THE GENUS STAPHYLOCOCCUS

Dr.T.V.Rao MD

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What are Staphylococcus

Sir Alexander Ogston, a Scottish surgeon, first showed in 1880 that a number of human pyogenic diseases were associated with a cluster-forming micro-organism. He introduced the name

'staphylococcus' (Greek: staphyle = bunch of grapes; kokkos = grain or berry), now used as the genus name for a group
of facultatively anaerobic, catalase-positive, Grampositive cocci.
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Staphylococci: Gram positive

cocci ( from Greek staphyle, means bunch of grapes ) that occur singly and in pairs, short chains and irregular grape-like clusters.

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Morphology of Staphylococcus

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S. aureus morphology

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Classification
Family
Micrococcaceae

Genus
Micrococcus and Staphylococcus

Species
more than 20 species

S. aureus S. saprophyticus S. epidermidis M. luteus

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The genus Staphylococcus contains about forty species and subspecies today.
Only some of them are important as human pathogens:

Staphylococcus aureus Staphylococcus epidermidis Staphylococcus hominis Staphylococcus haemolyticus Staphylococcus saprophyticus others
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Structure and Physiology

Gram-positive cocci, nonmotile, facultative anaerobes Cells occur in grapelike clusters because cells division occurs along different planes and the daughter cells remain attached to one another Salt-tolerant: allows them to tolerate the salt present on human skin Tolerant of desiccation: allows survival on environmental surfaces (fomites)
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The species Staphylococcus aureus Morphology

Gram-positive, spherical cells, mostly arranged in irregular grape like clusters. Polysaccharide capsule is only rarely found on cells. The peptidoglycan layer is the major structural component of the cell wall. It is important in the pathogenesis of staphylococcal infections. Other important component of cell wall is teichoic acid. Protein A is the major protein component of the cell wall. It is located on the cell surface but is also released into the culture medium during the cell growth. A unique property of protein A is its ability to bind to the Fc part of all IgG molecules except IgG3. It is not an antigen-antibody specific reaction.
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S aureus A Unique Organism

PVL

Dr.T.V.Rao MD Adapted from: Lowy. N Engl J Med. 1998;339:520-532.

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Virulence Factors in Staphylococcus

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The genus Staphylococcus can be divided into two subgroups (on the basis of its ability to clot blood plasma by enzyme coagulase):

coagulase-positive,

coagulase-negative.
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Physiology and structure

Capsule or polysaccharide slime layer
Peptidoglycan layer Teichoic acid Protein A Cytoplasmatic membrane

Clumping factor
Cytoplasma

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Capsule or polysaccharide slime layer

A loose-fitting, polysaccharide layer (slime layer) is only occasionally found in staphylococci cultured in vitro, but is believed to be more commonly present in vivo. Eleven capsular serotypes have been identified in S. aureus, with serotypes 5 and 7 associated with majority of infections.

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Capsule helps Staphylococcus

The capsule protects the bacteria by inhibiting the chemotaxis and phagocytosis of staphylococci by polymorph nuclear leukocytes, as well as by inhibiting the proliferation of mononuclear cells. It is also facilitates the adherence of bacteria to catheters and other synthetic material.

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Staphylococcal enzymes
Coagulase Catalase Hyaluronidase Fibrinolysin Lipases Nuclease Penicillinase

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Enzymes
1. Coagulase
Triggers blood clotting

2. Hyaluronidase
Breaks down hyaluronic acid, enabling the bacteria to spread between cells

3. Staphylokinase
Dissolves fibrin threads in blood clots, allowing Staphylococcus aureus to free itself from clots
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Enzymes (cont.)
4.

Lipases
Digest lipids, allowing staphylococcus to grow on the skins surface and in cutaneous oil glands

5. -lactamase Breaks down penicillin Allows the bacteria to survive treatment with -lactam antimicrobial drugs
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Protein A
The surface of most S. aureus strains (but not the coagulase-negative staphylococci) is uniformly coated with protein A. This protein is covalently linked to the peptidoglycan layer and has a unique affinity for binding to the Fc receptor of immunoglobulin IgG. The presence of protein A has been exploited in some serological tests, in which protein A-coated S. aureus is used as a nonspecific carrier of antibodies directed against other antigens. Additionally, detection of protein A can be used as a specific identification test for S. aureus.
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Peptidoglycan
Half of the cell wall by weight is peptidoglycan, a feature common to gram-positive bacteria. The subunits of peptidoglycan are N-acetylmuramic acid and N-acetylglucosoamine. Unlike gram-negative bacteria, the peptidoglycan layer in gram-positive bacteria consists of many cross-linked layers, which makes the cell wall more rigid.

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Teichoic acid
Teichoic acid is species-specific, phosphatecontaining polymers that are bound covalently to the peptidoglycan layer or through lipophilic linkage to the cytoplasmic membrane (lipoteichoic acid). Teichoic acid mediates the atachment of staphylococci to mucosal surfaces through its specific binding to fibronectin.

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Coagulase and other surface proteins The outer surface of most strains of S. aureus contains clumping factor (also called bound coagulase). This protein binds fibrinogen, converts it to insoluble fibrin, causing the staphylococci to clump or aggregate. Detection of this protein is the primary test for identifying S. aureus.

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 Other surface proteins that appear to be important for adherence to host tissues include:

collagen-binding protein elastin-binding protein fibronectin-binding protein

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Cytoplasmic membrane

The cytoplasmic membrane is made up of a

complex of proteins, lipids, and small amount
of carbohydrates.

It serve as an osmotic barrier for the cell and

provides an anchorage for the cellular

biosynthetic and respiratory enzymes.

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Staphylococcal toxins
S. aureus produces many virulence factors, including at least five cytolytic or membrane-damaging toxins: alpha toxin beta toxin delta toxin gamma toxin Panton-Valentin toxin two exfoliative toxins eigth enterotoxins (A-E, G-I) Toxic Shock Syndrome Toxin 1 (TSST-1)

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Toxin-mediated
Toxic shock syndrome toxin (TSST-1) is a super-antigen capable of activating large number of T cells Was associated with use of tampons but is also known to be associated with postoperative wound or soft tissue infections
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TSST and other Toxins

The enterotoxins and TSST-1 belong to a class of polypeptide known as super antigens. Staphylococcus aureus strains produce several other extracellular, biologically active substances, including proteases, phosphatases, lipases, lysozyme etc.
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Exfoliative toxins
Staphylococcal scalded skin syndrome (SSSS), a spectrum of diseases characterized by exfoliative dermatitis, is mediated by exfoliative toxins. The prevalence of toxin production in S. aureus strains varies geographically but is generally less than 5% to 10%.
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 Preformed, heat-resistant enterotoxin mediates staphylococcal food poisoning (symptoms in 2-6 hours; usually self-limiting) Exfoliative toxins A and B results in staphylococcal scalded skin syndrome; usually in infants and neonates

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Enterotoxins
Eigth serologically distinct staphylococcal enterotoxins (A-E, G-I) and three subtypes of enterotoxin C have been identified. The enterotoxin are stable to heating at 100 C for 30 minutes and are resistant to hydrolysis by gastric and jejunal enzymes.

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Enterotoxins
Thus, once a food product has been contaminated with enterotoxin-producing staphylococci and the toxin have been produced, neither reheating the food nor the digestive process will be protective. These toxins are produced by 30% to 50% of all S. aureus strains. Enterotoxin A is most commonly associated with disease. Enterotoxins C and D are found in contamined milk products, and enterotoxin B causes staphylococcal pseudomembranous enterocolitis.

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Toxic Shock Syndrome Toxin - 1

TSST-1, formerly called pyrogenic exotoxin C and enterotoxin F, is a heat and proteolysis resistant, chromozomally mediated exotoxin. The ability of TSST-1 to penetrate mucosal barriers, even though the infection remains localized in the vagina or at the site of a wound, is responsible for the systemic effects of TSS. Death in patients with TSS is due to hypovolemic shock leading to multiorgan failure.

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Catalase

All staphylococci produce catalase,

which catalyzes the conversion of toxic hydrogen peroxide to water and oxygen. Hydrogen peroxide can accumulate during bacterial metabolism or after phagycytosis.
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 S. aureus strains possess two forms of coagulase:

Coagulase

bound, free.

Coagulase bound to the staphylococcal cell wall can directly convert fibrinogen to insoluble fibrin and cause the staphylococci to clump. The cell-free coagulase accomplishes the same result by reacting with a globulin plasma factor.
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Staphylococcus aureus coagulation of plasma* Free staphylocoagulase

Fibrinogen + CRF Fibrin

*EDTA Rabbit plasma is preferred for the free or tube coagulase test because it contains a large amount of CRF
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Normal coagulation of plasma Thrombin Fibrinogen (soluble )

Coagulase

Fibrin (insoluble)

What is the role of Coagulase

The role of coagulase in the pathogenesis of disease is speculative, but coagulase may cause the formation of fibrin layer around a staphylococcal abscess, thus localizing the infection and protecting the organisms from phagocytosis.
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Hyaluronidase

Hyaluronidase hydrolyzes hyaluronic

acid, the acidic mucopolysaccharides present in the acellular matrix of connective tissue. This enzyme facilitates the spread of S. aureus in tissues. More than 90% of S. aureus strains produce this enzyme.
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Fibrinolysin

Fibrinolysin, also called staphylokinase, is

produced by virtually all S. aureus strains and can dissolve fibrin clots.

Staphylokinase is distinct from the

fibrinolytic enzymes produced by streptococci.
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The genus Staphylococcus can be divided into two subgroups (on the basis of its ability to clot blood plasma by enzyme coagulase):

coagulase-positive,

coagulase-negative.

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Subgroup of coagulase-negative species contains from human staphylococci:

Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri and other.
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The species Staphylococcus aureus Morphology

Gram-positive, spherical cells, mostly arranged in irregular grape like clusters. Polysaccharide capsule is only rarely found on cells. The peptidoglycan layer is the major structural component of the cell wall. It is important in the pathogenesis of staphylococcal infections. Other important component of cell wall is teichoic acid. Protein A is the major protein component of the cell wall. It is located on the cell surface but is also released into the culture medium during the cell growth. A unique property of protein A is its ability to bind to the Fc part of all IgG molecules except IgG3. It is not an antigen-antibody specific reaction.
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The species Staphylococcus aureus culture characteristics

Colonies on solid media are round, regular, smooth, slightly convex and 2 to 3 mm in diameter after 24h incubation. Most strains show a -hemolysis surrounding the colonies on blood agar. S. aureus cells produce cream, yellow or orange pigment.
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Blood agar plate, S. aureus

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The species Staphylococcus aureus resistance

Like most of medical important nonsporeforming bacteria, S. aureus is rapidly killed by temperature above 60 C S. aureus is susceptible to disinfectants and antiseptics commonly used. S. aureus can survive and remain virulent long periods of drying especially in an environment with pus.
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The species Staphylococcus aureus pathogenicity

S. aureus is pathogenic for human as

well as for all domestic and freeliving warm-blooded animals. S. aureus causes disease through the production of toxin or through direct invasion and destruction of tissue.
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Clinical Manifestations
The clinical manifestations of some staphylococcal diseases are almost exclusively the result of toxin activity (e.g. staphylococcal food poisoning and TSS), whereas other diseases result from the proliferation of the staphylococci, leading to abscess formation and tissue destruction (e.g. cutaneous infection, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis).
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Staphylococcal Diseases
Systemic Disease
Toxic shock syndrome-TSS toxin is absorbed into the blood and causes shock Bacteremia-presence of bacteria in the blood Endocarditis-occurs when bacteria attack the lining of the heart Pneumonia-inflammation of the lungs in which the alveoli and bronchioles become filled with fluid Osteomyelitis-inflammation of the bone marrow and the surrounding bone

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Factors predisposing to S. aureus infections

Host factors Breach in skin
Chemotaxis defects Opsinization defects Neutrophil functional defects Diabetes mellitus Presence of foreign bodies

Pathogen Factors
Catalase (counteracts host defenses) Coagulase Hyaluronidase Lipases (Imp. in disseminating infection) B lactamase(ass. With antibiotic resistance)
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Clinical diseases
Staphylococcal pyogenic infections:
folliculitis, furuncle, carbuncle, bullous impetigo, panaritia and paronychia, wound infections, mastitis, osteomyelitis, staphylococcal pneumonia and other.

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Defining Staphylococcal Diseases

Range from localized to systemic Localized cutaneous infections invade skin through wounds, follicles, or glands
Folliculitis superficial inflammation of hair follicle; usually resolved with no complications but can progress Furuncle boil; inflammation of hair follicle or sebaceous gland progresses into abscess or pustule Carbuncle larger and deeper lesion created by aggregation and interconnection of a cluster of furuncles Impetigo bubble-like swellings that can break and peel away; most common in newborns
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Clinical diseases
Stahylococccal intoxications:
staphylococcal food poisoning (enterotoxicosis),
exfoliative intoxications (Ritters disease or SSSS),

staphylococcal toxic shock syndrome (TSS).

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Boil (Furuncle )

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Stye

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Surgical wound infections: many causes including S. aureus

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Toxic Shock Syndrome Toxin

Super antigen Non-specific binding of toxin to receptors triggers excessive immune response
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TSS Symptoms
8-12 h post infection Fever Susceptibility to Endotoxins Hypotension Diarrhea Multiple Organ System Failure Erythroderma (rash)
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Treatment
Antistaphylococcal antibiotics of the first choice:
oxacillin (methicillin) cephalosporins of I. generation (cefazolin, cephalotin)

Antistaphylococcal antibiotics of the second choice:

lincosamides (e.g. clindamycin) glycopeptides (vancomycin, teicoplanin) linezolid tigecyklin daptomycin and others

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Antibiotic sensitivity pattern

Very variable and not predictable
Very imp. In Pt. Management Mechanisms 1.B lactamase production - plasmid mediated
Has made S. aureus resistant to penicillin group of antibiotics 90% of S. aureus (Gp A) B lactamase stable penicillin's (cloxacillin, oxacillin, methicillin) used

2. Alteration of penicillin binding proteins

(Chromosomal mediated) Has made S. aureus resistant to B lactamase stable penicillins 10-20% S. aureus Gp (B) GH Colombo/THP resistant to all Penicillins and Cephalosporins) Vancomycin is the drug of choice
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MRSA
Methicillin-resistant S. aureus Resistant to all penicillins, cephalosporins, and penems Usually multiply-resistant Vancomycin resistance is very rare so far Hospital-acquired Community-acquired cases now (CA MRSA)
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Panton-Valentine Leukocidin
Panton-Valentine Leukocidin (PVL) consists of 2 components S and F, together with exotoxin lyses WBC resulting in massive release of inflammatory mediators responsible for necrosis and severe inflammation PVL is an important virulence factor in MRSA infections
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Diagnosis
Microscopy smears of clinical materials are stained according to Gram stain Cultivation on solid media (agar, usually blood agar) Biochemical tests Phage typing susceptibility of S. aureus strains to various temperature phages

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MRSA
Methicillin-resistant S. aureus Resistant to all penicillins, cephalosporins, and penems Usually multiply-resistant Vancomycin resistance is very rare so far Hospital-acquired Community-acquired cases now (CA MRSA)
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Coagulase-negative staphylococcal spp (Cons)

S. epidermidis most frequently isolated staphylococcal spp. Colonizes moist body areas such as axilla, inguinal and perianal areas, anterior nares and toe webs Important cause of nosocomial infection esp. S. epidermidis Usually causes nosocomial infections in patients with predisposing factors such as immunodeficiency/ immunocompromised or presence of foreign bodies
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Diagnosis
Specimen Smear Culture

Coagulase test
Biochemical Reactions Antibiograms Typing
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Epidemiology
Staphylococci are ubiquitous. All persons have coagulasenegative staphylococci on their skin, and transient colonization of moist skin folds with S. aureus is common. Colonization of the umbilical stump, skin and perineal area of neonates with S. aureus is common. S. aureus and coagulase-negative staphylococci are also found in the oropharynx, gastrointestinal and urogenital tract. Because staphylococci are found on the skin and in the nasopharynx, shedding of the bacteria is common and is responsible for many hospital-acquired infections.

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. Staphylococcus epidermidis
Skin commensal Has predilection for plastic material Ass. With infection of IV lines, prosthetic heart valves, shunts Causes urinary tract infection in catheterized patients Has variable ABS pattern Treatment should be aided with ABST
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Importance of S.saprophyticus
S. saprophyticus frequently isolated in rectum and genitourinary tract of young women Can be causative agent in UTI in young healthy women 2nd most common urinary pathogen (other than E. coli) in uncomplicated cystitis in young women Colony counts of 105 CFU/ml usu. indicative of significant Bacteriuria
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. Staphylococcus saprophyticus
Skin commensal Imp. Cause of UTI in sexually active young women Usually sensitive to wide range of antibiotics
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Peptococcus species
Members of the genus Peptococcus are strictly anaerobic cluster-forming gram-positive cocci. These microorganisms grow only under anaerobic conditions. They are part of the normal microflora of the mouth, upper respiratory tract, bowel, and female genital tract. They often participate with many other bacterial species in mixed anaerobic infections in the abdomen, pelvis, lung, or brain.
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Related microorganisms
The genus Micrococcus (the genus contains two species)
Micrococcus luteus Micrococcus lyla

Both species are found in nature and colonize humans, primarily on the surface of the skin. Although micrococci may be found in patients with opportunistic infections, their isolation in clinical specimen usually represents clinically insignificant contamination with skin flora.
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Related microorganisms
The genus Stomatococcus Stomatococcus mucilaginosus, the only species in this genus, is a commensal microorganism that resides in the oropharynx and upper respiratory tract.

In recent years, this microorganism has been reported to be the cause of an increasing number of opportunistic infections (endocarditis, septicemia, catheter-related infections) in immunocompromised patients.

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Clinical Concerns and Treatment

95% have penicillinase and are resistant to penicillin and ampicillin MRSA methicillin-resistant S. aureus carry multiple resistance Some strains have resistance to all major drug groups except vancomycin Abscesses have to be surgically perforated Systemic infections require intensive lengthy therapy

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Antimicrobial susceptibility
MRSA can be due to 3 different resistance mechanisms
Production of penicillin-binding protein 2a (PBP2a) encoded by mecA gene Production of beta-lactamase Production of modified intrinsic PBPs

Resistance due to mecA can be detected via cefoxitin disk diffusion or dilution methods according to CLSI breakpoints ( 21mm resistant, 8g/ml resistant, respectively) Resistance due to beta-lactamase production can be detected via the use of beta-lactamase inhibitor such as clavulanic acid which would result in an increase in zone size (disk diffusion method) or decrease of 2 dilutions
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Prevention
Prevention
Hand antisepsis is the most important measure in preventing nosocomial infections Also important is the proper cleansing of wounds and surgical openings, aseptic use of catheters or indwelling needles, an appropriate use of antiseptics
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