Microbiological Considerations in Diagnosing S. aureus Bacteremia

1. Microbiological Considerations in Diagnosing S. aureus Bacteremia Patrick R. Murray, Ph.D. NIH Clinical Center Chief, Microbiology Laboratories

2. 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

3. 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

4. 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.

5. 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

6. 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

7. 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.

8. 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

10. 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