Microbiology Lab 9

DNase

a nuclease in S. aureus that digests DNA

API Staph System

a reliable method for identifying 23 species of gram-positive cocci, including 20 clinically important species of staphylococci. consists of 19 microampules that contain dehydrated substrates and/or nutrient media which are important in the indentification of Staphylococcus, Kocuria, and Micrococcus when a positive reaction occurs, there is a color change final species determination must take into consideration other factors such as the source of the specimen, the catalase reaction, colony characteristics, and antimicrobial susceptibility pattern

alpha-prime hemolysis

a small area of intact red blood cells around a colony surrounded by a wider zone of complete hemolysis displayed by some viridans group

pathogenesis of S. aureus

skin infections, wound infections, bone tissue infections, scalded skin infections, scalded skin syndrome, toxic shock syndrome, and food poisoning coagulase production (may form around staphylococcal infection protecting bacterium from host defenses)

staphylococci

"bunch of grapes" gram-positive, spherical bacteria that divide in more than one plane to form irregular clusters of cells. 40 different species; 24 subspecies non-motile, non-spore-forming and able to grow in media containing high salt concentrations; facultative anaerobes normal microbiota of nasal membranes, hair follicles, skin, and the perineum in healthy individuals

What is the role of alpha toxin in the pathogenesis of S. aureus?

Alpha toxin is an exotoxin that destroys red blood cells to release nutrients and growth factors that increase S. aureus multiplication in the host bloodstream

Characteristics to look for in differentiation of staphylococci species:

1. beta-type hemolysis (a-toxin) 2. mannitol fermentation 3. coagulase production if positive for all three >>> S. aureus

Estimate of population that carries Staphylococcus aureus

1/3 of US population

Differentiate between alpha and beta hemolysis

Alpha hemolysis is partial lysis of red blood cells and will create a halo of greenish discoloration around colonies on the blood agar. Beta hemolysis is complete lysis of red blood cells and will create a clear halo around colonies on the blood agar.

Why might hospital patients be tested for nasal carriage of S. aureus?

At-risk patients are tested for S. aureus nasal carriage because of the risk for endogenous infection.

Describe the selective and differential properties of mannitol salt agar (MSA) for the isolation and identification of staphylococci.

As a selective medium, MSA has a high concentration of salt (7.5% NaCl), which is inhibitory to most bacteria other than the staphylococci. As a differential medium, MSA contains the sugar mannitol as a substrate for fermentation and phenol red as a pH indicator to detect the production of acid. S. aureus ferments mannitol and changes the color of the medium from red to yellow. Most other staphylococci do not ferment mannitol.

What test can be performed to differentiate the enterococci from other group D streptococci?

Enterococci are tolerant to 6.5% NaCl whereas other group D streptococci are not.

Beta-hemolytic groups

Group A Streptococci Group B Streptococci Group C Streptococci

What was Rebecca Lancefield's contribution to the study of streptococci?

Lancefield classified streptococci into several major groups (e.g. group A, B, and C) based upon surface carbohydrate antigens (Lancefield antigens).

Two tests that are useful in differentiating S. pyrogens and S. agalactiae

Lancefield-antigen testing (S. pyogenes is group A, S. agalactiae is group B), bacitracin susceptibility (S. pyogenes is susceptible, S. agalactiae is resistant), and CAMP reaction (S. pyogenes is negative, S. agalactiae is positive)

Describe results from a coagulase, DNase, and novobiocin test that would suggest a mixed culture was used for the tests, as opposed to a pure culture.

Mixed cultures of S. aureus and S. saprophyticus would produce positive results for coagulase and DNase and be resistant to novobiocin. A pure cultures of S. aureus would be positive for coagulase and DNase but sensitive to novobiocin. A pure culture of S. saprophyticus would be negative for coagulase and DNase but resistant to novobiocin.

What is the most common cause of hospital-acquired urinary tract infections?

S. epidermidis

coagulase-negative staphylococci

S. epidermidis and S. saprophyticus also do not produce DNase or alpha-toxin unpigmented and appear opaque when grown on blood agar and staphylococcus 110 plates

What are health care acquired infections?

Nosocomial infections are infections acquired in a hospital or healthcare setting.

Why are staphylococcal infections becoming increasingly difficult to treat?

Numerous strains of S. aureus have developed multiple resistances to common antibiotics including penicillins and aminoglycosides and the resulting infections have become difficult to treat effectively.

What is the second most common cause of urinary tract infections in sexually active young women?

S. saprophyticus

What Staphylococci species is resistant to novobiocin?

S. saprophyticus Some strains also ferment mannitol to acid

What test can be performed to differentiate between group A and group C streptococci?

SXT sensitivity (group A strep are resistant, group C strep are sensitive)

Name two tests that are useful in differentiating of pneumococci and oral viridian's streptococci

Optochin susceptibility (S. pneumoniae is susceptible, oral viridans streptococci are resistant) and hemolysis (oral viridians streptococci are weakly alpha hemolytic, while S. pneumonia cultures exhibit strong alpha hemolysis.

When bacteria from a throat swab are streaked on blood agar, why is the agar stabbed several times with the loop?

Stabbing of the blood agar creates an anaerobic environment for the streptococci and improves the development of hemolysis.

Vaginal swabs are taken from the pregnant women in their third trimester. Which streptococcal species is the focus of the investigation?

S. agalactiae (group B strep)

Describe the appearance of an S. agalactiae colony grown on blood agar. Describe how that colony would differ in appearance from a colony of S. pyrogenes

S. agalactiae colonies on blood agar are large with a narrow zone of β- hemolysis S. pyogenes colonies are small with a large zone of β-hemolysis

Group B Streptococci

S. agalactiae is the only recognized species of Lancefield group B.. Found in pharynx, skin, and rectum but more likely to be found in genital and intestinal tracts of healthy adults and infants. important cause of serious neonatal infection involving sepsis and meningitis infections consist of abscesses, endocarditis, septicemia, bone and soft tissue infections, and pneumonia. colonies are large, with a narrow zone of beta-hemolysis. cells are spherical to ovoid preliminary identification of this species relies heavily on a positive CAMP reaction

Staphylococci associated with human disease

S. aureus S. epidermidis S. saprophyticus S. haemolyticus S. lugdunensis

S. aureus ferments...?

S. aureus ferments mannitol to produce acid When grown on mannitol salt sugar (MSA), the production of acid lowers the pH of the medium, causing the phenol red indicator to turn from red to yellow

Why are the staphylococci among the leading causes of nosocomial infections?

S. aureus is an opportunistic pathogen that causes infections of compromised hosts (e.g. surgical patients, burn and trauma patients) in the hospital. Thirty percent of the normal population carries S. aureus so endogenous infections in patients and transmission from healthcare worker to patient are common.

What is the role of coagulase in the pathogenesis of S.aureus?

S. aureus produces coagulase during infection to surround itself in a coat of clotted blood protein, which protects it against host defenses as the bacterium multiplies in the host.

Group D Nonenterococci

S. bovis is found in the intestinal tracts of humans as well as other animals human pathogen that has been implicated as causative agent of endocarditis and meningitis, and is associated with malignancies of the gastrointestinal tract colonies appear large, mucoid, and are either nonhemolytic or alpha-hemolytic in culture, grow in pairs or short chains positive bile esculin test and negative salt broth test

Describe the differential property of blood agar for the isolation and identification of staphylococci.

Staphylococcus aureus produces a toxin that lyses red blood cells whereas Staphylococcus epidermidis and S. saprophyticus do not produce the toxin. Colonies of S. aureus will produce β-hemolysis on blood agar.

In the CAMP reaction, which organism produces the CAMP factor? What substance does the CAMP factor react with to cause enhanced breakdown of RBCs?

Streptococcus agalactiae, the CAMP factor acts synergistically with staphylococcal hemolysins to cause an enhanced breakdown of blood cells

Alpha-hemolytic groups

Streptococcus pneumoniae Viridans Streptococci group Group D Enterococci Group D Nonenterococci

Group A Streptococci

Streptococcus pyogenes is the main representative and is by far the most serious streptococcal pathogen. Humans are primary reservoir. Most likely in pharynx but also isolated from skin and rectum. infections range from pharyngitis and skin infections to scarlet fever, rheumatic fever, and acute glomerlonephritus. can cause childbirth fever (puerperal sepsis) when grown on blood agar, colonies are small (0.5 mm diameter), transparent to opaque, and domed produces clear zone usually two to four times the diameter of the colony Sensitive to bacitracin

Which streptococci are implicated in the development of dental caries? What is the mechanism of their formation?

Viridans streptococci, such as S. mutans, create dental caries by fermenting sugars in the oral cavity and producing acids that erode tooth enamel.

Why is the coagulase test considered to be the definitive test for S. aureus?

Virtually all strains of S. aureus are coagulase positive whereas all other species are negative.

Humans may carry both staphylococci and streptococci as normal microbiota. How might you differentiate between the two genera?

a. Staphylococci occur in clusters and are catalase positive. b. Streptococci occur in chains and are catalase negative.

bile esculin (BE) hydrolysis

all group D streptococci are positive for this test able to hydrolyze esculin in the agar slant, causing slant to blacken (differentiation between two types of group D streptococci, enterococcus and S. bovis, depends on salt tolerance test) if less than half of slant is blackened or if no blackening occurs within 24 to 48 hours, the test is negative

Gram-stain test

all staphylococci will be gram positive

DNase test

clearing on DNase methyl green agar indicates DNA breakdown >>> S. aureus negative DNase test >>> no clearing around streak >>> CNS

beta-hemolysis

complete lysis of red blood cells around a colony and results in clear zone surrounding colonies.

Streptococcus pneumoniae

does not possess Lancefield antigen significant human pathogen; causative agent of bacterial pneumonia and can cause meningitis and otitis media in children normally colonizes the pharynx but can spread to lungs, sinuses, or middle ear virulent strains are covered with a polysaccharide capsule when grown on blood agar, colonies appear smooth, mucoid, and surrounded by a zone of greenish discoloration usually grow as diplococci (also singularly or short chains) presumptive identification can be made with a positive optochin susceptibility test

bacitracin sensitivity

if sensitivity is present, organism is probably S. pyogenes any zone of inhibition is considered postive 2 limitations: 1) the disks must be of the differential type 2) the test should not be applied to alpha-hemolytic streptococci

coagulase test

forms a clot after several hours >>>> coagulase positive >>> S. aureus does not form a clot >>>> coagulase negative >>>> CNS (only valid for gram-positive, staphylococcus-like bacteria because some gram-negative rods can cause clotting like Pseudomonas)

salt tolerance

group D enterococci are salt-tolerant. Other group D organisms are not (S. bovis) a positive result shows up as turbidity within 72 hours. a color change of purple to yellow may also be present If the organism is salt tolerant and BE positive, it is considered to be enterococcus 80% of group B streptococci will grow in this medium

a-toxin

hemolysin in S. aureus that causes a wide, clear sone of beta-hemolysis on blood agar damages red blood cells, leukocytes, heart muscle, and renal tissue

Viridans Streptococci Group

heterogeneous cluster of alpha-hemolytic and nonhemolytic streptococci. colonize the oral cavity, pharynx, gastrointestinal tract, and genitourinary tract oppurtunists with low pathogenicity 2 species are though to be primary cause of dental caries (can cause severe infections) most serious complication is subacute endocarditis on blood agar, colonies appear very small, gray to whitish gray, and opaque in culture, they appear rod-like and grown in chains differentiated by negative result in the bile escullin hydrolysis test, the salt-tolerance test, and the optochin susceptibility test

When differentiating species of staphylococci species, what inhibits growth of other bacteria?

high salt concentration (7.5% sodium chloride)

Novobiocin susceptibility test

if S. saprophyticus does not ferment lactose, it can be identified with novobiocin susceptibility test because it is resistant

CAMP test

if positive, organism is very likely S. agalactiae (B test) If you have an unknown that produce an enlarged arrowhead-shaped hemolytic zone at the juncture where the unknown meets the Staphylococcus aureus streak, the organisms is S. agalactiae. The CAMP factor produce by S. agalactiae acts synergistically with staphylococcal hemolysins, causing an enhanced breakdown of red blood cells and therefore producing the arrowhead zone of clearing. if incubated anaerobically, a positive CAMP reaction can occur on S. pyogenes incubated plates

Rebecca Lancefield

in the 1930s developed a method that uses an alphabetic system to designate different groups of bacteria. Serological tests are used to differentiate antigenic differences in cell wall carbohydrates that occur in the bacteria

fomite

inanimate object

MRSA

methicillin resistant S. aureus a major epidemiological problem in hospitals and recently in the community estimated that 1-2% of population now carries MRSA

streptococci and enterococci

most isolates occur in chains rather than clusters they lack the enzyme catalase, which degrades hydrogen peroxide to form water and oxygen gram-positive facultative anaerobes and generally non-motile

gamma-hemolysis

no effect on red blood cells in a blood agar plate

Colonies on SM110 plate

organisms are salt tolerant S. aureus colonies will appear yellow or orange CNS colonies will appear colorless

alpha hemolysis

partial breakdown of hemoglobin on blood agar, producing greenish discoloration around colonies

staphyloxanthin

pigment in S. aureus that has antioxidant properties which prevent reactive oxygen (superoxide) produced by the host immune system from killing the bacteria responsible for golden color

optochin sensitivity

pneumococci are sensitive and viridans organisms are resistant to these

color changed on mannitol sugar plate from red to yellow

presumed either S. aureus or S. saprophyticus

SXT sensitivity

this test together with bacitracin sensitivity test, is used for identification of group C streptococci (sensitive) groups A and B are uniformly resistant disks contain 1.25mg of trimethoprim and 27.75mg of sulfamethoxazole (SXT) if a beta-hemolytic streptococcus proves to be bacitracin resistant and SXT susceptible, it is classified as being a non-group A or B beta-hemolytic streptococcus (usually group C)

Group C streptococci

uncommon human pathogens (may be involved in zoonoses) S. dysgalactiae, accounts for less than 1% of all bacteremias can cause pharyngitis, endocarditis, and meningitis (usually in patients with underlying illness) large colonies with large zone of beta-hemolysis on blood agar presumptive differentiation of S. dysgalactiae from other beta-hemolytic streptococci is based primarily on resistance to bacitracin and negative CAMP test

Group D Enterococci

variably hemolytic two principle species- E. faecalis and E. faecium predominantly inhabitants of the gastrointestinal tract, although they have been isolated from the genitourinary tract and oral cavity important pathogens in hospitalized patients where they cause urinary tract infections, bacteremia, and endocarditis on blood agar, form large colonies that appear nonhemolytic, alpha-hemolytic, or rarely beta-hemolytic in culture, grow as diplococci in short chains can grow under extreme conditions isolates able to grow in presence of 6.5% NaCl and are able to hydrolyze bile esculin are presume to be enterococci

Colonies on blood agar

wide clear zone represents beta-hemolysis and S. aureus