for testing have
been picked from a media with high salt concentration (Mannitol Salt Agar). A homogenous
reaction is a Negative test.
STAPH EPIDERMIDIS
a.k.a. Coagulase Negative Staphylococci (CoNS)
CHARACTERISTICS:
- normal flora on upper respiratory tract, skin, intestinal tract, mucous membranes
- causes Sub-acute Bacterial Endocarditis (SBE), Urinary Tract Infections (UTI), shunt infections, stitch
abscesses, heart valve infections, prosthesis infections, bacteremia in compromised host (premature
infants, burn patients, trauma patients, osteomyelitis
- produce a virulence factor referred to as “SLIME”. Slime appears to inhibit neutrophil chemotaxis and
phagocytosis and inhibits the antimicrobial action of Vancomycin. Slime also appears to have other
effects on immune function. Slime has been shown to enhance the adherence of Staph to a wide
variety of plastic surfaces especially implanted medical devices. Slime producing strains of CoNS may
be more difficult to treat than non-slime producers. These infections usually arise as a complication of
invasive procedures common in modern medical care.
- Nosocomial” strains are more likely to be multiply-resistant but can be successfully treated with
combination therapy of Vancomycin plus Rifampin or an aminoglycoside.
- Methicillin-Resistant CoNS (MRCNS) have emerged as a clinical problem in patients with prosthetic
heart valves or who have undergone other forms of cardiac surgery
Coagulase Negative Staphylococci (CoNS)
REACTIONS:
- catalase positive
- coagulase negative
- DNA’se negative
- Mannitol Salt Agar (MSA): pink
(tolerates high salt concentrations up to 15 %;
ferments glucose but NOT mannitol)
www.microbeworld.org
Most labs do not speciate CoNS except when recovered from UTI isolates or normally
sterile body sites.
Staph saprophyticus
• Staph saprophyticus, a coagulase negative staph, is usually associated with urinary
tract infections not only causing cystitis but pyelonephritis and can occasionally cause
acute urethral syndrome in women of child bearing age. It also causes catheter
associated UTI in elderly men.
• Since Staph saprophyticus resembles Staph epidermidis it needs to be differentiated
on isolates recovered from urine cultures. Two key
reactions/tests to do this are Novobiocin susceptibility and the Phosphatase test.
• ** Staph saprophyticus is RESISTANT to Novobiocin (<= 16 mm zone size)
•
Other CoNS species are susceptible to Novobiocin ( >= 16 mm zone size)
• An agar may also be employed to accomplish this task. It is Trehalose-MannitolPhosphatase Agar (TMPA) and assists in the differentiation of Staph epidermidis or
other CoNS from Staph saprophyticus by a color change in the media.
•
• Staph saprophyticus is NEGATIVE for Trehalose-Mannitol-Phosphatase
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Staph aureus
• CHARACTERISTICS:
• Catalase positive
• Coagulase positive: identified on the basis of presence
of the enzyme coagulase, (the single most reliable test
for identification) which binds plasma fibrinogen,
causing the organisms to agglutinate or plasma to clot *
• DNAse positive
• Ferments glucose AND Mannitol and tolerates high salt
concentrations thus MSA a good medium to recover
Staph aureus (yellow colonies on pink/red agar) **
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Staph aureus continued:
• normal flora of nasopharynx, anterior part of nose, perineal area and skin; may colonize mucosal surfaces
• cell wall contains peptidoglycans and teichoic acids that aid in providing rigidity and resilience to the cell wall
• produces a creamy to yellow pigment called “lipochrome” (occurs only under aerobic conditions; does
not exhibit lipochrome anaerobically)
• produces an exotoxin that causes necrosis enabling the organism to get into tissue
• produces leucocidin which kills WBC’s
• usually beta hemolytic
• is fibrinolytic (may break down fibrin clot and allow spread of infection to tissues)
• produces enzyme “hyaluronidase” known as the “spreading factor” that contributes to the spread of infection
virulence factors: enterotoxins, cytolytic toxins and Protein A
Sidebar:
* NOTE: the slide Coagulase test using rabbit plasma with ethylene-diamine-tetraacetic acid
(EDTA) detects bound coagulase or “clumping factor” on the surface of the cell wall, which reacts
with the fibrinogen in the plasma. This test is not positive for all strains of Staph aureus, and a
negative result must be confirmed by the tube method for detecting “free coagulase” or
extracellular coagulase. The tube test is usually positive within 4 hours at 35o C; however, a
negative result must then be incubated at room temperature for the remainder of 18 –24 hours.
Some strains produce coagulase slowly or produce fibrinolysin, which dissolves the clot at 35o C.
*MSA- Mannitol Salt Agar contains a high concentration of salt (up to 10%), the sugar mannitol
and phenol red as the pH indicator. On this medium, Staph aureus can grow in the presence of
salt and ferment mannitol producing yellow colonies surrounded by a yellow halo.
Treatment:
- usually resistant to Ampicillin and Penicillin due to enzymes (beta lactamase) produced that
destroy beta lactam ring of antibiotic or Penicillin-Binding -Proteins (PBP) rendering it
ineffective for treatment.
- many strains are beta lactamase producers thus Methicillin becomes drug of choice
- in the last decade many strains are now Methicillin-Resistant (MRSA) leaving Vancomycin as
the drug of choice
Treatment:
Originally, penicillin was the drug of choice for the treatment of serious Staph aureus infections. Resistance
to penicillin emerged due to the acquisition of plasmid-borne genetic elements coding for β-lactamase
production. Strains of Staph aureus produce up to four different β-lactamase enzymes. Semi-synthetic
penicillins like Methicillin and Oxacillin became available from pharmaceutical companies that were
designated the drug of choice for treating these penicillin resistant strains of Staph aureus. Then in the
1980’s resistance to even these drugs emerged due to the presence of an altered penicillin-binding protein
called “PBP2a” (or PBP2 ) that results from the acquisition of a chromosomal gene called mecA. Staph
aureus strains expressing the mecA determinant are termed “Methicillin Resistant Staph aureus or
“MRSA”. The mecA gene may be expressed by some or all of the cells in a given population , so resistance
mediated by altered PBPs is termed “heteroresistance”. (This actually refers to two subpopulations
coexisting within a culture, one that is susceptible and the other resistant to antibiotics (s). The resistant
population grows more slowly than the susceptible one and can be overlooked. Therefore, the more
resistant subpopulation should be promoted growthwise). MRSA’s tend to be resistant to all the β-lactam
agents, cephalosporins and some macrolides like clindamycin and erythromycin. MRSA’s have emerged as
an epidemiological problem in hospitals in the United States today. They are usually isolated from
extremely ill patients in large tertiary care hospitals .To manage and control the spread of MRSA within
healthcare institutions is a real challenge. Some hospitals have instituted routine nasal cultures of
personnel to detect and treat MRSA carriers in order to reduce the number of patient exposures.
A new glycopeptide agent called “Vancomycin” has emerged as the drug of choice for infections caused by
MRSA. However, since 1996, some Staph aureus strains have become intermediate (in between
susceptible and resistant) to Vancomycin and are now termed “GISA’s” (Glycopeptide Intermediate Staph
aureus. In the United States these are called “VISA’s” Bancomyci Intermediate Staph aureus. Prompt and
accurate detection and confirmation of these strains is necessary and critical for optimal treatment of
individual patients and for prevention of transmission of these strains to other patients.
Infections:
1. Food Poisoning: caused by an enterotoxin (A-E, G - I ), heat stable toxin. Exact mode of action of these
enterotoxins is unknown, but shown to increase intestinal peristalsis. Ingestion of preformed enterotoxins in food
supporting Staph growth (bakery goods, custard, potato salad, processed meats, ice cream) results in vomiting with or
without diarrhea with in 2-8 hours. The disease is self-limited (24-48 hours) and usually only requires supportive therapy
for treatment.
2. Skin Diseases:
a. Scalded Skin Syndrome: produces an “exfoliative toxin” which cleaves the middle
layers of the epidermis allowing the surface skin to peel having a burn-like effect.
Sometimes referred to as “Ritter disease”. Usually afflicts neonates.
b. Toxic Shock Syndrome: disease which is elaborated by certain
strains growing in a localized focus of infection or colonization.
Most cases have occurred in menstruating young females
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who were vaginal carriers of Staph aureus and who used highly
absorbent tampons. The toxin is now designated Toxic Shock
Syndrome Toxin 1 (TSST-1)
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Skin Diseases continued:
c. Impetigo: acute inflammatory skin disease, caused by Staph or Strep,
characterized by vesicles and bullae (small pustules) that rupture,
spread and form yellow crusts
d.
Cellulitis: purulent infection of cells
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e. Acne-Boils-Folliculitis: superficial infections surrounding the hair follicles;
Furuncles and Carbuncles: involve subcutaneous tissue and cause systemic
symptoms (like fever)
f.
Post Surgical Wounds: serve as a nidus (central point or focus) for the
development of systemic infections like bacteremia, endocarditis or osteomyelitis.
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Staph aureus: Cellulitis
This child developed a secondary staphylococcal infection at the smallpox vaccination site.
Note: the signs of cellulitis including spreading erythema that envelopes the smallpox vaccination
site, swelling, and accompanying areas of cutaneous purulency. (Picture & text from CDC/PHIL)
Other Staph aureus Infections Continued:
3. Osteomyelitis: disease of the bone especially the marrow
precipitated by pain or pressure over affected area, fever,
leukocytosis, and suppuration. Very serious infection needing
immediate attention. If left untreated surgery, amputation,
or death can occur.
The infecting microorganism (generallv Staph aureus) enters the bloodstream
via a skin wound or an infection (usually in the nose or throat) and is carried to
the bone in the blood. The infected bone and marrow become inflamed and pus
forms, causing fever, severe pain and tenderness in the infected bone, and
inflammation and swelling of the skin over the affected area. The diagnosis may
be confirmed by blood culture, bone scanning, and bone X rays. Treatment is with
high doses of antibiotic drugs over several weeks or months.
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4. Endocarditis: inflammation of the inner layer of the heart,
the endocardium. The most common structures involved are the heart valves.
5. Respiratory pneumonia- causative agent of community acquired pneumonia
6. Cystitis – lower urinary tract infection
Factors that May Predispose one to a Staph aureus Infection:
1. Defects in leukocyte chemotaxis (either congenital like Down’s
Syndrome, or acquired like diabetes or Rheumatoid Arthritis)
2. Chronic granulomatous disease
3. Skin injuries (burns, surgical incisions, eczema)
4. Presence of foreign bodies (sutures, IV catheters, prosthetic devices)
5. Infections with other agents (like viruses especially influenza)
6. Underlying diseases (malignancy, alcoholism and heart disease)
7. Therapeutic antimicrobial therapy
To be discussed in more detail in Chapter 14 in book:
METHODS FOR DETECTING MRSA and GISA/VISA:
MRSA:
Successful detection of MRSA (and MRSE) depends largely on fostering the growth of the more resistant subpopulation. This can be accomplished by:
a. neutral pH (7.0-7.4)
b. cooler temperatures (30-35o C)
c. Prolonged incubation (minimal 24 hours up to 48 hours)
d. Addition of 2% - 4% NaCl to susceptibility test media
1) Microdilution MIC (minimal inhibitory concentration) tests using Mueller-Hinton broth supplemented with divalent cations and 2% sodium chloride incubated at
35o C for a FULL 24 HOURS is the most reliable way of recognizing MRSA. (An alternative screening procedure that is being phased out is the use of Mueller-Hinton
agar supplemented with 4% sodium chloride containing either oxacillin or nafcillin. Growth on these plates indicates MRSA. Methicillin for in vitro testing has
been proven to be unstable thus Oxacillin or Nafcillin being in the same class of antibiotics has been chosen for testing purposes).
2. Using the antibiotic Cefoxitin (30 mcg) is now used in place of Oxacillin which has recently proven to be more sensitive thus a better indicator for detecting mecAmediated resistance in staphylococci.
Zone Interpretation:
Staph aureus
Coagulase-Negative Staphylococci
< 22 = RR (MRSA)
< 24 = RR (MRCNS)
> 22 = SS (MSSA)
>25 = SS (MSCNS)
3. Penicillin-Binding-Protein 2 (PBP2): commercially prepared, rapid latex agglutination tests are available. Latex particles sensitized with a monoclonal antibody
against “penicillin-binding-proteins” will specifically react with methicillin resistant staphylococci to cause agglutination to the unaided eye. (See Procedure)
MICROCOCCUS
- gram positive cocci in tetrads, or clusters
- aerobic, generally a little larger than Staph
- catalase positive
- coagulase negative
- oxidizes glucose or not at all
- modified oxidase positive
- found in the environment
- normal flora on skin and mucous membranes
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- most strains produce pigments (yellow, orange, red, white)
- lysostaphin resistant
- benzidine positive
- bacitracin susceptible
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MICROCOCCUS continued:
Tests to differentiate Micrococcus from Staphylococcus:
1. OF Dextrose: a two-tube medium system (one tube over-layed with oil) used to determine the
fermentative, oxidative or non-utilizing properties of an organism on glucose.
2. Microdase Disk Test (Modified Oxidase Test): this test is only for catalase-positive grampositive cocci. Smear the organism on top of the filter paper impregnated with the substrate
[tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide
(DMSO)] and watch for possession of Cytochrome C as evidenced by a reaction within 2 minutes.
Positive reaction will be blue or purple-blue and no change in color indicates a negative result.
Micrococcus is positive and Staph species are negative.
3. Furazolidone (100g/disk) Susceptibility: Micrococcus species are resistant to furazolidone
whereas both Coagulase-Negative staphylococci and Rothia/ Stomatococci are susceptible (>15
mm).
ROTHIA formerly “Stomatococcus”
- catalase positive (weakly)
- gram positive cocci in clumps or tetrads
- Rothia mucilaginosus (“gummy”) is the only species of clinical significance
- risk factors include the presence of foreign devices especially
indwelling vascular catheters, immunocompromised conditions,
intravenous drug use and cardiac valve disease
(as an agent of endocarditis and sepsis)
- normal flora of oral cavity
- bacitracin: resistant
- microdase/modified oxidase: negative
- furazolidone: susceptible
- colonies are grey to white and may be mucoid in appearance.
They tend to adhere to the agar due to capsular
properties thus difficult to pick up and emulsify (called “sticky Staph”)
- will not grow in increased NaCl media
- Penicillin is usually drug of choice
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MACROCOCCUS
- very large, gram positive cocci (3X Staph size)
- modified oxidase: positive
- clinical significance unknown
PLANOCOCCUS
- motile, yellow, salt-loving
- mostly associated with plants
ALLOIOCOCCUS
- grows very tiny and slowly after 48 hours
- strict aerobe
- catalase: weakly +
- usually recovered from ear; cause of chronic otitis media; best specimen is tympanocentesis
- modified oxidase (microdase): negative
References
Most of the lecture notes produced in this power point presentation were acquired from:
Required Textbook:
Mahon, Connie R., Lehman, Donald C., Manuselis, George,(2015). Textbook of
diagnostic microbiology (5th edition). Saint Louis, Missouri: Saunders-Elsevier
Journals/Publications: Notes have also been ascertained using the following:
a) Laboratory Medicine
b) Advance for Medical Laboratory Professionals
Laboratory Procedures: The procedures used in the following notes with and without pictures were obtained with permission from
Carolinas Medical Center-Main, Microbiology Laboratory Procedure Manuals, to be used for educational purposes only by the faculty of
Carolinas College of Health Sciences, School of Clinical Laboratory Sciences, Medical Laboratory Science Program.
Hand-outs and Lecture Notes from various members and lecturers of the South Eastern Association for Clinical Microbiologists (SEACM) Annual
Meeting Conference
Pictures Obtained from:
www.bacteriainphotos.com
www.microbeworld.org
www.cdc.gov
www.medical-labs.net
www.flickr.com
www.dermapproved.com
www.studyblue.com
www.tabletsmanual.com
www.mddk.com
www.heartline24X7.com
www.medicaljournal.se
www.quizlet.com
The End