Download - Staphylococci microbiology

Frances Angelique A. Tequillo, MDCebu Doctors’ University

Staphylococci& Micrococci

Monday, June 27, 2011

The Staphylococci: An Overview

gram-positive spherical cells

arranged in grape-like irregular clusters

grow readily on many types of media

active metabolically

ferment carbohydrates

produce pigments from white to deep yellow


members of the normal flora of the skin & mucous membranes

may cause suppuration, abscess formation, and fatal septicemia

pathogenic types often hemolyze blood, coagulate plasma, & produce extracellular enzymes and toxins

rapidly develop resistance to antimicrobials

35 species

3 most important (clinically):

1.Staphylococcus aureus2.Staphylococcus epidermidis3.Staphylococcus saprophyticus


1. Coagulase positive Staphylococci

• S. aureus

2.Coagulase negative Staphylococci

• S. epidermidis

• S. saprophyticus

Grouping for Clinical Purposes


Staphylococcus aureus

major human pathogen

habitat: part of normal flora in humans and animals

usual site: skin, nasopharynx & perineum

can enter underlying tissue when there is a breach in mucosal barriers

forms characteristic abscesses


Diseases caused by S. aureus

due to direct effect of organism

local lesions of skin

deep abscesses

systemic infections

osteomyelitis, septic arthritis, infective endocarditis

toxin-mediated

food poisoning

toxic shock syndrome

scalded skin syndrome


Staphylococcus epidermidis

skin commensal

has predilection for plastic material

associated with infections of IV lines, prothetic heart valves, AV shunts

causes urinary tract infection in catheterized patients


Staphylococcus saprophyticus

skin commensal

important cause of urinary tract infection in sexually active young women

usually sensitive to a wide range of antibiotics


& IdentificationMorphology


Typical Organisms

spherical cells

about 1 um in diameter

arranged in irregular clusters

young cocci stain strongly Gm+

on aging, many cells become Gm-

nonmotile, non-sporeforming


grow readily on most media

under aerobic or microaerophilic conditions

grow most rapidly at 37C

form pigment best at room temperature (20-25C)

form round, smooth, raised, & glistening colonies on solid media

Culture


S. aureus form gray to deep golden yellow colonies

S. epidermidis form gray to white colonies

many colonies develop pigment only upon prolonged incubation

Peptostreptococcus sp. resemble staphylococci morphologically

S. aureus

S. epidermidis


produce catalase (unlike streptococci)

slowly ferment carbohydrates

producing lactic acid

resistant to drying, heat, and 9% NaCl

readily inhibited by some chemicals (3% hexachlorophene)

variably sensitive to many antimicrobials

Growth Characteristics


Resistance:

1. penicillins

2.nafcillin, methicillin, & oxacillin

3.vancomycin

4.tetracyclines, erythromycins, & aminoglycosides

Resistance


Variation

colony size, pigment, & hemolysis

enzyme elaboration

drug resistance

pathogenicity


Antigenic Structures


polysaccharide polymer w/c provides the rigid exoskeleton of the cell wall

destroyed by strong acid or exposure to lysozyme

elicits interleukin-1 production to cause fever and opsonic antibodies by monocytes

serves as chemoattractant for PMN leukocytes

have endotoxin-like activity

activates complement

Peptidoglycan


Teichoic acid

polymers of glycerol or ribitol phosphate

linked to the peptidoglycan layer

can be antigenic

antiteichoic acid antibodies detected in patients with active endocarditis (S. aureus)


Protein A

cell wall component of many S. aureus strains

binds to the Fc portion of IgG molecules, except IgG3

important reagent in immunology and diagnostic laboratory technology

protein A with attached IgG directed against a specific bacterial antigen will agglutinate bacteria that have that antigen (“coagglutination”)


other antigenic structures

capsules - inhibit phagocytosis by PMN leukocytes unless specific antibodies are present

coagulase (clumping factor) - binds nonezymatically to fibrinogen, leading to aggregation of bacteria


Enzymes & Toxins


Catalase

converts H2O2 into H2O and O2

catalase test differentiates staphylococci (+) from streptococci (-)


Coagulase & Clumping Factor

enzyme-like protein that clots oxalated or citrated plasma

binds to thrombin to initiate fibrin polymerization

deposits fibrin on the surface of bacteria and alters their ingestion by phagocytes

essential in invasive pathogenic potential

clumping factor: responsible for adherence of organisms to fibrinogen & fibrin

S. aureus tends to form clumps when mixed with plasma


Other Enzymes

hyaluronidase (spreading factor)

staphylokinase

proteinases

lipases

β-lactamase


Exotoxins

α-toxin: potent hemolysin

β-toxin: degrades sphingomyelin; toxic to many cells, including erythrocytes

δ-toxin: disrupts biologic membranes; plays a role in S. aureus diarrheal disease

γ-toxin: lyses white blood cells by causing pore formation in the cellular membranes that increase cation permeability


Leukocidin

S. aureus

has 2 components

act synergistically on the white blood cell membrane

important virulence factor in community associated methicillin resistant S. aureus infections


Exfoliative Toxins

epidermolytic toxins of S. aureus (superantigens)

toxin A: heat-stable (resists boiling for 20 mins)

toxin B: heat-labile

yield the generalized desquamation of the staphylococcal scalded skin syndrome by dissolving the mucopolysaccharide matrix of the epidermis


Toxic Shock Syndrome Toxin

TSST-1: produced by most strains of S. aureus isolated from patients with toxic shock syndrome; prototypical superantigen

binds to MHC class II molecules, stimulates T cells, leading to symptoms of TSS

fever, shock, multisystem involvement; desquamative rash

enterotoxin F


Enterotoxin

(A-E, G-I, K-M); superantigens

heat-stable and resistant to the action of gut enzymes

important cause of food poisoning

produced when S. aureus grows in carbohydrate and protein foods

ingestion of 25 μg of enterotoxin B leads to vomiting and diarrhea

emetic effect is due to CNS stimulation (vomiting center) after the toxin acts on neural receptors in the gut


Pathogenesis


members of the normal flora of human skin and respiratory & GI tracts

also found in clothing, bed linens & other fomites in human environments

pathogenicity is due to combined effect of extracellular factors and toxins together with the invasive properties of the strain

S. aureus (pathogenic, invasive) produce coagulase and forms a yellow pigment;hemolytic

S. epidermidis (nonpathogenic, non-invasive) are coagulase negative & non-hemolytic.


Pathology


prototype lesion: furuncle

S. aureus in hair follicles cause tissue necrosis

coagulase coagulates fibrin around the lesion & within the lymphatics

formation of a wall that limits the process, plus presence of inflammatory cells & fibrous tissue

liquefaction of necrotic tissue occurs at the center of the lesion

organisms may spread via lymphatics & bloodstream


Clinical Findings


localized infection appears as a “pimple”, hair follicle infection, or abscess

intense, localized, painful inflammatory reaction that undergoes central suppuration & heals quickly when the pus is drained

wall of fibrin & inflammatory cells around the core limits the spread of organisms

should not be broken down by manipulation or trauma


infection can result from direct contamination of a wound (e.g.) postoperative infections

secondary localization within or organ or system is accompanied by signs & symptoms of organ dysfunction and intense focal suppuration

food poisoning - violent nausea, vomiting & diarrhea; rapid convalescence; (-) fever


TSS - abrupt onset of high fever, vomiting, diarrhea, myalgia, scarlatiniform rash (erythematous skin rash which desquamates); hypotension with cardiac & renal failure (severe); can recur

seen in women who use tampons (5 days ff onset of menses)

also in men & children with staphylococcal wound infections


Staphylococcal Scalded Skin Syndrome

disease of young children

mediated through minor staphylococcal infection by “epidermolytic toxin”-producing strains

mild erythema and blistering of skin followed by shedding of sheets of epidermis

children are relatively healthy and most eventually recover


Diagnostic Laboratory Tests


Specimens

surface swab pus

blood

tracheal aspirate

spinal fluid for culture

depends on localization of the process


Smears

gram positive cocci in clusters in Gram-stained smears

(pus or sputum)

impossible to distinguish saprophytic from pathogenic staphylococci on smears


Culture

specimen placed on blood agar plates give rise to colonies in 18 hours at 37C

hemolysis & pigment color takes days (RT)

S. aureus ferments mannitol


Catalase Test

used to detect presence of cytochrome oxidase enzymes

a drop of 3% H2O2 is placed on a slide + small amount of bacteria

presence of bubbles (release of O2) indicates a (+) test


Coagulase Test

Citrated rabbit (or human) plasma diluted 1:5 is mixed with broth culture / growth

incubated at 37C

clot formation in 1-4 hours is (+)

indicates pathogenicity in humans


Susceptibility Testing

broth microdilution or disk diffusion

should be done routinely for isolates of clinically significant lesions

resistance to penicillin G can be predicted by a (+) test for β-lactamase (produced by 90%)

resistance to nafcillin (& oxacillin & methicillin) occurs in 35% of S. aureus & 75% of S. epidermidis isolates


Serologic & Typing Tests

little practical value

molecular typing techniques used to document spread of epidemic disease-producing clones of S. aureus

pulsed-field gel electrophoresis & multilocus sequence typing are highly discriminatory


Clues

in all pus-forming lesions

Gram stain and culture of pus

in all systemic infections

blood culture

in infections of other tissues

culture of relevant tissue or exudate


Treatment


tetracyclines - used for long term treatment of multiple skin infections (acne, furunculosis)

drainage - for abscess & other closed suppurating lesions; + antimicrobial therapy

Penicillin G - drug of choice for non-β-lactamase producing S. aureus

Vancomycin - for nafcillin-resistant staphylococci (eg S. epidermidis infection in patients with prosthetic devices)


Epidemiology & Control


chief sources of infection: shedding human lesionsfomites contaminated from shed lesionsrespiratory tractskin

cleanliness, hygiene & aseptic management can control spread of lesionsin hospitals: NICU, ICU, OR, and cancer chemotherapy units are at highest risk for severe staphylococcal infectionsrecent development: dissemination of CA-MRSA


So, if we were to answer...

species coagulase catalase Gram stain β-hemolysis

S. ____ + + ? ?

S. ____ - - ? ?

S. ____ - + ? ☺


The Micrococci


comprise the normal flora of the upper layers of the epidermis & hair follicles (w/ S. epidermidis)

Gram+ spherical cells that appear in tetrads

cell wall comprises ~50% of cell mass

rich in guanine & cytosine (GC)

M. luteus, M. roseus

produce yellow & pink colonies when grown on mannitol salt agar


thought to be saprophytic or commensal organisms

may be opportunistic pathogens, esp. in immunocompromised hosts

pulmonary infections (in immunocompromised), recurrent bacteremia, septic shock, septic arthritis, endocarditis, meningitis

difficult to identify as cause of infection

part of normal skin flora

not usually identified with disease


M. lutea


“Blessed is the man who, having nothing to say,abstains from giving us wordy evidence of the fact.”

GEORGE ELIOT, Impressions of Theophrastus Such
