MIDTERMS C1 - Catalase - Positive, Gram-Positive Cocci Flashcards
Coagulase-Positive Staphylococci
Staphylococcus aureus
Coagulase-Negative Staphylococci
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus saprophyticus
Staphylococcus lugdunensis
Staphylococcus schleiferi
Staphylococcus capitis
Staphylococcus caprae
Staphylococcus warneri
Staphylococcus hominis
Staphylococcus auricularis
Staphylococcus cohnii
Staphylococcus xylosus
Staphylococcus simulans
Micrococcus spp. and related
genera
Alloiococcus
Staphylococcus is a catalase-producing gram (+) cocci that belong to the family
Staphylococcaceae
Staphylococcus is aerobic or facultative anaerobic except what species?
S. aureus subsp. anaerobius and S. sacchrolyticus which are obligate anaerobes
nonmotile, non–spore-forming
spherical cells (0.5 to 1.5 μm) that appear singly, in pairs, and in clusters
normal inhabitants of skin, mucous membranes and intestines
Staphylococcus
In Blood Agar Plate, what are the colonies size and characteristics?
medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “buttery looking”, other spp. may have gray colonies; some may be β-hemolytic (S. aureus
(Staphylococci/Micrococci)
Bacitracin Test Resistant
Staphylococci
(Staphylococci/Micrococci)
Bacitracin Test Susceptible
Micrococci (>10mm)
(Staphylococci/Micrococci)
Furazolidone Susceptibility Test (100ug) - SUSCEPTIBLE
Staphylococci (>15mm)
(Staphylococci/Micrococci)
Furazolidone Susceptibility Test (100ug) - RESISTANT
Micrococci (6-9 mm)
(Staphylococci/Micrococci)
Lysostaphin Sensitivity Test - Susceptible
Staphylococci (10-16 mm)
(Staphylococci/Micrococci)
Lysostaphin Sensitivity Test - Resistant
Micrococci
(Staphylococci/Micrococci)
Modified Oxidase Test/Microdase Test - NEGATIVE
Staphylococci
(Staphylococci/Micrococci)
Modified Oxidase Test/Microdase Test - POSITIVE
Micrococci
(Staphylococci/Micrococci)
Growth on Furoxone-Tween 80-oil Red O Agar - NEGATIVE
Staphylococci
(Staphylococci/Micrococci)
Growth on Furoxone-Tween 80-oil Red O Agar - POSITIVE
Micrococci
(Staphylococci/Micrococci)
Aid production from Glycerol (with erythromycin) - POSITIVE
Staphylococci
(Staphylococci/Micrococci)
Aid production from Glycerol (with erythromycin) - NEGATIVE
Micrococci
(Staphylococci/Micrococci)
OF reaction - Fermenter
Staphylococci
(Staphylococci/Micrococci)
OF reaction - Oxidizer
Micrococci
true coagulase positive and most virulent species of staphylococci
grow well on most routine media like NA and TSB
on solid media, round, smooth, opaque and butyrous
Staphylococcus aureus
on BAP, colonies have golden yellow color and β-haemolytic
responsible for various skin, wound and deep tissue infection
Staphylococcus aureus
Staphylococcus aurreus is cultivated by adding
7.5 to 10% NaCl—HALOPHILIC
VIRULENCE FACTORS of Staphylococcus Aureus
A. Antigenic Structure
B. Enzymes
C. Toxins
contain ribitol teichoic acid in cell wall
Teichoic Acid
together with teichoic acid, it protects the
bacteria from lysis and probably aids in
adherence
Peptidoglycan
group specific antigen unique to S. aureus
prevents antibody-mediated phagocytosis by
PMN—competes for the Fc portion
Protein A
component on cell wall responsible for clumping of the whole staphylococci in the presence of plasma
Clumping Factor
protects fro phagocytosis
Capsular Polysaccharide
coagulates fibrinogen in the plasma
promotes fibrin layer formation around the staphylococcal abscess protecting the bacteria from phagocytosis
Coagulase (Staphylocoagulase)
2 types of Coagulase (Staphylocoagulase)
Cell-bound Coagulase or Clumping Factor
Unbound or Free Coagulase
bound to the cell wall and clots human, rabbit or pig plasma
Cell-bound Coagulase or Clumping Factor
extracellular enzymes not bound to the cell wall and cause clot formation when bacterial cells are incubated with plasma
Unbound or Free Coagulase
hydrolyzes hyaluronic acid present in the intracellular ground substance, permitting the spread of infection
Hyaluronidase (Spreading Factor)
fibrinolytic activities by dissolving fibrin clot
Staphylokinase (Fibrinolysin)
produced by both coagulase (+) and coagulase (-) staphylococci
act on lipids present on the surface of the skin, particularly fats and oil secreted by the sebaceous glands
Lipase (Fat-splitting Enzyme)
important in the formation of furuncles, carbuncles and boils
Lipase (Fat-splitting Enzyme)
lowers viscosity of exudates giving the pathogen more mobility
destroys DNA
Deoxyribonuclease (DNAse) and Phosphatase
breaks down Penicillin and β-lactam drugs
β-LACTAMASE
Cytolytic Toxins
Hemolysins and Leukocidins
4 types of Hemolysins
a. α-Hemolysin
b. β-Hemolysin (Sphingomyelinase C)
c. δ-Hemolysin
d. γ-Hemolysin
damage RBC, platelets and macrophages and cause severe tissue damage
Predominant hemolysin
α-Hemolysin
acts on sphingomyelin in the plasma membrane of RBC
aka“hot-cold” lysine : enhanced hemolytic activity on incubation at 37° C (heat labile) and subsequent exposure to cold (4° C)
β-Hemolysin (Sphingomyelinase C)
exhibited in the CAMP test
lethal and dermonecrotic
β-Hemolysin (Sphingomyelinase C)
less toxic to cells than either α-hemolysin or β-hemolysin
produced by all S. aureus strain that cause RBC injury in culture an produce edematous lesions
δ-Hemolysin
associated with Panton-Valentine leukocidin (PVL)
γ-Hemolysin
exotoxin lethal to polymorphonuclear leukocytes
Pore forming exotoxin that suppress phagocytosis and associated with severe cutaneous infections and necrotizing pneumonia
associated with community-acquired staphylococcal infections
Staphylococcal Leukocidin/ Panton-Valentine leukocidin
heat-stable exotoxin: 100° C for 30 minutes
resistant to hydrolysis by gastric and jejunal enzymes
Enterotoxins
act as neurotoxins that stimulate vomiting through the vagus nerve
produced by 30% to 50% of S. aureus isolates
Enterotoxins
Staphylococcal food poisoning
Enterotoxins A, B, and D
TSS (Enterotoxins)
Enterotoxins B and C and sometimes G and I
Staphylococcal Pseudomembranous
Enterocolitis (contaminated milk products)
Enterotoxin B
aka ENTEROTOXIN F or PYROGENIC EXOTOXIN C
menstruating-associated TSS= TSS associated with tampon use
chromosomal-mediated toxin
TOXIC SHOCK SYNDROME TOXIN-1(TSST-1)
SUPERANTIGEN stimulating T-cell proliferation and production of a large amount of cytokines
low concentrations= leakage by endothelial cells; higher concentrations= cytotoxic
TOXIC SHOCK SYNDROME TOXIN-1(TSST-1)
aka EPIDERMOLYTIC TOXIN A and B or EXFOLIATIN serotypes A and B
Serine protease that divides the intercellular bridges of the epidermis and causes excessive sloughing of the epidermis (stratum granulosum)
Exfoliative Toxins
causes STAPHYLOCOCCAL SCALDED SKIN SYNDROME referred to as RITTER’S DISEASE
implicated in BULLOUS IMPETIGO
Exfoliative Toxins
mild inflammation of a hair follicle or oil gland; infected area is raised and red
Folliculitis
focal suppurative lesions which has resulted from an infection (folliculitis) that extend into subcutaneous tissue; large, raised, superficial abscesses
Furuncles (Boils)
larger, more invasive lesions develop from multiple furuncles, which can progress into deeper tissues; present with fever and chills, indicating systemic infection
Carbuncles
larger pustules surrounded by a small zone of erythema
highly contagious infection that spread by direct contact, fomites, or autoinoculation
Bullous Impetigo
superficial cutaneous infection commonly seen in newborns and young children characterized by the formation of encrusted pustules surrounded by red border
Impetigo
bullous exfoliative dermatitis that occurs primarily in newborns and previously healthy young children
localized skin lesion: few blisters, pemphigus neonatorum, Ritter disease; generalized form: cutaneous erythema, profuse peeling of the epidermis
Scalded Skin Syndrome
clinical manifestation with multiple causes; symptoms are due to hypersensitivity reaction
Toxic Epidermal Necrolysis (TEN)
rare but potentially fatal, multisystem disease characterized by sudden onset of fever, chills, vomiting, diarrhea, muscle aches, and rash, which can quickly progress to hypotension and shock
Toxic Shock Syndrome
intoxication resulting from ingestion of a toxin formed outside the body
symptoms appear rapidly (2 to 8 hours after ingestion) and resolve within 24 to 48 hours: nausea, vomiting, abdominal pain, and severe cramping, diarrhea
Food Poisoning
Percentage of Enterotoxins A, D, and B in Food Poisoning
Enterotoxins A (78%), D (38%), and B (10%)
perfuse and watery diarrhea due to water and electrolyte loss
Food Poisoning
Enterotoxin A and (leukocidins) LukE and LukP
Enterocolitis
leads to secondary pneumonia and endocarditis observed among IV drug users
Staphylococcal Bacteremia
secondary to bacteremia
Staphylococcal Osteomyelitis
secondary to influenza virus infection
multiple abscesses and focal lesions in the pulmonary parenchyma
Staphylococcal Pneumonia
frequent in children and occur in patients with a history of rheumatoid arthritis or IV drug abuse
Septic Arthritis
JOINT INFECTIONS/SEPTIC ARTHRITIS
Bacteria in Both Adults and Neonates/ Children
S. aureus
S. agalactiae
S. pyogenes
Enterobacteriaceae
JOINT INFECTIONS/SEPTIC ARTHRITIS
Bacteria in Neonates / Children Only
H. influenzae type b (Hib)
Kingella kingae
JOINT INFECTIONS/SEPTIC ARTHRITIS
Bacteria in Sexually Active
N. gonorrhoeae
Specimen for Laboratory Diagnosis
Pus, Purulent Fluids, Sputum, Urine, Blood
Laboratory Diagnosis
Gram Stain
Gram (+) cocci in irregular clusters
Lab Diagnosis - Culture Media
BAP, PEA, MSA, CNA, Chapman Stone Agar, Vogel-Johnson
Medium
Columbia Colistin–Nalidixic acid (CNA) -purulent exudates
MSAandPEAheavilycontaminated specimen
CHROMAgarselective-differential for MRSA
Lab Diagnosis - Biochemical Test (S. aureus)
a. Catalase Test
b. Coagulase Test
c. Mannitol Fermentation Test
d. Growth on Tellurite Glycine Agar
e. Polymyxin B Sensitivity
f. Lysostaphin Sensitivity Test (2ug/mL)
g. Voges-Proskauer (VP) Test
h. Deoxyribonuclease (Dnase) Test
i. Pyrrolidonyl Arylamidase (PYR) Test
j. Rapid Methods of Identification
k. Molecular Methods
Aerobic Catalase Test Reagent
3% H2O2
Anaerobic Catalase Test Reagent
15% H2O2
Catalase Test RESULT
Staphylococcus: catalase +
Streptococcus: catalase -
best single criterion of recognition and pathogenicity of S. aureus
Coagulase Test
Reagent for Coagulase Test
Rabbit plasma with EDTA
Methods for Coagulase Test
- Slide Method
- Tube Method
rapid screening test
detects cell-bound coagulase or clumping factor
Slide Method
sensitive but definitive; confirm all slide negative results
detects extracellular or free coagulase
Tube Method
Other Slide Coagulase Positive:
S. lugdunensis and S. schleiferi
Other Tube Coagulase Positive:
S. hyicus, S. intermedius, S. lutrae, S. delphini and S. schleiferi subsp. coagulans
In tube method incubate it for ___________________: if no clot forms after 4 hours reincubate at room temperature for additional ______ hours
1-4 hours at 35°C-37°C ; 20 hours
In Coagulase Test Result should be read within 4 hours to prevent
False (-) Reaction
lyses the clot formed
FIBRINOLYSIN
Coagulase plasma with _____ is not suitable
Citrate
Pseudomonas and Enterococci use citrate and release calcium forming clot in the absence of coagulase
False (+) Result
can ferment mannitol and can tolerate high salt concentrations (7.5-10%)
S. aureus
Culture Medium for Mannitol Fermentation Test
Mannitol Salt Agar
Mannitol Salt Agar
pH indicator:
Positive Result:
: Phenol Red
: yellow-colored colonies surrounded by a yellow halo
S. aureus reduce tellurite producing what?
JET-BLACK COLONIES
In Tellurite Glycine Agar, Other Staphylococci is ________
inhibited; if growth occurs gray colonies are seen
Polymyxin B Sensitivity
S. aureus = Resistant
Other Staphylococci = Susceptible
Lysostaphin Sensitivity Test (2ug/ml)
S. aureus = Sensitive
Micrococci = Resistant
differentiate S. aureus (+) from S. intermedius (-)
VP Test
Culture Medium for VP Test
VP Broth with 5% glucose
Reagent for VP Test
α-naphthol and KOH
Other VP Positive:
S. lugdunensis, S. haemolyticus, and S. schleiferi
Culture Medium for Dnase test
DNA-Methyl Green Agar
T or F
S. aureus = Dnase Negative
False; Dnase Positive si S. aureus (like pag nagegets)
differentiates coagulase(+) staphylococci by slide method
Pyrrolidonyl Arylamidase (PYR) Test
Substrate for PYR Test
Pyroglutamyl-β-naphthylamide (L-pyrrolidonyl-β-naphthylamide; PYR)
Reagent for PYR Test
p-dimethylaminocinnamaldehyde
End Product for PYR Test
L-pyrrolidone and β-naphthylamine
Result of PYR Test
Cherry Red
Positive for PYR Test
S. lugdunensis, S. intermedius S. schleiferi, S.
haemolyticus
Negative for PYR test
S. aureus
Particle Agglutination Test
Staphyloslide - use sensitized sheep RBC
Staphaurex
BACTiStaph
Staphylochrome
Sero-STAT
Bacto Staph Latex
Accu-Staph
Hemostaph
Staphylatex
plasma-coated carrier particles
latex
plasma detects clumping factor with?
fibrinogen
plasma detects protein A in the cell wall of S. aureus with?
with IgG
identifying both MRSA and MSSA
Real-time PCR
staphylococci from prepared smears in
blood cultures
identification of mecA gene
Qualitative Nucleic Acid Hybridization Assays
Specimen for Molecular Methods
Anterior Nares Swabs
Advantage of Molecular Methods
rapid detection test for MRSA
indigenous microbiota of the skin
contaminant of medical instruments, catheters, CSF shunts and prosthetic heart valve implants (implanted medical devices), hip prostheses
Staphylococcus epidermidis
Diseases caused by S. epidermidis
Stitch abscess, Health care-acquired UTIs, Endocarditis, Bacteremia
adherence of S. epidermidis
Poly-γ-DL-Glutamic Acid (PGA)
S. epidermidis in BAP
gray to white, opaque, small to medium-sized pin heads and non-hemolytic colonies
S. epidermidis - Biochemical Tests
Coagulase(-),
CNA (+),
DNase (-),
Mannitol Fermentation (-)
S. epidermidis is susceptible with
5-ug NOVOBIOCIN (16mm-27mm)
present on the normal skin and in the periurethral and urethral flora
adheres effectively to the epithelial cells lining the urogenital tract
Staphylococcus saprophyticus
Disease associated with S. saprophyticus
common cause of UTI in young sexually active women
urine culture <10,000 CFU/ml = significant
S. saprophyticus in BAP
white, opaque, slightly larger than pin-heads, non-hemolytic colonies although some strains produce yellow pigments
S. saprophyticus - Biochemical Test
Coagulase (-),
Dnase (-)
Mannitol Fermentation (-)
Resistance to NOVONIOCIN (5ug; 6mm-12mm) and NALIDIXIC ACID
Absence of Phosphatase production
Staphylococcus saprophyticus
clumping factor (+), tube coagulase (-)
contain mecA gene that encodes oxacillin resistance
more aggressive than other CoNS in ineffectivity
Staphylococcus lugdunensis
Disease associated with S. lugdunensis
infective endocarditis, septicemia, meningitis, skin and soft tissue infections, UTIs, and septic shock
Other Coagulase-Negative Staphylococci
S. warneri, S. capitis, S. simulans, S. hominis, and S. schleiferi
S. haemolyticus
causes endocarditis, septicemia, and wound infections
S. warneri, S. capitis, S. simulans, S. hominis, and S. schleiferi
causes
wounds, bacteremia, endocarditis, and UTIs
medium-sized colonies, with moderate or weak hemolysis and variable pigment production
S. haemolyticus
NOVOBIOCIN SUSCEPTIBLE CoNS
S. epidermidis
S. capitis
S. haemolyticus
S. hominis subsp. hominis
S. lugdunensis
S. saccharolyticus
S. warner
NOVOBIOCIN RESISTANT CoNS
S. saprophyticus
S. cohnii
S. kloosii
S. xylosus
RESISTANT GENES PRODUCED BY STAPHYLOCOCCI
a. Erythromycin Ribosomal Methylase (ERM) Gene
b. Methionine Sulfoxide Reductase (MSR) A Gene
Class of enzyme inactivating genes
Codes for the methylation of the 23s rRNA
Results in resistance to erythromycin
Inducible or constitutive resistance to clindamycin
Erythromycin Ribosomal Methylase (ERM) Gene
May not bedetected in routine susceptibility testing
Confer cross-resistance to macrolides (erythromycin) and streptogramins (quinupristin)
Erythromycin Ribosomal Methylase (ERM) Gene
Codes for efflux mechanism resistance to
erythromycin but susceptibility to clindamycin
Methionine Sulfoxide Reductase (MSR) A Gene
type of S. aureus that is resistant to methicillin, nafcillin, and oxacillin
Methicillin-Resistant Staphylococci aureus (MRSA)
acquired after prolonged stay in the hospital, close contact with carriers, effects of broad spectrum antibiotic treatments and exposure to nasal secretions
Methicillin-Resistant Staphylococci aureus (MRSA)
treatment of choice for MRSA
VANCOMYCIN
Types of MRSA
Community-Associated MRSA (CA-MRSA),
Health Care–Associated Community-Onset (HACO-MRSA),
Hospital-Associated (HA-MRSA)
codes for altered Penicillin-Binding
Protein (PBP) = PBP2a or PBP2’
mecA
used to screen for MRSA in clinical samples
differentiate MRSA from hyperproducers of β-lactamase, or Borderline Oxacillin-Resistant Staphylococcus aureus (BORSA)
Oxacillin-Salt Agar Plate
BORSA
Borderline Oxacillin-Resistant Staphylococcus aureus (BORSA)
Chromogenic Selective Differential Media
MRSA Select
Spectra MRSA
CHROMagar MRSA
inhibit non-MRSA
Cefoxitin
Result of Chromogenic Test
Changes in color of MRSA colonies within 24 to 48 hours using CHROM Agar against colonies of non-MRSA
Mauve-colored colonies
Methicillin-Resistant Staphylococci aureus (MRSA)
detect altered PBPs
alternative method for testing and confirmation of oxacillin resistance
performed on both CoNS and S. aureus
Latex Agglutination
“gold standard” for MRSA detection
Molecular Nucleic Acid Probes or PCR Amplification
Screening for Vancomycin-Resistant Staphylococci
Vancomycin Agar Plate
= discrepant macrolide test results (erythromycin resistant and clindamycin susceptible)
Modified Double Disk Diffusion Test
Beta-Lactamase Test
a. Cephalosporinase Test
b. Acidimetric Method
c. Iodometric Method
uses cephalosporin or cefinase disk
Cephalosporinase Test
Cephalosporinase Test
Substrate:
(+) result:
Substrate: nitrocefin
(+) result: deep pink or red color within 10 minutes (60 minutes for Staphylococci)
Acidimetric Method
Reagent:
pH indicator:
(+)result:
Reagent: citrate-buffered penicillin
pH indicator: phenol red
(+)result: red to yellow (penicilloic acid = decrease pH)
Iodometric Method
Reagent:
(+) result:
(-) result:
Reagent: citrate-buffered penicillin and starch iodine complex
(+) result: colorless solution- penicilloic acid reduces iodine and prevents it to combine with starch
(-) result: purple (no color change)
ANTIMICROBIAL TESTING
-Treatment:
methicillin, oxacillin, nafcillin, cloxacillin, and dicloxacillin (penicillinase-resistant penicillin drugs)
most commonly used drug
Oxacillin
Drug for Cutaneous Infections
Oral oxacillin or dicloxacillin, if allergic, erythromycin may be substituted
Antimicrobial Testing
- Systemic
parenteral nafcilllin or oxacillin, if allergic, vancomycin or cephalosporin may be used
Antimicrobial Testing
- MRSA
vancomcin alone or in a combination with rifampicin
Screening Test for MRSA:
Oxacillin Screen Plate
Culture Media for Oxacillin Screen Plate
MHA with 4% NaCl and 6 ug/mL oxacillin
- spot inoculated with cotton swab and incubated for 24 hours at 35°C
oxacillin in cation-supplemented MH broth containing 2% NaCl
Microdilution Testing
Screening Test for MRSA: Oxacillin Screen Plate
Result:
CoNS:
Result:
Resistant- growth of more than one colony
Susceptible- no growth on the agar plate
CoNS:
Resistant- 24 mm zone of inhibition
Susceptible- >25 mm zone of inhibition
Disadvantage of Oxacillin
Does not reliably detect oxacillin-resistant CoNS
preferred method for detection of oxacillin-resistance for both S. aureus and S. lugdunensis
improves detection of MRSA
Cefoxitin Disk Diffusion (30 ug)
serves to induce greater PBP2a in mecA-containing strains
Test reagent to detect resistance- both MIC and diffusion method
Cefoxitin Disk Diffusion (30 ug)
Interpretation for Cefoxitin Disk Diffusion
Resistant- <21 mm
Susceptible- >22 mm
detection of heteroresistant VISA because the test uses a higher concentration of organisms (1 x 108 bacteria/ mL)
Macro E Test
S. aureus should be screened with 6-ug/mL vancomycin incorporated into BHIA
Broth microdilution test
best method for detection of either Vancomycin-resistant S. aureus (VRSA) or VISA
Vancomycin Agar Screen Plate
Confirmatory test for oxacillin resistance
Broth dilution and E-test