# Presentation: 326695

## 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: 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

- 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

- 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: 247-251, 2002
- Oliveira et al. J Clin Microbiol 41:889-891, 2003
- Chapin and Musgnug. J Clin Microbiol 41:4324-4327, 2003

## Slide 9

## 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