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GRAM-POSITIVE BACTERIA

Genus Staphylococcus

Objectives: Upon completion of this lecture, the student will:

Outline the medically important staphylococci species.

Describing the morphology & physiology for

staphylococci.

Determine the virulence factors for staphylococci.

Analyze the diseases & pathogenicity for staphylococci.

Demonstrate the epidemiology/transmission for

staphylococci.

Outline the laboratory diagnosis for staphylococci.

State the drug of choice and prophylaxis where regularly

used.

Lec.1 Dr.Sarmad Zeiny 2013-2014 BCM

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GRAM-POSITIVE BACTERIA

Genus Staphylococcus

The 3 medical important species:

- Staphylococcus aureus. - Staphylococcus epidermidis. - Staphylococcus saprophyticus.

Important Properties of genus staphylococcus:

- The Shape: Staphylococci are spherical gram-positive cocci

- Arrangement: arranged in irregular grapelike clusters (Figure 1).

- Biochemically: All staphylococci produce catalase enzyme, whereas no streptococci

do (catalase degrades H2O2 into O2 and H2O), Catalase is an important virulence

factor because H2O2 is microbicidal and its degradation limits the ability of neutrophils

to kill.

- S.aureus is by far the most pathogenic species. S.aureus is distinguished from the others

primarily by coagulase production, for that reason, S.aureus called coagulase positive

Fig.1

Lec.1 Dr.Sarmad Zeiny 2013-2014 BCM

Gram positive cocci

Streptococci (catalase –ve) Staphylococci (catalase +ve)

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staphylococcus. Coagulase is an enzyme that causes plasma to clot by activating

prothrombin to form thrombin. Thrombin then catalyzes the activation of fibrinogen to

form the fibrin clot.

- S.epidermidis and S.saprophyticus are often referred to as coagulase-negative

staphylococci.

- All Staphylococci tolerate high salt concentration (up to 9.5% Nacl).

- S.aureus produces a pigment called staphyloxanthin, which imparts a golden color to

its colonies. This pigment enhances the pathogenicity of the organism by inactivating

the microbicidal effect of superoxides and other reactive oxygen species within

neutrophils.

- Sta. aureus usually ferments mannitol and hemolyzes red blood cells, whereas the

others do not.

- S.saprophyticus resistant to novobiocin (type of antibiotic) while others are sensitive.

- Staphylococci and streptococci are non-motile and do not form spores.

Staphylococcus aureus (Staphylococcus pyogenes):

Distinguishing Features:

• Small, yellow colonies on blood agar.

• β – hemolytic on blood agar (complete destruction of RBC).

• Coagulase positive (all other Staphylococcus species are negative).

• Ferments mannitol on mannitol salt agar.

Fig. 2: Staphylococci lie in grape-like clusters as seen on Gram

stain. Visualize this cluster of hospital staff posing for a group

photo. Staphylococcus aureus is catalase-positive, thus

explaining the cats in the group photo. Staphylococcus aureus

(aureus means "gold") can be differentiated from the other beta-

hemolytic cocci by their elaboration of a golden pigment when

cultured on sheep blood agar. Notice that our hospital Staff

(Staph) all proudly wear gold medals around their necks. Note

how all the Gold-Medalists (Staphylococcus aureus) hang out

together to show each other their gold medals. You can think of

them as coagulating together.

Reservoir:

• Normal flora

- Nasal mucosa (25% of population are carriers)

- Skin

Fig.2

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Transmission:

• Hands

• Sneezing

• Surgical wounds

• Contaminated food

- Custard pastries

- Potato salad

- Canned meats

Virulent factors and pathogenesis:

S.aureus has a microcapsule surrounding its huge peptidoglycan cell wall, which in turn

surrounds a cell membrane containing penicillin binding protein (also called transpeptidase).

Numerous powerful defensive and offensive protein weapons stick out of the microcapsule or

can be excreted from the cytoplasm to wreak havoc on our bodies:

a) Proteins That Disable Our Immune Defenses:

1) Protein A: This protein has sites that bind the Fc portion of IgG. This may protect the

organism from opsonization and phagocytosis. See fig.3

2) Coagulase: This enzyme can lead to fibrin formation around the bacteria, protecting it from

phagocytosis. See fig.3

3) Hemolysins (4 types): Alpha, beta, gamma, and delta. They destroy red blood cells,

neutrophils, macrophages, and platelets. See fig.4

4) Leukocidins: They destroy leukocytes (white blood cells). See fig.4

5) Penicillinase: This is a secreted form of beta-lactamase. It disrupts the beta-lactam portion

of the penicillin molecule, thereby inactivating the antibiotic.

6) Novel penicillin binding protein: This protein, also called transpeptidase, is necessary for

cell wall peptidoglycan formation and is inhibited by penicillin. Some strains of Staphylococcus

aureus have new penicillin binding proteins that are resistant to penicillinase - resistant

penicillins and cephalosporins.

Fig. 3: Staphylococcus aureus wielding protein A and coagulase shields,

defending itself from attacking antibodies and phagocytosis.

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b) Proteins to Tunnel Through Tissue:

1) Hyaluronidase ("Spreading Factor"): This protein breaks down proteoglycans in

connective tissue.

2) Staphylokinase: This protein lyses formed fibrin clots (like streptokinase).

3) Lipase: This enzyme degrades fats and oils, which often accumulate on the surface of

our body. This degradation facilitates Staphylococcus aureus colonization of sebaceous

glands.

4) Protease: destroys tissue proteins. See fig.5

Fig.4: Luckless red blood cell following a neutrophil, running to destruction at the

hands of Staphylococcus aureus and its hemolysis and leukocidin dynamite.

Fig.5: Staphylococcus aureus produces proteins that

allow the bacteria to tunnel through tissue.

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c) Exotoxin Assault Weaponry: (see fig.6)

1) Exfoliatin: A diffusible exotoxin that causes the skin to slough off ( SSS “scalded skin

syndrome”). 2) Enterotoxins (heat stable): Exotoxins which cause food poisoning 2-6h after eating

preformed toxin, resulting in vomiting and diarrhea. (See fig.7)

3) Toxic Shock Syndrome toxin (TSST-1): This exotoxin is analogous to the pyrogenic

toxin produced by Lancefield group A beta-hemolytic streptococci, but is far more

deadly. This exotoxin causes toxic shock syndrome and is found in 20% of S. aureus

isolates. These pyrogenic toxins are called superantigens which bind to the MHC class II

molecules on antigen presenting cells (such as macrophages). The toxin-MHC II complex

causes a massive T cell response and outpouring of cytokines, resulting in the toxic

shock syndrome described below. (See Fig.8).

Fig.6: Staphylococcus aureus produces exotoxin assault weaponry.

Fig. 8: Toxic shock syndrome caused by Staphylococcus

aureus releasing TSST-1.

Fig.7: Staphylococcus aureus

gastroenteritis. "I told you not to eat

the mayonnaise, sweetheart!"

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Diseases:

Disease caused by exotoxin release:

1) Gastroenteritis (food poisoning).

2) Toxic shock syndrome.

3) Scalded skin syndrome(SSS).

Disease resulting from direct organ invasion: see fig.9

1) Pneumonia

2) Meningitis

3) Osteomyelitis (most common cause).

4) Acute bacterial endocarditis (rapid and destructive, emergency case)

5) Septic arthritis (most common cause in pediatric age and adult above 50, emergency case)

6) Skin infections (Folliculitis, Pustule, Boil (furuncle): e.g. Stye, Carbuncle, Impetigo,

cellulites).

7) Bacteremia/sepsis

8) Urinary tract infection.

Predisposing Factors for Infection:

• Surgery/wounds

• Foreign body (tampons, surgical packing,

sutures)

• Severe neutropenia (<500/ µl)

• Intravenous drug abuse.

• Chronic granulomatous disease

• Cystic fibrosis

Fig.9: Diseases caused by direct organ invasion by

Staphylococcus aureus. Visualize the Staph-wielding

wizard. (Note the cluster of staphylococci at the head

of his staff.)

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Staphylococcus aureus and antibiotics (very important):

More than 90% of Sta. aureus strains contain plasmids that encode β-lactamase

(penicillinase), the enzyme that degrades many, but not all, penicillins. Rx. With

β-lactamase–resistant penicillins, e.g., nafcillin or cloxacillin.

Some strains of Sta. aureus are resistant to the β -lactamase–resistant penicillins, such

as methicillin and nafcillin, by feature of changes in the penicillin-binding protein (PBP)

in their cell membrane. Genes on the bacterial chromosome encode these altered PBPs.

These strains are commonly known as methicillin-resistant Sta. aureus (MRSA) or

nafcillin-resistant Sta. aureus (NRSA). MRSA currently accounts for more than 50% of

Sta. aureus strains isolated from hospital patients in the United States Rx. With

vancomycin.

Strains of Sta. aureus with intermediate resistance to vancomycin (VISA) and with full

resistance to vancomycin (VRSA) have also been detected. The gene that encodes

vancomycin resistance in Sta. aureus are located on a plasmid. MRSA, NRSA, VRSA &

VISA can be treated with either Linezolid, quinupristin/dalfopristin or daptomycin.

Mupirocin is very effective as a topical antibiotic in skin infections caused by Sta. aureus.

It has also been used to reduce nasal carriage of the organism in hospital personnel and in

patients with recurrent staphylococcal infections.

S.aureus can resist antibiotics by:

1) β –lactamase production, encoded by plasmid.

2) Change in molecular structure of PBP, encoded by bacterial chromosome.

3) Resistant to Vancomycin, transmitted from other bacteria, encoded by plasmid.

Staphylococcus epidermidis (Staphylococcus albus):

Normal bacterial flora of the human skin.

It is coagulase-negative.

Infections are almost always hospital-acquired: compromised hospital patients with

Foley urine catheters or intravenous lines can become infected when this organism

migrates from the skin along the tubing.

Diseases: Infections of prosthetic devices in the body, such as prosthetic joints,

prosthetic heart valves, CSF shunts, and peritoneal dialysis catheters. In fact,

Staphylococcus epidermidis is the most frequent organism isolated from infected

indwelling prosthetic devices.

The organisms have a polysaccharide capsule (slime layer) that allows adherence to

these prosthetic materials.

Sta. epidermidis is highly antibiotic resistant. Most strains produce -lactamase and

many are methicillin-/nafcillin-resistant due to altered penicillin-binding proteins. The

drug of choice is vancomycin.

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Staphylococcus saprophyticus

This organism is a leading cause (second only to E.coli) of urinary tract infections

(UTI) in sexually active young women "Honeymoon cystitis".

It is most commonly acquired by females in the community (NOT in the hospital).

This organism is coagulase-negative. Urinary tract infections can be treated with a quinolone, such as norfloxacin, or with

trimethoprim-sulfamethoxazole. See table1.

Laboratory features for genus Staphylococcus:

Slide: (microscopic characteristics) :A Gram’s stained film will shows spherical G+ve

cocci arranged in grape like irregular

Culture: (macroscopic characteristics):

- Aerobic or facultatively anaerobic,

- Grow in ordinary media as (nutrient agar or blood agar).

- Tolerate high salt conc. (9%NaCl).

- The colonies are round, smooth, raised & glistening,

- Producing pigments: S. epidermidis & S.saprophyticus white, S. aureus appear

golden-yellow colonies.

- Only S.aureus produces β-hemolysis, a zone of β-hemolysis will appear as clear zone

around the colony on blood agar.

- Only S.aureus can ferment mannitol.

The selective & differential medium used is called (Mannitol salt agar), this medium

contain: Mannitol +7.5% NaCL + Phenol red (indicator), the pink color of the indicator

turn to yellow because of mannitol fermentation & acid production (only by S.aureus), &

the salt inhibit most other normal flora.

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Steps of laboratory Diagnosis:

1) Specimens: according to type of infection, such as pus, blood, urine…etc.

2) Gram Stain: Gram’s positive cocci, Grape-like arrangement (clusters).

3) Culture: on both

- Blood agar: see large, round, smooth, raised & glistening white or golden color, β-

hemolysis if S.aureus.

- Mannitol Salt Agar: S.aurues: yellowish discoloration of media, other Staphylococci:

no discoloration (remains pink media)

4) Biochemical tests:

a) Coagulase test: is recognized as the most important test for testing the virulence of

S. aureus which is the only coagulase +ve staphylococci. So this test used to detect the

ability of S. aureus to clot blood plasma (fibrinogen fibrin). There are two main

methods: Slide method and tube method.

a) Catalase test: This is done to differentiate Staphylococci from Streptococci. A drop

of 3% H2O2 solution placed on a slide, and a small amount of bacterial growth is in the

solution placed on the slide, The formation of bubbles indicates a positive test of oxygen

release.

5) Serological test: Latex agglutination test to detect S.aureus.

6) Antibiotic Sensitivity test.

7) For epidemiological purposes, Sta. aureus can be subdivided into subgroups based on

the susceptibility of the clinical isolate to lysis by a variety of bacteriophages. A person

carrying Sta. aureus of the same phage group as that which caused the outbreak may be

the source of the infections.

Prevention: (very important):

There is no vaccine against staphylococci.

Cleanliness, frequent hand washing, and aseptic management of lesions help to control

spread of Sta. aureus.

Persistent colonization of the nose by Sta. aureus can be reduced by intranasal

mupirocin or by oral antibiotics, such as ciprofloxacin or trimethoprim-

sulfamethoxazole, but is difficult to eliminate completely.

Shedders may have to be removed from high-risk areas, e.g., operating rooms and

newborn nurseries.

Cefazolin is often used perioperatively to prevent staphylococcal surgical-wound

infections.

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It is extremely important to know how to differentiate staphylococci from streptococci because

most staphylococci are penicillin G resistant.

Both staphylococci and streptococci are gram-positive cocci, but they are distinguished by

two main criteria:

1. Microscopically, staphylococci appear in grapelike clusters, whereas streptococci are in

chains.

2. Biochemically, staphylococci produce catalase (i.e., they degrade hydrogen peroxide),

whereas streptococci do not.

Summary:

• S.aureus is the most pathogenic species because of their virulent factors.

• S.aureus colonizes nasal mucosa (25% of population are carriers). • Staphylococci easily diagnosed in the lab. By using Gram ’s staining,

culture and biochemical tests. • S.aureus and S.epidermidis are highly antibiotic resistance and causing

nasty hospital acquired infections. • Staphylococci infection can be prevented by regular hygiene precautions

and by antibiotics, no vaccine.

Table 1: summarized the points of differences between 3 medically

important Staphylococci species

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References:

- Review of Medical Microbiology and Immunology11th edition 2010.

- Clinical Microbiology Made Ridiculously Simple, 6th ed, 2014.

- Baily & Scott diagnostic microbiology, 12th ed.

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