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 –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 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: 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 Time to detection of positive cultures –Most positive cultures detected in first 48 hours of incubation –S. aureus detected in 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 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 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 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 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: , 2002 Oliveira et al. J Clin Microbiol 41: , 2003 Chapin and Musgnug. J Clin Microbiol 41: , 2003

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: , 2002 Oliveira et al. J Clin Microbiol 41: , 2003 Chapin and Musgnug. J Clin Microbiol 41: , 2003