Staphylococcus: General Characteristics

• Staphylococcus (STAPHYLOCOCCI) is an important isolate
• Lecture is for the school year 2023-2024, by Sir. Glenford Monzon

General Characteristics

• Originally belonged to the genus Micrococcus in the family Micrococcaceae
• Now belongs to the order of Bacillales
• Micrococcus (M. luteus) are gram-positive, catalase positive, bacteria seen in clusters/tetrads and are strict aerobes.
• Distinguished by the use of Modified Oxidase test
• Catalase-producing GP Cocci
• Spherical shape, measuring 0.5-1.5 um, appearing singly, in pairs, or in clusters
• Non-motile and non-spore forming
• Aerobic or facultative anaerobe
• Can grow in most culture media that supports the growth of Gm (+) organisms
• Colonies are produced after 18-24 hours
• Medium sized, between 4-8 mm
• Round, smooth, white, creamy-colored, and rarely light gold and butyrous
• Small-colony variants are rare fastidious strains that require CO2, hemin or menadione for growth

Staphylococcus aureus

• Possesses a golden yellow pigment, soluble in ether and alcohol, termed lipochrome, variable from deep orange to pale yellow due to carotenoid
• Staphylococcus citreus has lemon yellow pigment
• Staphylococcus albus has porcelain white pigment

Virulence Factors

• Structural Defenses
• Toxins
• Enzymes
• Slime Layer provides protection and attachment
• Protein A is found in the cell wall of the bacteria and binds to the Fc portion of IgG, neutralizing the Ab

Virulence Factors: Enzymes

• Coagulase is also known as Staphylocoagulase
• Hyalurodinase hydrolyzes hyaluronic acid in the intracellular ground substances of connective tissues
• Lipase digests lipids on the surface of the skin
• Protease is capable of destroying tissue and facilitating spreading
• Staphylokinase helps digest its way out of the clot
• Beta Lactamase breaks down penicillin
• DNase breaks down NET

Virulence Factors: Toxins

• Enterotoxins are heat-stable exotoxins that cause a variety of symptoms, including diarrhea and vomiting
• Serologically distinct enterotoxins: A-E and G-J
• Enterotoxins A, B, and D are implicated in Staphylococcal food poisoning
• Enterotoxins B, C, G, and I are implicated in TSS
• Enterotoxin B has been linked to staphylococcal pseudomembranous enterocolitis
• Toxic Shock Syndrome Toxin-1 are superantigens that stimulates T-cell proliferation and subsequent cytokine release that are responsible for the symptoms
• Chromosomal mediated toxin causes leakage by endothelial cells and cytotoxicity in higher concentration
• Causes nearly all cases of menstruating-associated TSS and is absorbed through vaginal mucosa
• Referred to as Enterotoxin F, chromosomal mediated toxin associated with approximately 50% of non-menstruating associated TSS
• Exfoliative Toxin has two types, A and B, causes dissolution of the epidermal layer of the skin and is implicated in Ritter's Disease and Bullous Impetigo
• Cytolitic Toxin includes:
  • Alpha hemolysis which lyses RBC, damages PLT, Macrophages and tissues
  • Beta hemolysis also known as Hot-Cold Lysin, detected in CAMP test
  • Gamma hemolysis is associated with PVL, staphylococcal bone disease
  • Delta hemolysis is considered less toxic to cells and attacks RBC, macrophage, lymphocytes, neutrophils and PLT
• Panton-Valentine Leukocidin is an exotoxin lethal to PMN
• Contributes to the invasiveness of organism by suppressing phagocytosis
• Associated with severe cutaneous infections and necrotizing pneumonia
• Produced by relatively few strains and is often associated with CA-MRSA

Epidemiology

• Primary reservoir for staphylococci is human nares, colonizing the axillae, vagina, pharynx, and other skin surfaces
• Transmission through direct contact with unwashed, contaminated hands, and inanimate objects (fomites)
• Major HC concern: Nosocomial-acquired infections caused by MRSA

Staphylococcal Diseases

• Development of infection is determined by:
  • Virulence of the strain
  • Size of infection
  • Status of the patient
• Non-Invasive Disease: Food Poisoning
  • Type of intoxication resulting from ingestion of preformed toxins
• Enterotoxins associated gastrointestinal disturbances: Enterotoxin A (78%), Enterotoxin D (38%), and Enterotoxin B (10%)
• Symptoms appear rapidly, within 2-8 hours after ingestion, including nausea, vomiting, abdominal pain, and severe cramping; diarrhea and headaches can occur

Cutaneous Diseases

• Folliculitis/Furuncles/Carbuncles: inflammation of a hair follicle or oil gland; infected area raised and red; often presents as pimple.
• Sty is seen in base of eyelid and infects follicle of the eyelash
• Bullous Impetigo: pustules are larger and surrounded by a small zone of erythema; red patches on the face and limbs (with bacteria), common in children

Cutaneous Diseases

• Staphylococcal Scalded Skin Syndrome is caused by staphylococcal exfoliative toxin which is probably present at a lesion distant from the site of infection
• Symptoms includes reddening of the skin (erythema) followed by a profuse peeling of the epidermal layer of the skin
• Systemic Disease: Toxic Shock Syndrome: Multisystem disease, sudden onset of fever, chills, vomiting, dehydration due to diarrhea, muscle aches, red rash, hypotension, and shock; 2 categories: menstruating-associated and non-menstruating
• other systemic diseases are:
  • Bacteremia
  • Pneumonia
  • Endocarditis
  • Osteomyelitis
  • Septic arthritis

Staphylococcus lugdunensis

• CONS, but with clumping factors
• More virulent and can mimic S. aureus infections clinically
• Contains mecA gene that encodes resistance to oxacillin
• Can cause infective endocarditis, meningitis, skin and soft tissue infections, UTI, and septic shock

Other CONS

• St. warneri, S. capitis, S. simulans, S. hominis, S. schleiferi
• S. haemolyticus- commonly isolated CoNS in wounds, endocarditis, bacteremia and UTI

Resistant Genes Produced by Staphylococci

• Erythromycin Ribosomal Methylase (ERM) gene of enzyme inactivating gene
• Codes for the methylation of the 23s rRNA, which results in the resistance to erythromycin and either inducible or constitutive resistance to clindamycin
• Methionine Sulfoxide Reductase (MSR) A gene codes for an efflux mechanism, which results in the resistance to erythromycin but susceptibility to clindamycin

Specimen collection and handling

• Samples are collected from sites of infection after cleansing the surrounding area and must be transported immediately to the laboratory.
• Aspirates, Tissue samples, or blood cultures are preferred over swabs
• if a swab is used then 2 need to be submitted

Antimicrobial Resistant Strains

• Most isolates of S. aureus produce B-lactamase, therefore treatment of most infections relies on penicillinase- resistant drugs
• Methicillin Resistant Staphylococcus aureus (MRSA)- mecA (PBP2a)
• Vancomycin Resistant Staphylococcus aureus (VRSA)

Coagulase Negative Staphylococci

• Staphylococcus epidermidis is normal flora, most common cause of nosocomial UTI
• Contaminant of medical instruments, catheters and prosthetic heart valves and may cause endocarditis
• Sensitive to novobiocin and produces biofilm
• Staphylococcus saprophyticus causes UTI
• Adheres more effectively to the epithelial lining of urogenital tract
• Resistant to novobiocin and nalidixic acid

Culture

• Blood Agar Plate (SBA)
• Tellurite Lysine Agar (TLA)
• Mannitol Salt Agar (MSA)
• Columbia Colistin-Nalidixic Acid Agar (CAN)
• Phenylethyl Alcohol (PEA)
• Broth:
  • Thioglycolate
  • Brain Heart Infusion Broth
  • CHROMagar Staph aureus (BD Dx Systems)

Culture Characteristics

• S. aureus isolates produce round smooth, white, creamy or butyrous colony in SBA
• It may also appear as Golden yellow due to the production of Lipochrome
• May produce hemolytic zones around colonies
• Tellurite Medium = Jet Black Colonies
• Ferments Mannitol
• Produces acids from fermentation of glucose under anaerobic condition
• Negative on Mod. Oxidase Test
• S. epidermidis - small to mediums size, nonhemolytic and gray to white colonies
• S. saprophyticus- slightly larger colony and may produce yellow pigment
• S. lugdunensis- medium size, hemolytic

BioChemical Methods

• Catalase Test
• Coagulase Test
• Pyrolidonyl Arylamidase Test
• DNase Test
• Oxidase Test
• Phage Typing
• Sensitivity Test

Rapid Diagnostic Test

• Rapid Agglutination Test uses Staphyloslide (BD-BBL, Sparks, MD), BactiStaph (Remel) and Staphaurex (Remel)
• Molecular Method uses Polymerase Chain Reaction and MALDI-TOF

Phage typing

• A bacteriophage, or phage, is a virus that infects a bacterial cell, taking over the host cell's genetic material, reproducing itself, and eventually destroying the bacterium
• The word phage comes from the Greek word phagein, meaning "to eat"
• A lawn of the bacterium to be tested is spread over the surface of a suitable agar medium and the inoculum is allowed to dry
• A range of bacteriophage strains are then inoculated onto the lawn and the plate is incubated
• Some bacteriophage have no effect on the test strain, others can attack the bacterial cells causing the formation of plaques
• This is not an all or nothing effect and observations can be recorded in a semi- quantitatve manner:
  • • or ++ -strains of bacteriophage that give a moth-eaten appearance
  • +++ = those that destroy the test bacterium completely