SUPILANAS, IVY KEITH R.

DEC 1, 2015
ME-14O MS. JONEL MEDINA

Rapid Identification of Staphylococcus aureus:

FISH Versus PCR Methods
Qing Wu, MD, Yan Li, MD, PhD*, Huixia Hu, MS, Ming Wang,
MD,ZegangWu, MS and Wanzhou Xu, MS
+ Author Affiliations
Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China

To whom correspondence should be addressed. E-mail: [email protected]

Abstract
Staphylococcus aureus (S aureus) is the most important pathogen in the genus Staphylococcus.
The ability to rapidly and accurately distinguish between S aureus and non–S aureus bacteria (coagulase-
negative Staphylococcus species [CoNS]) is essential for the appropriate therapeutic use of antibiotics
and timely intervention for infection control. Several methods have been reported as being effective in
the rapid identification of S aureus, among which molecular biological methods have the most potential.
Methods In this study, 2 molecular techniques, fluorescence in situ hybridization (FISH) and polymerase
chain reaction (PCR), were compared in 1300 clinical specimens. After smear testing, specimens
containing gram-positive cocci in clusters were submitted for investigation. These specimens had been
subjected to FISH analysis and PCR examination. Overall, we gathered statistics on 131 specimens that
had been determined to be members of the Staphylococcus genus. We compared the effectiveness,
efficiency, and costliness of the 2 methods. Results Comparing the culture determination methods
revealed that the identification sensitivity, specificity, and positive and negative predictive values were
100%, 100%, 100% and 100%, respectively, for the FISH method and 98.5%, 100%, 100% and 98.5%,
respectively, for the PCR method. The FISH method took approximately 3 hours to complete per sample,
whereas PCR took approximately 4 hours. S aureus was differentiated from CoNS via the FISH method 1
hour faster than via PCR identification. Conclusion Although the FISH and PCR methods both allow for
the rapid and reliable identification of S aureus in clinical specimens, FISH is more appropriate for testing
a few specimens, whereas PCR is more appropriate for testing a large number of specimens at a lower
cost.
SUPILANAS, IVY KEITH R. DEC 1, 2015
ME-14O MS. JONEL MEDINA

Keywords:
Identification Staphylococcus aureus fluorescence in situ hybridization polymerase chain reaction

Staphylococci include Staphylococcus aureus (S aureus) and coagulase-negative Staphylococcus species

(CoNS). S aureus is more virulent and usually causes more serious infections than CoNS.1-3 Particularly
in the lower respiratory tract and bloodstream, CoNS may be a colonizing bacterium rather than a
pathogen.2-4 unfortunately, after Gram staining, the definitive identification of S aureus from
staphylococci is time consuming. Traditional methods require subculturing (for 12-24 hours) followed by
biochemical analysis. Rapid identification of S aureus is beneficial. Several molecular methods for the
rapid identification of pathogenic bacteria have been described.5-9 Fluorescence in situ hybridization
(FISH) and polymerase chain reaction (PCR) methods have the greatest potential for the rapid and
accurate identification of microorganisms.5-6 However, the 2 methods have not been evaluated
simultaneously. The time efficiency and costliness of these methods could affect their use. Anything that
interferes with the rapid and reliable identification of S aureus in clinical specimens would likewise
interfere with the ultimate choice of health care professionals as to which method to use. Further, the
FISH method is generally less sensitive than PCR, so it is difficult to determine which would be the
preferred method among health care professionals. In this study, we used the FISH and PCR methods for
the molecular identification of S aureus in clinical specimens. The primers and probes were based on 16S
ribosomal RNA (rRNA). These rRNA sequences were compared to all microorganisms by the National
Center for Biotechnology (NCBI) Basic Local Alignment Search Tool (BLAST) to find identical sequences
among diverse strains of the same bacterium that are unique to those strains. The two methods were
conducted simultaneously. We evaluated the identification sensitivity, specificity, positive predictive
values and negative predictive values of the 2 S aureus tests and recorded the time efficiency and
costliness of each method to determine the appropriate circumstances for the use of each type of test.

Materials and Methods

Clinical Samples and Reference Strains All 1300 specimens were obtained from patients in the Renmin
Hospital of Wuhan University, Wuhan, China, from October 2010 through September 2011. These
clinical specimens including 427 urine samples, 344 sputum samples, 342 blood samples, 96
cerebrospinal fluid samples, 47 joint fluid samples, and 44 pleural fluid samples. Each clinical sample was
divided and transferred into 2 parts, one for the conventional culture identification and the other for
FISH and PCR tests. The reference strains S aureus (ATCC 25923), Staphylococcus epidermidis (ATCC
12228), Staphylococcus Saprophyticus (ATCC 49907), and Staphylococcus capitis (ATCC 146) were
purchased from the American Type Culture Collection (ATCC). These strains were stored at -80°C until
testing.

Culture and Sample Preparation Reference strains stored at -80°C were inoculated onto 5% sheep blood
agar plates (bioMérieux, Marcy l'Etoile, France) and incubated at 37°C for 18 to 24 hours, according to
the manufacturer's recommendations. Colonies were removed with a sterile plastic loop and suspended
in 100 μl of sterile water. These bacterial suspensions were adjusted to 1 MacFarland standard (used to
visually approximate the concentration of cells in a suspension). Each suspension was divided; one part
was used for FISH examination and one part was used for PCR analysis.
SUPILANAS, IVY KEITH R. DEC 1, 2015
ME-14O MS. JONEL MEDINA

The urine and sputum specimens were first separately cultured for 18 to 24 hours. S aureus colonies
were then prepared for the bacterial suspension in the same manner. Each suspension was divided into
2 parts; one part was used for FISH examination and one part was used for PCR analysis. The clinical
samples collected for asepsis evaluation, including positive blood media, cerebrospinal fluid, joint fluid,
and pleural fluid, were centrifuged at 5000 × g, 4°C for 3 minutes; the supernatant was removed. The
cell pellets were washed 3 times with physiological saline. Finally, the pellets were suspended in
physiological saline (0.5 mL). Each suspension was divided into 2 parts: one was used for FISH
examination and the other was used for PCR analysis. All samples were identified by conventional Gram
staining, seeding in different subculture media, and using an API test system (bioMèrieux). Each blood
sample was divided and transferred into 2 bottles, one for aerobic growth and the other for anaerobic
growth. These bottles were incubated using the BacT/ALERT microbial detection system (bioMèrieux).
The positive cultures were further processed for identification by conventional culture methods
(described earlier herein).

FISH Methods
Sample Fixation and Preparation of Cell Smears Three μl of the bacterial suspension (from the sample
preparation step) was spotted onto a cleaned glass slide and allowed to air dry. Then, the glass slide was
immersed in paraformaldehyde (4% in sodium perborate [PBS]) and fixed for 10 minutes. The cell pellet
was washed 3 times with PBS for 1 minute. Finally, the glass slides were rinsed with deionized water and
allowed to air dry.

FISH Assay The FISH method has been described previously:10 glass slides bearing S aureus and CoNS
were incubated in 95% ethanol (for 5 minutes each). Staphylococci were incubated with lysozyme (1
mg/mL for 10 minutes, at 30°C), followed by lysostaphin (Sigma-Aldrich Co LLC, St. Louis, Missouri) (1
mg/mL for 5 min, at 30°C); each enzyme was dissolved in 10 mM Tris buffer (pH, 8.0). The slides were
washed and 5 ng of each oligonucleotide probe (5′-AGA GAA GCA AGC TTC TCG TCC GTT C) (25
monomerie unit; Life Technologies Corporation, Carlsbad, California) were added to 10 mL of
hybridization buffer (20 mM Tris-HCl, 0.9 M NaCl, 0.1% SDS, 20% formamide, pH, 7.2). The slides were
then incubated for 60 minutes at 48°C in a humidified chamber. The slides were washed, air dried, and
analyzed via fluorescence microscopy (×100 magnification; Olympus IX-70; Olympus Corporation, Tokyo,
Japan) with a fluoroscein isothiocyanate (FITC) filter (absorption wavelength, 494 nm; emission
wavelength, 518 nm).

PCR Detection A 50 μL bacterial suspension was prepared, as described in the previous paragraph.
Bacterial suspensions were centrifuged at 7000 × g for 3 minutes; then the supernatant was removed.
The precipitate was resuspended in 45 μL TE (10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid
[EDTA], pH, 8.0), and 5 μL of lysozyme (20 mg/mL) (Sigma-Aldrich Co LLC) was added. The mixture was
incubated in a water bath (30 minutes, at 37°C) and then incubated in a second water bath (10 minutes,
at 100°C). Finally, the suspension was centrifuged at 12 000 × g for 10 min at 4°C, and 3 μL aliquots were
used as the template for the PCR reaction.

Primers for PCR Amplification The primers used for S aureus were forward, 5′-AAC TCT GTT ATT AGG
GAA GAA CA-3′ (23 mer) and reverse, 5′-CCA CCT TCC TCC CCG TTG TCA CC-3′ (23 mer). These primers
were purchased from Life Technologies Corporation.
SUPILANAS, IVY KEITH R. DEC 1, 2015
ME-14O MS. JONEL MEDINA

PCR Amplification The final concentration of the primer pairs was 10 μmol/L. The reaction conditions
used were 25 mmol/L MgCl2, 2 mmol/L deoxynucleotide triphosphates (dNTP) and 1 U/μL Taq DNA
polymerase in KCl buffer. For the reaction mixture, 2.5 μL of extracted DNA template were added to
each tube. Sterilized deionized water was added to produce a final volume of 25 μL. The bacterial
samples were amplified with the previously described species-specific primers. A GeneAmp PCR System
2400 (F. Hoffman-La Roche Ltd, Basel, Switzerland) was used; the thermocycling conditions were 94°C
for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 90 seconds,
with a final extension at 72°C for 10 minutes. Each PCR product was analyzed by electrophoresis using
1.5% agarose gels containing 0.5 mg/mL of EtBr.

Discussion
Conventional identification of a microorganism from a clinical specimen requires 24 to 48 hours. A rapid,
sensitive, and specific method is crucial for the identification of pathogens. Kempf et al5 reported
probes with a sensitivity and specificity of 100% using the FISH method; Pechorsky13 concluded that
PCR is a specific and precise method for identifying pathogenic bacteria in blood samples. Our study
evaluated 2 molecular assays, FISH and PCR; our results were in agreement with those of other
studies.5, The FISH and PCR methods used in these published studies were based on rRNA, which, as a
combination of highly conserved ribosomal rna and highly variable regions, enables the synthesis of
specific probes and primers targeted to various phylogenetic levels without performing cultures.14-16 A
total of 161 gram-positive clinical samples were identified by conventional microbiological identification
methods and FISH and PCR molecular methods. Compared with the standard culture method, the total
diagnostic accuracy of all clinical specimens of the 2 molecular methods is high (Table 2, Table 3). The
FISH and PCR methods both satisfied requirements for S aureus detection. One S aureus specimen in our
series tested negative via PCR; this error resulted from a blood sample that was collected from a patient
with multiple myeloma. Immunoglobulin G, which is a major inhibitor of diagnostic PCR, was elevated in
this specimen. After twice washing with Tris/EDTA (TE) buffer, purifying the isolate, and retesting it via
PCR, a positive result was obtained. This omission was not observed for the other types of microbial
samples, such as those from pleural fluid and cerebrospinal fluid. The turnaround time of FISH was
approximately 4 hours for the blood culture media, cerebrospinal fluid, and other sterile specimens
collected body sites. This turnaround time is 1 hour less than the PCR assay and 8 to 44 hours less than
the conventional culture method. For the sputum and urine specimens, culture is required for the FISH
and PCR methods. Thus, the turnaround time for sputum and urine specimens is 12 to 24 hours longer.
Nevertheless, the time required for the 2 molecular methods was still 8 to 20 hours less than the culture
method.

Cost reduction is essential when performing a large number of tests. The cost of the FISH method (RMB
7.60¥ [US$1.20]) was higher than that of PCR (RMB 3.04¥ [US$0.48]). The equipment required to
perform the FISH assay is costly. A narrow space or poor room conditions may restrict the
implementation of PCR. For a small clinical laboratory, the FISH method is much more appropriate than
the alternatives. However, for a laboratory with standard room conditions, the costs of the reagents are
lower with PCR, making this method preferable.
SUPILANAS, IVY KEITH R. DEC 1, 2015
ME-14O MS. JONEL MEDINA

In conclusion, FISH and PCR methods are promising laboratory tools for the rapid and accurate
identification of S aureus in clinical samples. However, considering the cost of materials and variability in
room conditions, FISH is more appropriate for the testing of a few specimens, whereas PCR is more
appropriate for a large number of specimens.

Acknowledgments
This work was supported by the Fundamental Research Funds for the Central Universities, Wuhan,
China.

References
↵ Gómez-Pavón J, Rodríguez Salazar J, Fernández de la Puente E, et al. Staphylococcus aureus infection
in an acute geriatric unit [in Spanish]. Rev Esp Geriatr Gerontol. 2010;45:5-9. MedlineGoogle Scholar

↵ Reighard A, Diekema D, Wibbenmeyer L, Ward M, Herwaldt L. Staphylococcus aureus nasal

colonization and colonization or infection at other body sites in patients on a burn trauma unit. Infection
Control and Hospital Epidemiology. 2009;30:721-726. CrossRefMedlineGoogle Scholar

↵ Liu C, Bayer A, Cosgrove SE, et al. Clinical practice guidelines by the Infectious Diseases Society of
America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and
children. Clin Infect Dis. 2011;52:e18-e55. Abstract/FREE Full Text

↵ Tokars JI. Predictive value of blood cultures positive for coagulase-negative staphylococci: implications
for patient care and health care quality assurance. Clin Infect Dis. 2004;39:333-341. Abstract/FREE Full
Text

↵ Kempf VAJ, Trebesius K, Autenrieth IB. Fluorescent in situ hybridization allows rapid identification of
microorganisms in blood cultures. J Clin Microbiol. 2000;38:830-838. Abstract/FREE Full Text

↵ Yamamoto Y. PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids. Clin Diagn Lab
Immunol. 2002;9:508-514. MedlineGoogle Scholar

Yeh CH, Chang YH, Chang TC, Lin HP, Lin YC. Electro-microchip DNA-biosensor for bacteria detection.
Analyst. 2010;135:2717-2722. MedlineGoogle Scholar

Ellerbrok H, Nattermann H, Ozel M, et al. Rapid and sensitive identification of pathogenic and
apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol Lett. 2002;214:51-59.
CrossRefMedlineWeb of ScienceGoogle Scholar

↵ Ratnayake L, Olver WJ. Rapid PCR detection of methicillin-resistant Staphylococcus aureus and
methicillin-sensitive S. aureus samples from charcoal-containing blood culture bottles. J Clin Microbiol.
2011;49:2382. FREE Full Text