Combined automated screening for detection of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) in broth enriched clinical samples

Introduction

Rapid, reliable, and sensitive methods for screening of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) are warranted in a low-endemic area. The aim of this study was to develop an automated screening solution for both heterogenic MRSA as well as VRE in selective broth enriched clinical samples.

Material & Methods

Primary tubes of clinical samples enriched in selective broth for MRSA(1) (cefoxitin 3 mg/L) and VRE (vancomycin 4 mg/L, aztreonam 60 mg/L), were cultured over night in 36°C for at least 15 hour. The samples were automated pooled with up to three samples (only MRSA) from one patient and automated DNA prepared by a NorDiag Bullet with the Bullet BUGS’n BEADS™ kit (NorDiag ASA).
A real-time multiplex PCR for detection of MRSA using S. aureus specific orfX- and SCCmec-regions as well as a duplex PCR for VRE with the vanA and vanB genes were automated prepared in a Qiagility (Qiagen) and run on a RotorgeneQ6000 (Qiagen), see Figure 1&2 and the Table. All positive results in the screening were confirmed by subcultivation from the primary tubes and then tested in specific PCRs for MRSA (nuc and mecA) as well as VRE (ddlE.faecalis, ddlE.faecium, vanA and vanB), see Table.
The screening PCRs for MRSA and VRE were compared with routine methods at the laboratory for nuc screening1 as well as culturing using the broth enriched clinical  samples. The method was tested on 54 MRSA, 25 VRE and 4 other enterococci isolates that were inoculated in the respectively selective broth. Also 264 pools of clinical broth of MRSA screening samples (27 MRSA and 237 non-MRSA) and 71 clinical broth of VRE screening samples (7 VRE and 64 non VRE) were tested.

Results

All isolates were correctly identified. Of the MRSA and VRE clinical broth samples only 5% were false positive in the screening but negative in further analysis.
The screening was fully automated from primary tubes and a PCR result could be seen within 3 hours. Consequently a negative result could be reported the day after admission in 95% of the cases and a positive result after 2-3 days.

Figure 1. Schematic illustration of the staphylococcal cassette chromosome mec (SCCmec) and location of primers used in the present study.4

 
Figure 2. Representative amplification curves from the real-time MRSA multiplex PCR and the duplex VRE PCR using Taqman probe detection.

MRSA

vanA

vanB

a PCRs were run with Rotogene™ SYBR® Green or Rotogene™ Probe® kit (Qiagen) in the same PCR program of 95°C 3min; 40 cycles of 95°C 3sec and 60°C 10sec; followed by melting analysis 90sec, 70°C - 99°C (1°C, 5sec/step).
bSix primers were used, see Figure 1.
c Kindly provided by Ingegerd Sjögren at the Department of Clinical Microbiology and Infection Control, The County Hospital, Halmstad, Sweden.

Conclusions

Screening is warranted in a low endemic area such as Sweden and the automation facilitates handling of large amount of samples, e.g. outbreaks. A rapid negative result is also very important to facilitate the infection control management of patients and hospital staff.

References
1. Nilson P et al. Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach. Clin Microbiol Infect. 2005;11:1027-34
2. Huletsky A et al. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol 2004;42:1875-84.
3. Ornskov D et al. Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method. Clin Microbiol Infect. 2008;14:22-8.
4. Bo Söderquist et al. Real-time multiplex PCR for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in broth enriched clinical samples. 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsingfors, Finnland, 16-19 maj 2009. Abstract/P1563.
5. Berglund C et al. Predominance of staphylococcal cassette chromosome mec (SCCmec) type IV among methicillin-resistant Staphylococcus aureus (MRSA) in a Swedish county and presence of unknown SCCmec types with Panton-Valentine leukocidin genes. Clin Microbiol Infect 2005;11:447–56.