Staphylococcus Notes

Staphylococcus Aureus

Trigger Words

• Coagulase
• Cytotoxins
• Exfoliative toxins
• Enterotoxins
• Toxic shock syndrome toxin
• MRSA

Biology and Virulence

• Catalase-positive, gram-positive cocci arranged in clusters.
• Characterized by coagulase and protein A presence.
• Virulence factors:
  • Structural components for adherence to host tissues.
  • Mechanisms to avoid phagocytosis.
  • Toxins and hydrolytic enzymes (refer to Table 18.3).
• Hospital- and community-acquired infections with MRSA are a significant worldwide problem.

Epidemiology

• Normal flora on human skin and mucosal surfaces.
• Can survive on dry surfaces for extended periods due to a thickened peptidoglycan layer and the absence of an outer membrane.
• Spread through person-to-person contact or contaminated fomites such as bed linens and clothing.
• Risk factors:
  • Foreign bodies (splinter, suture, prosthesis, catheter).
  • Previous surgical procedures.
  • Antibiotics that suppress normal microbial flora.
• Patients at risk for specific diseases:
  • Infants (scalded skin syndrome).
  • Young children with poor hygiene (impetigo and cutaneous infections).
  • Patients with intravascular catheters (bacteremia and endocarditis) or shunts (meningitis).
  • Patients with compromised pulmonary function or a viral respiratory infection (pneumonia).
• MRSA is now the most common cause of community-acquired skin and soft-tissue infections.

Diseases

• Toxin-mediated diseases:
  • Food poisoning.
  • Toxic shock syndrome.
  • Scalded skin syndrome.
• Pyogenic diseases:
  • Impetigo.
  • Folliculitis.
  • Furuncles.
  • Carbuncles.
  • Wound infections.
• Other systemic diseases.

Diagnosis

• Microscopy is useful for pyogenic infections but not for blood or toxin-mediated infections.
• Staphylococci grow rapidly on nonselective media.
• Selective media (e.g., chromogenic agar, mannitol-salt agar) can recover $S. aureus$ from contaminated specimens.
• Nucleic acid amplification tests screen for MSSA and MRSA carriage.
• Identification via biochemical tests (e.g., coagulase), molecular probes, or mass spectrometry.

Treatment, Prevention, and Control

• Localized infections are managed by incision and drainage; systemic infections require antibiotic therapy.
• Empirical therapy targets MRSA strains.
• Oral antibiotics: trimethoprim-sulfamethoxazole, doxycycline/minocycline, clindamycin, or linezolid.
• Intravenous therapy: vancomycin (drug of choice), daptomycin, tigecycline, or linezolid.
• Treatment for food poisoning is symptomatic.
• Proper wound cleansing and disinfectants prevent infections.
• Thorough hand washing and covering exposed skin prevent spread.

Coagulase-Negative Staphylococci

Trigger Words

• Opportunistic
• Slime layer
• Subacute

Biology and Virulence

• Catalase-positive, coagulase-negative, gram-positive cocci arranged in clusters.
• Relatively avirulent, but slime layer production allows adherence to foreign bodies (e.g., catheters, grafts, prosthetic valves/joints, shunts) and protection from phagocytosis and antibiotics.

Epidemiology

• Normal human flora on skin and mucosal surfaces.
• Can survive on dry surfaces for long periods.
• Spread through direct contact or contaminated fomites, but most infections involve the patient’s own organisms.
• Patients are at risk when a foreign body is present.
• Ubiquitous, with no geographic or seasonal limitations.

Diseases

• Infections include:
  • Subacute endocarditis.
  • Infections of foreign bodies.
  • Urinary tract infections.

Diagnosis

• Similar to $S. aureus$ infections.

Treatment, Prevention, and Control

• Oxacillin (or other penicillinase-resistant penicillin) is used, or vancomycin for oxacillin-resistant strains.
• Removal of the foreign body is often required.
• Prompt treatment is necessary to prevent further tissue damage or immune complex formation.

General Characteristics of Staphylococci

• Gram-positive cocci, spherical shape, no endospores.
• Catalase-positive (differentiates from Streptococcus and Enterococcus).
• Grow in grape-like clusters.
• Size: 0.5 to 1.5 µm in diameter.
• Can grow in aerobic and anaerobic conditions, high salt concentrations (e.g., 10% sodium chloride), and temperatures from 18°C to 40°C.
• Over 80 species and subspecies found on human skin and mucous membranes.
• Specific niches:
  • $S. aureus$: anterior nares.
  • $S. capitis$: sebaceous glands (e.g., forehead).
  • $S. haemolyticus$ and $S. hominis$: apocrine glands (e.g., axilla).
• Important human pathogens causing opportunistic infections and systemic diseases.
• Most common species associated with human diseases: $S. aureus$, $S. epidermidis$, $S. lugdunensis$, and $S. saprophyticus$.
• MRSA causes serious infections in hospitalized patients and healthy individuals.
• $S. aureus$ colonies can have a yellow or gold color due to carotenoid pigments.
• $S. aureus$ produces coagulase, which converts fibrinogen to fibrin, forming a clot when suspended in plasma.
• Coagulase-negative staphylococci are less virulent and cause opportunistic infections.

Physiology and Structure of Staphylococci

Capsule and Slime Layer

• Polysaccharide capsule covers the cell wall.
• Serotypes 1 and 2: thick capsules, mucoid colonies, rarely associated with human disease.
• Serotypes 5 and 8: associated with ~75% of human infections.
• Capsule protects bacteria by inhibiting phagocytosis by polymorphonuclear leukocytes (PMNs).
• Slime layer (biofilm): water-soluble film of monosaccharides, proteins, and small peptides produced by staphylococci.
• Extracellular substance binds bacteria to tissues and foreign bodies, important for coagulase-negative staphylococci survival.

Peptidoglycan and Associated Enzymes

• Peptidoglycan forms half the cell wall by weight.
• Layers of glycan chains with N-acetylmuramic acid and N-acetylglucosamine subunits.
• Oligopeptide side chains are attached to N-acetylmuramic acid subunits and cross-linked with peptide bridges.
• Gram-positive bacteria have many cross-linked layers for rigidity.
• Penicillin-binding proteins (PBPs) catalyze peptidoglycan layer construction and are targeted by penicillins and β-lactam antibiotics.
• Resistance to methicillin and related antibiotics is mediated by the mecA and mecC genes encoding PBP2a, which has low affinity for these antibiotics.
• mecA gene is on the staphylococcal cassette chromosome mec (SCCmec).
• MRSA strains are now present in the community, not just hospitals.
• Peptidoglycan has endotoxin-like activity, stimulating endogenous pyrogens, complement activation, IL-1 production, and PMN aggregation.

Teichoic Acids and Lipoteichoic Acids

• Teichoic acids are species-specific, phosphate-containing polymers bound either covalently to N-acetylmuramic acid residues of the peptidoglycan layer or to lipids in the cytoplasmic membrane (lipoteichoic acids).
• Stimulate specific antibody response when bound to peptidoglycan.

Surface Adhesion Proteins

• Important virulence factors that adhere to host matrix proteins.
• Designated microbial surface components recognizing adhesive matrix molecules (MSCRAMM).
• Examples:
  • Staphylococcal protein A (spa): binds to the Fc receptor of IgG1, IgG2, and IgG4.
  • Fibronectin-binding protein A: binds fibronectin.
  • S. aureus surface protein A: undetermined function.
• MSCRAMM proteins include staphylococcal protein A, fibronectin-binding proteins A and B, and clumping factor proteins A and B.
• Clumping factor proteins (coagulase) bind fibrinogen and convert it to insoluble fibrin, causing staphylococci to clump/aggregate.

Cytoplasmic Membrane

• Made up of a complex of proteins, lipids, and a small amount of carbohydrates.
• Serves as an osmotic barrier for the cell and provides an anchorage for the cellular biosynthetic and respiratory enzymes.

Pathogenesis and Immunity

• Ability to evade immune clearance.
• Produce surface proteins that mediate adherence to host tissues during colonization.
• Produce disease through specific toxins and hydrolytic enzymes, leading to tissue destruction.
• These properties (immunologic evasion, adherence, tissue destruction) are common to most pathogenic organisms.

Regulation of Virulence Genes

• Expression of virulence factors and biofilm formation is controlled by the accessory gene regulator (agr) operon.
• Quorum-sensing system allows expression of adhesion proteins and promotes tissue colonization and intracellular growth when bacterial density is low, and tissue invasion/toxin production when density is high.
• Operon encodes autoinducer peptides (AIP1 to 4) that bind to cell-surface receptors and regulate protein expression based on population density.
• Innate immune regulation of bacterial virulence is mediated by apolipoprotein B, which binds to AIPs and suppresses agr signaling.
• Bacterial density is maintained at a low concentration, providing the benefits of immune stimulation by colonizing staphylococci without tissue invasion/destruction.

Defenses against Innate Immunity

• Opsonins bind to encapsulated staphylococci, but the capsule inhibits phagocytosis by PMNs.
• Specific antibodies increase C3 binding, leading to phagocytosis.
• Extracellular slime layer interferes with phagocytosis.
• Protein A binds immunoglobulins, preventing antibody-mediated immune clearance and forming immune complexes.

Staphylococcal Toxins

• $S. aureus$ produces:
  • 5 cytolytic or membrane-damaging toxins (alpha, beta, delta, gamma, and P-V leukocidin).
  • 2 exfoliative toxins (A and B).
  • Numerous enterotoxins (A to E, G to X, plus multiple variants).
  • TSST-1.
• Cytolytic toxins = hemolysins, but activity is not restricted to red blood cells, and P-V leukocidin cannot lyse erythrocytes.
• Cytotoxins can lyse neutrophils, resulting in the release of lysosomal enzymes that subsequently damage surrounding tissues.
• P-V leukocidin is linked to severe pulmonary and cutaneous infections.
• Exfoliative toxin A, enterotoxins, and TSST-1 are superantigens that bind to class II MHC molecules on macrophages, interacting with T-cell receptors (VβTCR).
• This results in massive cytokine release by macrophages (IL-1β and TNF-α) and T cells (IL-2, IFN-γ, and TNF-β).
  • IL-1β is associated with fever.
  • TNF-α and TNF-β are associated with hypotension and shock.

Cytotoxins

• Alpha toxin, encoded on the bacterial chromosome and a plasmid, is a 33,000-Da polypeptide produced by most strains causing human disease.
  • Disrupts smooth muscle in blood vessels and is toxic to erythrocytes, leukocytes, hepatocytes, and platelets.
  • Binds to the cell surface, aggregates into a heptamer with a 1- to 2-nm pore, causing rapid efflux of $K^+$ and influx of $Na^+$, $Ca^{2+}$, and other small molecules, leading to osmotic swelling and cell lysis.
  • Important mediator of tissue damage in staphylococcal disease.
• Beta toxin (sphingomyelinase C) is a 35,000-Da heat-labile protein produced by most strains.
  • Specificity for sphingomyelin and lyso-phosphatidylcholine, toxic to erythrocytes, fibroblasts, leukocytes, and macrophages.
  • Catalyzes hydrolysis of membrane phospholipids, with lysis proportional to sphingomyelin concentration.
  • Effect on erythrocytes occurs primarily at low temperatures.
• Delta toxin is a 3000-Da polypeptide produced by almost all $S. aureus$ strains and other staphylococci.
  • Wide spectrum of cytolytic activity, disrupting cellular membranes through detergent-like action.
• Gamma toxin and P-V leukocidin:
  • Bicomponent toxins composed of two polypeptide chains: S (slow-eluting proteins) and F (fast-eluting proteins) components.
  • Three unique S proteins (HlgA, HlgC, and LukS-PV) and two F proteins (HlgB and LukF-PV) have been identified.
  • All six toxins can lyse neutrophils and macrophages.
  • Greatest hemolytic activity is associated with HlgA/HlgB, HlgC/HlgB, and HlgA/LukF-PV.
  • PV leukocidin toxin (LukS-PV/LukF-PV) is leukotoxic but has no hemolytic activity.
  • Cell lysis is mediated by pore formation, with increased permeability to cations and osmotic instability.

Exfoliative Toxins

• SSSS is mediated by exfoliative toxins, prevalent in less than 5% of $S. aureus$ strains.
• Two distinct forms: ETA (heat stable, phage-associated) and ETB (heat labile, plasmid-located).
• Serine proteases split desmoglein-1, responsible for intercellular bridges in the stratum granulosum epidermis.
• No cytolysis or inflammation, neither staphylococci nor leukocytes are present in the involved layer of the epidermis (diagnostic clue).
• Protective neutralizing antibodies develop after exposure.
• Seen mostly in young children.

Enterotoxins

• Enterotoxin A is most commonly associated with food poisoning.
• Enterotoxins C and D are found in contaminated milk products.
• Enterotoxin B causes staphylococcal pseudomembranous enterocolitis.
• Stable to heating at 100°C for 30 minutes and resistant to hydrolysis by gastric and jejunal enzymes.
• Produced by 30% to 50% of all $S. aureus$ strains.
• Superantigens capable of inducing nonspecific activation of T cells and massive cytokine release.
• Histologic changes include neutrophil infiltration and loss of the brush border in the jejunum.
• Stimulation of inflammatory mediator release from mast cells causes emesis.

Toxic Shock Syndrome Toxin-1

• TSST-1 is a 22,000-Da heat- and proteolysis-resistant, chromosomally mediated exotoxin.
• Produced by 90% of $S. aureus$ strains responsible for menstruation-associated TSS and half of the strains responsible for other forms of TSS.
• Enterotoxin B and enterotoxin C are responsible for approximately half the nonmenstruation-associated TSS cases.
• Requires elevated oxygen concentration and neutral pH.
• Superantigen stimulating cytokine release, producing leakage of endothelial cells and cytotoxic effects.
• Penetrates mucosal barriers, causing systemic effects.
• Death is caused by hypovolemic shock leading to multiorgan failure.

Staphylococcal Enzymes

• $S. aureus$ strains possess bound and free coagulase.
  • Bound coagulase directly converts fibrinogen to insoluble fibrin causing clumping.
  • Free coagulase reacts with a globulin plasma factor to form staphylothrombin, catalyzing the conversion of fibrinogen to insoluble fibrin.
• Coagulase may cause fibrin layer formation, localizing infection and protecting organisms from phagocytosis.
• Hyaluronidase hydrolyzes hyaluronic acids in connective tissue.
• Fibrinolysin (staphylokinase) dissolves fibrin clots.
• Lipases hydrolyze lipids.
• Thermostable nuclease hydrolyzes deoxyribonucleic acid (DNA).

Epidemiology

• Staphylococci are ubiquitous.
• Coagulase-negative staphylococci are present on the skin, and transient colonization of moist skinfolds 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 found in the nares, oropharynx, gastrointestinal tract, and urogenital tract.
• Approximately 15% of normal healthy adults are persistent nasopharyngeal carriers of $S. aureus$, higher incidence for hospitalized patients, medical personnel, and those with eczematous skin diseases.
• Adherence of the organism to the mucosal epithelium is regulated by the staphylococcal cell-surface adhesins.
• Shedding of bacteria is common and is responsible for many hospital-acquired infections.
• Staphylococci are susceptible to high temperatures and disinfectants but can survive on dry surfaces.
• Transferred to susceptible persons through direct contact or fomites.
• MRSA spread rapidly in hospitalized patients starting in the 1980s.
• Community-acquired MRSA outbreaks were reported in 2003, with genetically unique strains.

Clinical Diseases

Staphylococcus Aureus

• Clinical manifestations result from toxin activity (SSSS, staphylococcal food poisoning, and TSS) or proliferation of organisms (cutaneous infections, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis).
• Introduction of small numbers of staphylococci can establish disease in the presence of a foreign body.
• Patients with impaired chemotactic or phagocytic response are more susceptible to staphylococcal diseases.

Staphylococcal Scalded Skin Syndrome

• Ritter disease or SSSS is characterized by the abrupt onset of perioral erythema that spreads over the entire body within 2 days.
• Slight pressure displaces the skin (positive Nikolsky sign), and large bullae form, followed by desquamation of the epithelium.
• Blisters contain clear fluid but no organisms or leukocytes.
• The epithelium becomes intact again within 7 to 10 days, when antibodies against the toxin appear.
• Scarring does not occur because only the top layer of epidermis is affected.
• Primarily a disease of neonates and young children, with a mortality rate less than 5%.
• Infections in adults usually occur in immunocompromised hosts or patients with renal disease.
• Bullous impetigo is a localized form of SSSS, with specific strains of toxin-producing $S. aureus$, associated with formation of superficial skin blisters.
• $S. aureus$ is present in the localized blisters of patients with bullous impetigo.
• Erythema does not extend beyond the borders of the blister, and the Nikolsky sign is not present.
• Occurs primarily in infants and young children and is highly communicable.

Staphylococcal Food Poisoning

• Intoxication rather than an infection, caused by bacterial toxin present in food.
• Contaminated foods: processed meats, custard-filled pastries, potato salad, and ice cream.
• Results from contamination of food by a human carrier.
• After staphylococci are introduced into the food, the food must remain at room temperature or warmer for the organisms to grow and release the toxin.
• The contaminated food will not appear or taste tainted, and subsequent heating will kill the bacteria but not inactivate the heat-stable toxin.
• Abrupt and rapid onset, with a mean incubation period of 4 hours.
• Severe vomiting, diarrhea, and abdominal pain or nausea are characteristic.
• Sweating and headache may occur, but fever is not seen.
• Diarrhea is watery and non-bloody.
• Treatment is for relief of abdominal cramping and diarrhea and for fluid replacement; antibiotic therapy is not indicated.
• Short-lived immunity means that second episodes can occur, particularly with serologically distinct enterotoxins.
• Certain strains can also cause enterocolitis, manifested by watery diarrhea, abdominal cramps, and fever.
• Enterocolitis occurs primarily in patients who have received broad-spectrum antibiotics.
• Diagnosis is confirmed after more common causes have been excluded, and abundant $S. aureus$ is detected in the stool.

Toxic Shock Syndrome

• Initiated with the localized growth of toxin-producing strains in the vagina or a wound, followed by release of the toxin into the blood.
• Toxin production requires an aerobic atmosphere and neutral pH.
• Clinical manifestations start abruptly and include fever, hypotension, and a diffuse, macular, erythematous rash.
• Multiple organ systems are involved, and the entire skin desquamates.
• Purpura fulminans is a particularly virulent form of TSS, characterized by large purpuric skin lesions, fever, hypotension, and disseminated intravascular coagulation.
• Risk of recurrent disease is as high as 65% unless the patient is specifically treated with an effective antibiotic.
• More than 50% of patients fail to develop protective antibodies after their disease resolves.

Cutaneous Infections

• Localized pyogenic cutaneous infections include impetigo, folliculitis, furuncles, and carbuncles.
• Impetigo is a superficial infection that mostly affects young children, occurring primarily on the face and limbs.
• Folliculitis is a pyogenic infection in the hair follicles.
• Furuncles (boils) are an extension of folliculitis.
• Carbuncles occur when furuncles coalesce and extend into the deeper subcutaneous tissue, accompanied by chills and fevers.
• Staphylococcal wound infections occur after a surgical procedure or trauma when organisms colonizing the skin or from an external source are introduced into the wound.
• Infections are characterized by edema, erythema, pain, and an accumulation of purulent material.
• MRSA strains are now the most common cause of skin and soft-tissue infections in patients presenting to U.S. hospital emergency departments.

Bacteremia and Endocarditis

• $S. aureus$ is a common cause of bacteremia.
• The initial foci of infection are not known in approximately a third of patients.
• Acute endocarditis caused by $S. aureus$ is a serious disease, with a mortality rate approaching 50%.
• Symptoms can deteriorate rapidly and include disruption of cardiac output and peripheral evidence of septic embolization.
• The exception is $S. aureus$ endocarditis in parenteral drug abusers, whose disease normally involves the right side of the heart.

Pneumonia and Empyema

• $S. aureus$ respiratory disease can develop after the aspiration of oral secretions or from the hematogenous spread of the organism from a distant site.
• Radiographic examination reveals the presence of patchy infiltrates with consolidation or abscesses.
• Community-acquired MRSA is responsible for a severe form of necrotizing pneumonia with massive hemoptysis, septic shock, and a high mortality rate.
• Empyema occurs in 10% of patients with pneumonia, and $S. aureus$ is responsible for a third of all cases.

Osteomyelitis and Septic Arthritis

• Osteomyelitis results from hematogenous dissemination to bone or a secondary infection resulting from trauma or the extension of disease from an adjacent area.
• Hematogenous spread in children generally results from a cutaneous staphylococcal infection and usually involves the metaphyseal area of long bones.
• In adults, it commonly occurs in the form of vertebral osteomyelitis.
• With appropriate antibiotic therapy and surgery, the cure rate for staphylococcal osteomyelitis is excellent.
• $S. aureus$ is the primary cause of septic arthritis in young children and in adults.
• Staphylococcal arthritis is characterized by a painful erythematous joint, with purulent material obtained on aspiration.

Staphylococcus Epidermidis and Other Coagulase-Negative Staphylococci

• $S. epidermidis$, $S. lugdunensis$, can infect prosthetic and less commonly, native heart valves.
• Infections of native valves are believed to result from the inoculation of organisms onto a damaged heart valve.
• Catheter and shunt infections: infections of catheters and shunts are caused by coagulase-negative staphylococci.
• Prosthetic joint infections: infections of artificial joints caused by coagulase-negative staphylococci.
• Urinary Tract Infections: $S. saprophyticus$ causes urinary tract infections in young, sexually active women.

Laboratory Diagnosis

Microscopy

• Staphylococci are gram-positive cocci.
• Successful detection depends on the type of infection and the quality of the material submitted for analysis.

Nucleic Acid–Based Tests

• Used to detect nasal carriage of methicillin-sensitive $S. aureus$ (MSSA) and MRSA.

Culture

• Clinical specimens should be inoculated onto nutritionally enriched agar media supplemented with sheep blood.
• Staphylococci grow rapidly on nonselective media incubated aerobically or anaerobically.

Identification

• Biochemical tests used to identify $S. aureus$. Colonies resembling $S. aureus$ are identified in most laboratories by mixing a suspension of organisms with a drop of plasma. Mass spectrometry has been used to identify the staphylococci. Analysis of genomic DNA by pulsed-field gel electrophoresis a method for characterizing isolates. Whole genome sequencing is becoming. the preferred tool for subtyping organisms.

Antibody Detection

• Antibodies to cell wall teichoic acids are present in many patients with long-standing $S. aureus$ infections.

Treatment, Prevention, and Control

• Less than 10% of the strains are susceptible to penicillin.
• Currently, the majority of $S. aureus$ responsible for hospital- and community-acquired infections are resistant to semisynthetic penicillins, and these MRSA strains are resistant to all β-lactam antibiotics.
• Patients with localized skin and soft-tissue infections can generally be managed by incision and drainage of the abscesses.
• Oral therapy can include trimethoprim-sulfamethoxazole, a long-acting tetracycline such as doxycycline or minocycline, clindamycin, or linezolid.
• Vancomycin is the drug of choice for intravenous therapy, with daptomycin, tigecycline, or linezolid acceptable alternatives.
• Isolates of $S. aureus$ have been found with low-level resistance which is observed in strains with a thicker, more disorganized cell wall and high-level resistance to vancomycin. Staphylococci are ubiquitous organisms present on the skin and mucous membranes.
• The number of organisms required to establish an infection is generally large unless a foreign body is present in the wound.
• The spread of staphylococci from person to person is more difficult to prevent.