Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach.
A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed and evaluated. The method utilised an MRSA-selective enrichment broth for 16 h, followed by PCR quantification of the nuc gene. Samples below a quantitative PCR threshold were reported as MRSA-negative. Broths from PCR-positive samples were subcultured for MRSA isolation. Clinical samples (n = 334) in a constructed high prevalence population were analysed in parallel with a selective plating method. The new broth-PCR assay increased the number of positive samples by 35% (49 vs. 66), and 94% of negative samples were reported within 24 h. To reduce costs and workload, 665 clinical samples were grown separately in enrichment broth and then pooled in the PCR step. The broth-PCR assay increased the number of MRSA positive samples from 11 to 15 compared with selective plating. Most (89%) of the culture-negative samples were also PCR-negative and could be reported within 24 h. The growth of 25 European EMRSA strains was tested in the selective enrichment broth. On average, the MRSA strains showed a 300 000-fold increase in CFU, compared with 30-fold for the eight methicillin-sensitive Staphylococcus aureus strains tested.
Nilsson
P
P
Department of Clinical Microbiology and Infection Control, The County Hospital of Halmstad, Sweden. peter.a.nilsson@lthalland.se
Alexandersson
H
H
Ripa
T
T
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D003470:Culture Media; D004720:Endonucleases / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D016106:Methicillin Resistance; D008836:Micrococcal Nuclease / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*
D001426:Bacterial Proteins; D003470:Culture Media; C074002:nuc protein, staphylococcus; D004720:Endonucleases; D008836:Micrococcal Nuclease