Microbiological considerations in diagnosing s aureus bacteremia
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Microbiological Considerations in Diagnosing S. aureus Bacteremia. Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories. Staphylococcus aureus Bacteremia Microbiological Considerations. Overview of Blood Culture Systems Skin Antisepsis

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Microbiological Considerations in Diagnosing S. aureus Bacteremia

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Microbiological considerations in diagnosing s aureus bacteremia

Microbiological Considerations in Diagnosing S. aureus Bacteremia

Patrick R. Murray, Ph.D.

NIH Clinical Center

Chief, Microbiology Laboratories


Staphylococcus aureus bacteremia microbiological considerations

Staphylococcus aureus BacteremiaMicrobiological Considerations

  • Overview of Blood Culture Systems

    • Skin Antisepsis

    • Volume of blood and blood-to-broth ratio

    • Number and timing of cultures

    • Methods for detection of microbial growth

  • Interpretation of Culture Results

    • Time to detection of positive cultures

    • Spectrum of organisms recovered in blood cultures

    • Significant vs. contaminated culture

  • Identification of Staphylococci

    • Direct identification vs. use of subculture

    • Coagulase test - slide test, tube test, commercial tests

    • Protein A - commercial tests

    • Genetic probes for S. aureus

    • Fluorescent in situ hybridization (FISH) test


Overview of blood culture systems

Overview of Blood Culture Systems

  • Skin antisepsis

    • 70% alcohol followed by 2% tincture of iodine, povidone-iodine, or chlorhexidine

    • <3% considered good practice

  • Volume of blood and blood-to-broth ratio

    • Most septic patients with <1 org/ml of blood; therefore, the volume of blood cultured is related to % positive cultures

    • Volume: 20-30 ml for adults, proportionally less for children

    • Dilution of blood in broth by at least 1:5 ratio (except resin media)

  • Number and timing of cultures

    • Continuous vs. intermittent bacteremia

    • 2-3 cultures over 24-hour period

  • Methods for Detection of Microbial Growth

    • Manual methods - obsolete

    • Lysis-centrifugation system - quantitative culture

    • Automated methods - measure of microbial metabolism, e.g., carbon dioxide production, oxygen consumption


Interpretation of culture results

Interpretation of Culture Results

  • Time to detection of positive cultures

    • Most positive cultures detected in first 48 hours of incubation

    • S. aureus detected in <24 hours; other staph >24 hours

    • Culture routinely held 5-7 days

  • Spectrum of organisms recovered in blood cultures

    • 10-15% of cultures typically positive

    • Most common isolates are: CNS, S. aureus, E. coli, Enterococcus, Klebsiella, S. pneumoniae

  • Significant vs. contaminated culture

    • Most isolates of S. aureus, S. pneumoniae, ß-hemolytic streptococci, Enterococcus, Enterobacteriaceae, Ps. aeruginosa, gram-neg. anaerobes, and yeasts are significant

    • Most isolates of CNS, Corynebacterium, Proprionibacterium, and Bacillus are insignificant

    • Significant isolates of CNS are typically associated with a contaminated line or other foreign body.


Identification of staphylococci

Identification of Staphylococci

  • Direct identification vs. use of subculture

    • Subculture plate - growth of S. aureus by 4-6 hours

    • Serum separater/clot tube (SST) used to concentrate bacteria

    • Positive broth can be used for the FISH (fluorescent in situ hybridization) test and molecular probes, but a heavier inoculum (e.g., from subculture plate) is needed for the coagulase and protein A tests


Identification of staphylococci1

Identification of Staphylococci

  • Coagulase test

    • Measures the ability of Staph to clot plasma; EDTA rabbit plasma is recommended

    • Coagulase can be bound to the surface of the bacteria or freely excreted; bound coagulase (clumping factor) is detected by the “slide” test and commercial latex agglutination tests (rapid), free coagulase is detected by the “tube” test (4-24 hours)

    • All S. aureus isolates are positive by the tube test; 85% are positive by the slide or commercial tests

    • S. schleiferi and S. lugdunensis are positive by the slide test but not the tube test

    • S. intermedius and S. hyicus (animal strains) are positive by the tube test (generally a delayed reaction)

  • Protein A test

    • Can be detected in combination with coagulase by commercial tests


Identification of staphylococci2

Identification of Staphylococci

  • Genetic probes for S. aureus

    • The GenProbe AccuProbe system uses a single-stranded DNA probe with a chemiluminescent label that is complementary to rRNA of S. aureus.

    • The test inoculum is prepared from a subculture plate or from a broth culture with a turbidity of a McFarland 1 standard.

    • The total test time for cell lysis, hybridization, and detection takes less than 1 hour.

    • Marlowe et al (JCM 41:1266, 2003) reported the limit of detection with seeded blood cultures was approximately 10,000 CFU/ml with this method. This is at least 10- to 100-fold more sensitive than the limit of detection for the blood culture instrument.


Identification of staphylococci3

Identification of Staphylococci

  • Fluorescent in situ hybridization (FISH)

    • Applied Biiosystems (Boston Probes) developed a FISH method using Peptide Nucleic Acid (PNA) probes that target mRNA of specific bacteria (e.g., S. aureus).

    • PNA is a synthetic pseudopeptide that hybridizes to complementary nucleic acid targets. These probes have a higher specificity and more rapid hybridization kinetics compared to traditional DNA probes. Fluorescent labels are attached to the probe to help detect the target organisms.

    • The total test time is approximately 2.5 hours.

    • In three studies using the probes with positive blood culture broths, the sensitivity and specificity was virtually 100%.

      • Oliveira et al. J Clin Microbiol 40: 247-251, 2002

      • Oliveira et al. J Clin Microbiol 41:889-891, 2003

      • Chapin and Musgnug. J Clin Microbiol 41:4324-4327, 2003


Identification of staphylococci4

Identification of Staphylococci

  • Fluorescent in situ hybridization (FISH)

    • Applied Biiosystems (Boston Probes) developed a FISH method using Peptide Nucleic Acid (PNA) probes that target mRNA of specific bacteria (e.g., S. aureus).

    • PNA is a synthetic pseudopeptide that hybridizes to complementary nucleic acid targets. These probes have a higher specificity and more rapid hybridization kinetics compared to traditional DNA probes. Fluorescent labels are attached to the probe to help detect the target organisms.

    • The total test time is approximately 2.5 hours.

    • In three studies using the probes with positive blood culture broths, the sensitivity and specificity was virtually 100%.

      • Oliveira et al. J Clin Microbiol 40: 247-251, 2002

      • Oliveira et al. J Clin Microbiol 41:889-891, 2003

      • Chapin and Musgnug. J Clin Microbiol 41:4324-4327, 2003


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