BACTE (STAPHYLOCOCCI)

• Micrococcus

  ○ (M. luteus) are gram-positive catalase producing bacteria

  ○ they appear in clusters or tetrads

  ○ distinguished by the use of modified oxidase

  tests.
• Staphylococci

  ○ Gram positive catalase producing bacteria.

  ○ They can be seen as spherical in size that appear singly in pairs, or clusters during gram stain.

  ○ Non-motile and non-spore forming

  ○ Aerobic or facultative anaerobes except for

  Staphylococcus saprophyticus because it is an obligate anaerobe
• COLONIAL MORPHOLOGY

  • Staphylococcus grows in most culture media that support the growth of Gram positive organisms such as Blood agar & Chocolate agar.
  • Colonies are produced after 18-24 hours of incubation.
  • Colonies are medium sized (4-8um) that appear round, smooth, white, creamy colored or light gold with butyrous appearance.
  • Some “small-colony variants” are rare, fastidious strains that require increased CO2, hemin or menadione for growth.
• EPIDEMIOLOGY

  • Associated with various skin & mucosal related infections from cutaneous infection to systemic diseases that can cause death.
  • Considered as normal flora
  • They are normal microbiota of skin that cannot cause disease unless colonization/inoculation to normally sterile site occurs in untreated wounds, abrasions & surgical procedures that provide access to Staphylococci to colonize deeper tissue or organs.
• TREATMENT AND PREVENTION:

  • Oxacillin
  • Methicillin
  • Vancomycin

  For drug-resistant strains, CDC recommends fast recognition using rapid test to detect drug-resistant species & isolation of infected patient is necessary.
• CLINICALLY SIGNIFICANT SPECIES

  • Coagulase Positive Staphylococcus

  ○ Staphylococcus aureus - responsible for numerous diseases & most virulent species.

  • Coagulase Negative Staphylococci (CoNS) or Non-Staphylococcus aureus

  ○ S. epidermidis

  ○ S. saprophyticus

  ○ S. lugdunensis

  ○ Other CoNS

  S. (ESL)
• GENERAL CHARACTERISTICS OF

  Staphylococcus aureus

  • Most clinically significant specie of Staphylococci
  • Most virulent specie of Staphylococcus encountered
  • Responsible for various infection both mild and life-threatening
  • Mainly distinguish or differentiated from other Staphylococcus specie by the Positive Coagulase Test
  • Normally found in the skin including the axilla, vagina, pharynx and nasal surfaces specifically the nares
• GEN CHARACTERISTICS OF S. AUREUS

  • Infection occurs when the colonizing strain enters a normally sterile site by the result of abrasion or trauma to skin surface or mucosal surfaces
  • Important cause of nosocomial Infection
  • Diseases include Cutaneous infections, Pyogenic infections (such as abscess, endocarditis, and osteomyelitis) and toxin-induced diseases that render food poisoning and toxic shock syndrome.
• VIRULENCE FACTORS

  • Innate immunity against S. aureus is high
  • Classified as an Opportunistic pathogen

  1. Structural Defenses
  2. Enterotoxin
  3. TSST 1
  4. Exfoliative toxins
  5. Cytolytic toxins
• VIRULENCE FACTORS (structural defenses)

  • Slime layer

  ○ aids in invading the immune response; it also increases the capability of the bacteria to adhere to inorganic materials and avoid antibiotic action.

  • Protein A

  ○ This protein is very significant since it is responsible for binding into the Fc portion of immunoglobulin-G or IgG thereby blocking the phagocytosis and the oxidization and decreasing the clearance of the bacteria from the site of infection.
• VIRULENCE FACTORS (structural defenses)

  • Coagulase (Staphylocoagulase)

  ○ produced by S. aureus, only S. aureus can produce this enzyme

  • Hyaluronidase, Lipase, Protease

  ○ Most strains of S. aureus produce Hyaluronidase which has the ability to hydrolyze hyaluronic acid in the connective tissues, increasing the spread of the bacteria

  ○ Lipase, produced by both S. aureus and the CoNS acts on the lipids, fats, and oils (secreted by the sebaceous gland).

  ○ These three (3) enzymes are capable of destroying tissues and are responsible for the spread of bacteria to deeper tissues or organs
• VIRULENCE FACTORS (Structural defenses)

  • Staphylokinase

  ○ lyses fibrin thus allowing the bacteria to digest its way out of the clot

  • Beta-lactamase

  ○ Most isolate of S. aureus have the resistance to penicillin due to the production this enzyme

  ○ This enzyme inactivates beta-lactam antibiotics by breaking the beta-lactam ring of the drug
• VIRULENCE FACTORS (Enterotoxin)

  • One of the toxins produced by S. aureus 30-50% of the isolates demonstrate the production of these enterotoxin
  • There are 9 known distinct enterotoxin (A, B, C, D, E, G, H, I, and J)
  • ■ A, B, and D: associated with Staphylococcal food poisoning
  • ■ B and C, (and sometimes) G and I: toxic shock syndrome
  • ■ B: Pseudomembranous enterocolitis These enterotoxins are heat-stable and can survive up to 100°C for 30 minutes and cause a variety of signs and symptoms.
  • Reheating contaminated food does not prevent the disease
• VIRULENCE FACTORS (TSST 1)

  • Previously known as enterotoxin F or pyrogenic exotoxin C
  • A super-antigen released by S. aureus that causes nearly all case of menstruating TSS along with 50% of non-menstruating TSS
  • A chromosomal mediated toxin that causes leakage in the endothelial cells in lower doses and cytotoxicity in higher doses.
  • This toxin can be easily absorbed to their vaginal mucosa leading to a systemic disease
• VIRULENCE FACTORS (Exfoliative toxins)

  • Produced by some strains of S. aureus
  • causes the epidermal layer of the skin to sloughed-off
  • known to cause ritter's disease
• VIRULENCE FACTORS (CYTOLYTIC TOXINS)

  • Alpha-hemolysin: attaches to RBCs, damages the platelets and macrophages. It causes severe tissue destruction
  • Beta-hemolysin: aka Sphingomyelinase C acts on the sphingomyelin
  • It is known as the hot-cold lysin. This hemolysin can be seen in the CAMP test
  • Delta-hemolysin: is found in higher percentages among S. aureus and some CoNS.
  • Less toxic than the other two hemolysins but it attacks RBC, macrophages, lymphocyte, neutrophils, and platelets
  • Gamma-hemolysin: often found associated with PVL. Produced by all strains of S.aureus.
• VIRULENCE FACTORS (cytolytic toxins)

  • Panton Valentine Leukocidin (PVL)
  • An exotoxin lethal to polymorphonuclear cells (PMNs)
  • It suppresses the phagocytosis and has been associated with severe cutaneous infection and necrotizing pneumonia
• EPIDEMIOLOGY (S. aureus)

  • found in human nares
  • colonization also occurs in the axilla, vagina, pharynx, and other skin surfaces
  • Transmission:
  • Person-to-person via direct contact with a skin of a carrier individual
  • Fomites
  • 3 types of carrier state for the bacterium:
  • Persistent, Intermittent, Non-carriers
• Diseases associated w/ S. aureus (non invasive)

  • Food poisoning
  • Signs and symptoms appear rapidly 2-8 hrs
  • Intoxication from ingestion of preformed toxin
  • Resolves within 24-48 hours
  • Staphylococcus aureus produces enterotoxin. This toxin causes food poisoning.
• Diseases associated w/ S. aureus (cutaneous)

  • Folliculitis
  • Furuncles
  • Carbuncles
  • Bullous impetigo
  • SSS/ Ritter's disease
• Diseases associated w/ S. aureus (systemic)

  • Toxic Shock syndrome
  • 2 types: menstruating and non menstruating
  • Bacteremia
  • pneumonia
  • endocarditis
  • osteomyelitis
  • septic arthritis
• Treatment - S. aureus

  • Relies on the use of antibiotics
  • Most isolates of S. aureus produce beta-lactamase or penicillinase. Antibiotic choice would be penicillinase-resistant drugs such as Methicillin and Oxacillin
  • Recent laboratory analysis detects the rise in Methicillin Resistant S. aureus (MRSA) isolates.
  • Recent studies revealed that a small percentage of S. aureus presents resistance to Vancomycin known as Vancomycin Resistant S. aureus (VRSA)
• CONS

  Staphylococcus epidermidis

  • One of the most significant CoNS
  • Infections by this bacteria are predominantly nosocomial
  • Common cause of nosocomial UTI and Prosthetic heart valve endocarditis
  • Sensitive to novobiocin
  • Normally found on the skin as a normal flora
• CONS

  • Staphylococcus saprophyticus

  ○ Causes acute UTI - associated with urinary tract infections in young women.

  ○ Adheres more effectively to the epithelial lining of urogenital tract

  ○ Rarely found on other skin surfaces/ mucus membranes.

  ○ Distinguished from other CoNS by its resistance to novobiocin and nalidixic acid
• CONS

  • Staphylococcus lugdunensis

  ○ Maybe mistaken as Staphylococcus aureus for it may produce a clumping factor but it has no free coagulase.

  ○ CoNS, but produce clumping factor

  ○ More virulent and it mimics Staphylococcus aureus infection

  ○ Contains mecA gene

  ○ Important pathogen of endocarditis, septicemia, meningitis, skin and soft tissue

  infections UTI

  ○ Endocarditis caused by this bacteria is more

  aggressive, requiring valve replacement and has a high mortality rate.
• LAB DIAGNOSIS

  • Gram stain smear
  • Staphylococci are gram positive cocci
  • In pairs, tetrads, clusters
  • Remember! Many bacteria may contaminate the specimen and will appear in the gram stain smear. That’s why regardless of the microscopic examination, it is important to culture and isolate the sample.
• LAB

  • Culture methods
  • Staphylococci can grow in most enriched media that supports the growth of gram-positive bacteria

  ■ Example: Sheep’s Blood Agar

  • For heavily contaminated sample

  ■ Example: Selective media: Mannitol Salt Agar, Columbia colistin-nalidixic acid agar and Phenyl ethyl Alcohol

  • Use Broth to increase the yield of the bacteria before plating

  ■ Examples: Thioglycolate, Dextrose, brain-heart infusion

  • CHROMagar is a selective media for staphylococcus aureus
• LAB (Cultural characteristics)

  Staphylococcus aureus

  ■ Isolates produce round smooth, white, creamy or butyrous colonies in sheep's blood agar (SBA).

  ■ It will produce a small zone of hemolysis around the colonies

  ■ It may also appear as Golden yellow (pigment) due to the production of Lipochrome

  • Yellow colonies of S. aureus in mannitol salt agar
  • There is a change in agar color due to the fermentation of mannitol
  • Coagulase negative staphylococcus does not ferment mannitol and can be seen in the original pink colored agar.
• Biochemical test to distinguish and differentiate staphylococci:

  1. Catalase Test
  2. Coagulase (Slide and Tube Method
  3. Pyrrolidonyl-arylamidase Test
  4. DNAse Test
  5. Oxidase Test
  6. Sensitivity Test