Staphylococcus

Characteristics

• morphology

• testing

• colony

• Gram pos cocci in clusters

• Catalase pos

• Some are β-hemolytic 

• Colony: cream, white, or rarely light gold 

Clinical significance of S. aureus

• qualities

• induces what

• coag-neg staph examples

• Opportunistic

• Acquires drug-resistance

• Toxin-induced conditions: food poisoning, SSS, TSS (toxic shock syndrome)

• Coagulase-neg staphylococci

  • Staphylococcus epidermidis

  • Staphylococcus saprophyticus 

  • Staphylococcus lugdunensis

Initial differentiation of staphylococcal species

• Tube coagulase test → most specific - takes too long

  • Positive = clot in plasma bc staphylocoagulase (secreted enzyme)

  • S.aureus has vWF protein which combines to staphylocoagulase and prothrombin → complex forms “staphylothrombin” (fibrinogen cleave to fibrin = clot)

• Slide coagulase test 

  • Detects “bound coagulase” (surface protein) that binds to fibrinogen → agglutination

  • Common false pos: S.lugdunensis 

Coagulase negative Staphylococci

• Staphylococci that do not produce coagulase → staphylocoagulase neg (neg tube test) + clumping factor neg (neg slide test)

• Examples

  • S.epidermidis: nosocomial (hospital-induced) infections

  • S.saprophyticus: UTIs

What might staphylocci be confused with

Staphylococci may be confused w/ 

• Micrococcaceae → micrococcus 

  • Gram-pos cocci → tetrads shaped

  • Catalase pos

  • Coagulase neg

  • Yellow pigment

  • Normal flora

  • Lysostaphin resistant

Staph vs micrococci

• modified oxidase

• anaerobic acid production from glucose

• resistance to bacitracin (0.04)

test	Staphylococci	Micrococci
Modified oxidase	negative	positive
Anaerobic acid production from glucose	positive	negative
Resistance to bacitracin (0.04 units)	R	S

Virulence factors in S. aureus

• enterotoxins

• toxic shock

• exfoliative toxin

• Enterotoxins

  • 9 distinct heat stable (100°C for 30 mins) enterotoxins that cause food poisoning, etc.

• Toxic shock syndrome toxin-1 (TSST-1)

  • Menstruating (almost all) and non-menstruating = TSS → absorbed through vaginal mucosa

  • Superantigen: polyclonal T cells proliferation + lots of cytokines release

    • Leakage by endothelial cells

    • Cytotoxic at higher concentration 

• Exfoliative toxin

  • Ritter disease -- SSSS (Staphylococcal Scalded Skin Syndrome) most common in newborns and infants

Virulence factors in S. aureus

• Cytolytic toxins

• Protein A

• Cytolytic toxins

  • Affects RBCs and WBCs 

    • Panton-valentine leukocidin (PVL) 

• protein A

  • Surface protein binds to fc portion of IgG → no phagocytosis

  • ↑ Protein A = ↑ virulence

Enzymes

• Enzymes: 

  • Staphylocoagulase (s.aureus)

  • protease → destroys tissue

  • Hyaluronidase: hydrolyzes hyaluronic acid in connective tissues = destroys tissue + promotes infection spreading

  • Lipase: destroys skin surface lipids

Epidemiology

• primary reservoir

• colonization areas

• outbreak areas

• transmission via

• specific type

• Epidemiology

  • Primary reservoir = humans

  • Colonization: axillae, vagina, pharynx, skin surfaces

  • Outbreaks: nurseries, burn units, surgical recovery units

  • Transmission: direct contact w/ unwashed contaminated hands + contact w/ inanimate objects 

  • MRSA: health care and community acquired

Skin and wound infections

• folliculitis

• furuncles

• carbuncles

• impetigo

1. Skin and wound infections

• Folliculitis: inflammation of hair follicle

• Furuncles: boils → may be extension of folliculitis

• Carbuncles: invasive lesions due to multiple furuncles in proximity — potential systemic spread

• Impetigo: red sores on the face that are highly contagious + autoinfection can occur

  • Bullous- large, surrounded by erythema

Scalded skin syndrome

• affected population

• causes?

• what toxin

• other info

1. Seen in newborns/young children

2. Bullous exfoliative dermatitis - rash

   1. Staphylococcus exfoliative or epidermolytic toxin → cleared by kidneys

   2. High rate of spontaneous recovery 

   3. Localized or ritter disease

   4. Adults → seen in chronic renal failure

Toxic shock syndrome

• toxin?

• hematologic effect

• treatment

Toxic epidermal necrolysis (TEN)

• diagnosis criteria

• what it causes

1. Toxic shock syndrome

   1. Localized infection; TSST-1 toxin = systemic

      1. ↑ WBC + ↓ PLT 

      2. Treatment is generally supportive → fluid replacement + antibiotics

Toxic epidermal necrolysis (TEN)

1. Rare + diagnosis when >30% of skin area affected

2. Severe Cutaneous Adverse Rxn (SCAR) w/

   • Adverse rxn to medication 

   • Disease state affects onset

NOT caused by S.aureus but similar to SSSS

Food poisoning

Other infections

• Staphylococcal pneumonia

• Staphylococcal bacteremia

• Staphylococcal osteomyelitis

• Septic arthritis

1. Food poisoning 

   1. Intoxication: mostly enterotoxin A

   2. Rapid onset symptoms → resolve within 24-48 hrs

      1. No fever → normal food poisoning symptoms

2. Other infections

   1. Staphylococcal pneumonia 

      1. Secondary to flu virus infection → rare + high mortality rate

   2. Staphylococcal bacteremia 

      1. IV drug users

   3. Staphylococcal osteomyelitis 

      1. Secondary to bacteremia

   4. Septic arthritis

      1. Trauma to extremities 

      2. RA 

      3. IV drug use

Infections caused by coag-neg staph

• S. epidermidis

• S. saphrophyticus

• S. lungdunensis

• S. haemolyticus

• CoNS

Infections caused by coagulase-neg staphylococci

• S.epidermidis 

  • Hospital acquired/nosocomial (w/ preexisting conditions/instrumentation procedures)

  • Causes prosthetic valve endocarditis 

• S. saprophyticus 

  • UTIs → adhere to epithelial cells lining

  • Low number are significant 

• S.lugdunensis

  • clumping factor pos 

  • community/hospital acquired 

    • Infective endocarditis → aggressive, high mortality rate

• S. haemolyticus 

  • Wounds, bacteremia, endocarditis, UTIs 

  • Vancomycin resistance 

• CoNS 

  • Most common contaminant of blood cultures → > 1 blood culture bottle positive = infection

Lab diagnosis

• microscopic appearance

• preferred specimen

• Microscopic 

  • Stained smear → GPC w/ lot PMNs 

  • Preferred specimens: aspirated or 2 swabs

Isolation and ID

• media types

• Sheep blood agar (SBA)

• CHROMagar Staph aureus: selective + differential 

• Selective 

  • Mannitol salt agar (MSA): 7.5% salt concentration

    • S.aureus = yellow; CoNS = red

  • CNA (w/ nalidixic acid): inhibits gram-neg 

  • Phenleythyl alcohol agar (PEA): inhibits facultative gram-neg anaerobes

S. aureus

S. epidermidis

S. saprophyticus

S. haemolyticus

S. lugdunensis

Grows at?

Colony characteristics?

• 35°C -37°C

  • S. aureus

    • Can produce hemolytic zones

    • Rarely exhibit pigment production (yellow)

  • S. epidermidis

    • Nonhemolytic, gray-to-white colonies

  • S. saprophyticus

    • 50% produce yellow pigment

  • S. haemolyticus

    • Moderate to weak hemolysis, variable pigment production

  • S. lugdunensis

    • Often hemolytic

ID methods

• slide coagulase test

• tube coagulase test

• CoNS in urine

• Slide coagulase test → screening test 

  • Agglutination = pos = S.aureus 

  • Neg → confirm w/ tube coagulase test

• Tube coagulase test 

  • Clot formation after 4 hrs at 37˚C

  • If neg → leave tube at room temp then reread

• CoNS in urine

  • S.saprophyticus → resistant to nocobiocin susceptibility w/ disk 

  • Most other CoNS are susceptible

Rapid methods of ID

Real-time PCR: MRSA vs MSSA

• Rapid agglutination test kits 

  • Differentiates S. aureus from CONS

  • Plasma detects both clumping factor and protein A in the cell wall of S. aureus

  • Caveat:  Some strains of S. saprophyticus, S. sciuri, S. lugdunensis, and Micrococcus spp. can produce positive tests

• Negative with tube coagulase

• Real-time PCR

  • MRSA → methicillin-resistant S.aureus

MSSA → methicillin-sensitive S.aureus

Antimicrobial susceptibility

• CoNS

• Resistance

• MRSA detection

• Vancomycin-resistant staphylococci

• Macrolide resistance

• Testing follow CLSI guidelines

• CoNS (Coagulase-negative staph)

  • Depend on source and if isolate is contaminant or pathogen 

  • S.saprophyticus from urine 

  • No routine testing needed → sensitive UTI treatment agent

• S. aureus + S. lugdunensis

  • Production of B-lactamases (penicillinases)

    • Break down B-lactam ring of penicillins

    • Most S. aureus isolates resistant to penicillin

• B-lactam resistance 

  • mecA gene → codes for an altered penicillin-binding protein (PBP)

    • PBP cross-link cell wall layer

    • PBP2a → does not bind to B-lactam antibiotics = ineffective 

• MRSA detection 

  • Gold standard → detect mecA gene use PCR

  • Screens: oxacillin-slat agar plate

  • Specialized media: chromogenic selective differential media

• Vancomycin-resistant staphylococci

  • vancomycin = last resort drug → limit spread of resistance

• Macrolide resistance

  • Erythromycin and clindamycin = same results 

  • D-zone test when discrepant results: sensitive to clindamycin but resistant to erythromycin

    • Put in agar → D = clindamycin resistant; circle = sensitive