# Shortened Time to IdentifyStaphylococcus Species from Blood Cultures and Methicillin Resistance Testing Using CHROMAgar

Authors: Shingo Chihara; Mary K. Hayden; Eileen Minogue-Corbett; Kamaljit Singh
Journal: International Journal of Microbiology
 (2009)

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## Abstract

The ability to rapidly differentiate coagulase-negative staphylococcus (CoNS) fromStaphylococcus aureus and to determine methicillin resistance is important as it affects the decision to treat empiric antibiotic selection. The objective of this study was to evaluate CHROMagar S. aureus and CHROMagar MRSA (Becton Dickinson) for rapid identification of Staphylococcus spp. directly from blood cultures. Consecutive blood culture bottles (BacT Alert 3D SA and SN, bioMérieux) growing gram-positive cocci in clusters were evaluated. An aliquot was plated onto CHROMagar MRSA (C-MRSA) and CHROMagar S. aureus (C-SA) plates, which were read at 12 to 16 hours. C-SA correctly identified 147/147 S. aureus (100% sensitivity); 2 CoNS were misidentified as S. aureus (98% specificity). C-MRSA correctly identified 74/77 MRSA (96% sensitivity). None of the MSSA isolates grew on C-MRSA (100% specificity). In conclusion, CHROMagar is a rapid and sensitive method to distinguish MRSA, MSSA, and coagulase-negative Staphylococcus and may decrease time of reporting positive results.


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## Body

## 1. Introduction

Staphylococcus species are the most commonly
isolated bacteria from blood cultures [1–3]. The ability
to rapidly differentiate coagulase-negative staphylococcus (CoNS) from S. aureus is important
as CoNS is a common skin contaminant whereas S. aureus bacteremia is associated with significant morbidity and
mortality [2, 3]. In addition, the increased prevalence of methicillin
resistance in Staphylococcus spp.  has led to the widespread use of vancomycin as empiric therapy, which may
provide selective pressure for the spread of vancomycin-resistant enterococcus
and the emergence of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) [4]. The
ability to rapidly differentiate CoNS from S.
aureus and to determine methicillin susceptibility, particularly from blood
cultures, is clearly desirable as it would allow more directed antibiotic
therapy and limit the unnecessary use of vancomycin.The objective of this study was to evaluate CHROMagarS. aureus (Becton Dickinson, Sparks, Md, USA) and CHROMagar MRSA (Becton Dickinson, Sparks, Md, USA) for rapid identification and susceptibility testing of Staphylococcus spp. directly from blood culture
broth media.


## 2. Materials and Methods

The study was performed in the
microbiology laboratory at Rush University Medical Center in Chicago (Ill,
USA) from November 2006 to August 2007. Consecutive blood culture bottles (BacT/Alert 3D SE and SN bottles, bioMérieux, Durham, NC, USA) growing gram-positive cocci in clusters were evaluated. Only one set of blood
cultures from each patient was included. A 0.05 mL aliquot of blood broth was plated onto CHROMagar  MRSA (C-MRSA) and CHROMagarS. aureus (C-SA) plates that were
divided into 4 quadrants to allow each plate to be used for 4 patient isolates.
Plates were incubated at 35°C in ambient air. Growth on plates was evaluated after 12 to 16 hours of incubation and
again after the manufacturer's recommended incubation time of 24 hours. Mauve
colonies on C-SA plate were classified as S. aureus. Mauve colonies on C-MRSA were classified as
MRSA. CoNS produced white colonies.Results were compared to
conventional microbiologic methods for identifyingStaphylococcus spp., that is, gram stain, catalase
reaction, and tube or slide coagulase testing (BactiStaph, Remel Inc. Lenexa, Kan, USA). Discrepant identification results were
resolved by an automated identification method (Microscan Walkaway, Dade Behring Inc., West Sacramento,
Calif, USA). Susceptibility testing was done by using the cefoxitin disk test according to
CLSI guidelines [5] Methicillin resistance in S. aureus was confirmed by the detection of PBP2a using the MRSA
screen slide agglutination test (Denka Seiken Co, LTD, Tokyo, Japan).


## 3. Results

Two hundred and
sixty three unique blood culture broths growing gram-positive cocci in clusters
were studied. Five bottles that yieldedMicrococcus spp. were excluded from further analysis; none of these produced mauve colonies
on C-MRSA or C-SA plates. There were 147 S. aureus and 111 CoNS
identified by conventional microbiologic methods. C-SA correctly identified
147/147 S. aureus after 16
hours of incubation (100% sensitivity). There were 2 CoNS isolates that
produced faint mauve colonies on C-SA; these were confirmed BactiStaph negative, and
were identified as Staphylococcus hominis and Staphylococcus haemolyticus. Therefore, the specificity of C-SA for
identification of S. aureus was 98%. The positive predictive value of the C-SA
plates for identification of S. aureus was 99% (147/149) while the negative predictive value was 100% (109/109).C-MRSA correctly
identified 74/77 MRSA (96% sensitivity) after 12 to 16 hours incubation. None
of the MSSA or CoNS isolates grew on C-MRSA (100% specificity). Both C-SA and
C-MRSA plates were read again after 24 hours incubation; the results were
completely concordant with those obtained after 12 to 16 hours incubation (data
not shown). The positive predictive value of the C-MRSA plates for
identification of MRSA was 100% (74/74) while the negative predictive value was
98% (181/184).


## 4. Discussion

Our study shows that the direct subculture of positive blood culture broths onto C-MRSA and
C-SA plates allows for the rapid identification ofS. aureus and determination of methicillin susceptibility. It is
important to distinguish among MSSA, MRSA, and CoNS as management changes
depending on the species isolated. It is a common practice among clinicians to
initiate empiric vancomycin therapy when informed of a positive blood culture
growing gram-positive cocci in clusters. The use of a beta-lactam antibiotic is
preferred therapy if MSSA is isolated but current methods of susceptibility
testing using oxacillin or cefoxitin disks require cultures to be held for 24
hours before susceptibility results can be reported. Hence, a decision to continue, stop, or
change therapy is generally not possible in the first 24 to 48 hours of culture
incubation using conventional microbiology methods. In addition, it is often
recommended to place patients with MRSA infection on contact isolation as soon
as possible; hence the rapid identification of MRSA is also an important
infection control consideration.CHROMagarS. aureus is a selective and differential medium developed to isolate and identify S. aureus. It has been used to identify S. aureus in clinical specimens such as
respiratory samples and wound swabs [6, 7]. CHROMagar MRSA is a selective and
differential medium developed to detect MRSA in nasopharyngeal specimens and
thereby ascertain MRSA carriage [8]. However, there are few data on the use of
these plates as primary media for subcultures of positive blood culture bottles.
In a study by Pape et al., only C-MRSA was utilized and the plates were read at
18 to 24 hours. This study showed that the medium was highly sensitive (97.6%)
and specific (99.9%) for identifying MRSA from blood culture broths [9]. In a
similar study by Colakoglu et al., MRSA ID chromogenic medium (Becton Dickinson, Sparks, Md,
USA) was
used to identify MRSA directly from blood culture bottles, wound swabs, and
abscesses. For blood cultures, the sensitivity was 97.8% and the specificity
was 99.7% at 24 hours incubation [10]. We elected to use both CHROMAgar C-SA and C-MRSA as we feel that it is
important to be able to rapidly identify both MSSA and MRSA bacteremia. We read
C-SA and C-MRSA plates at 12 to 16 hours rather than at 18 to 24 hours of
incubation as we surmised that there are significantly higher organism loads in
blood culture broths compared to nasal swab specimens and most positive blood
cultures are pure cultures compared to the mixed flora seen from nasal swabs.
There was no discrepancy between the 12- to 16-hour and 24-hour readings.While there are
alternative methods to rapidly identifyStaphylococcus spp., such as real-time PCR, they are more expensive, require specialized equipment
and sometimes batch testing of specimens [11]. Direct tube coagulase test is a simple, inexpensive, and rapid method to
test for coagulase-positive and coagulase-negative Staphylococcal spp.
in blood culture broths, but the sensitivity of the test has been reported as
low as 65% [12, 13]. In our study, the
direct tube coagulase test had a high specificity (100%), but a sensitivity of
only 71% (data not shown).In the current
study, there were 2 false-positive C-SA cultures that grew CoNS and these were
identified asS. hominis and S. haemolyticus. Previous reports have
suggested that other coagulase-negative Staphylococcus
spp. including S. cohnii, S.
intermedius, and S. schleiferi, may yield a faint mauve color on C-SA that with experience can be distinguished
from the deep mauve color of S. aureus [14]. We suggest performing a
gram-stain and coagulase test when there is a question as to the final
identification of the organism isolated.While none of the
MSSA isolates produced mauve colonies on C-MRSA (negative predictive value
100%), there were three false negative MRSA isolates which did not give mauve
colonies on C-MRSA (positive predictive value 98%).  This suggests that while a positive result by
C-MRSA can be reported as MRSA, a negative result should only be reported asS. aureus with susceptibility to be
followed by conventional testing.In conclusion,
CHROMagar is a rapid and sensitive method to distinguish MRSA, MSSA, and CoNS
directly from blood culture bottles. It is an easy method that can fit into the
routine workflow of most clinical microbiology laboratories and that does not
require any new or expensive equipment. Use of C-SA and C-MRSA may facilitate a
decreased time for reporting positive results for MSSA and MRSA
bacteremia.



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*Source: 636502-2009-01-11.xml*