Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: Methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR

Abstract

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Analysis of Antibiotic Resistance Genes and its Associated SCC mec Types among Nasal Carriage of Methicillin Resistant Coagulase Negative Staphylococci from Community Settings, Chennai, Southern India

“…S. epidermidis and S. haemolyticus were further confirmed by species specific PCR by using S. epidermidis specific PCR fragment and mvaA genes respectively [12].…”

The general status of patients and limited physical activity as risk factors of Methicillin-resistant Staphylococcus aureus occurrence in long-term care facilities residents in Krakow, Poland

“…In PPS and CS, in case of an infection, various diagnostic specimens including tracheal/bronchial secretions and others were collected for culture and assessment of the microbial aetiology of infections (nasal swabs were not done continuously). Isolates were identified as SA by polymerase chain reaction (PCR) [10]. …”

Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers

“…According to Pereira et al [13], the blood cultures were incubated until positive signal and processed for PCR within 12 h from positive signal. Bacterial DNA suspension (10 ml) extracted from spiked blood was used as template for PCR in 50 ml reactions.…”

“…Bacterial 16S rRNA and cns primer-probe oligonucleotides were designed for this study. Against the convention of single genome oligo-design common to PCR-based diagnostic assays [9,13], oligonucleotide primers and Taqman probes were defined following clustalW multiple alignments, considered a suitable approach for oligodesign [17,18]. Briefly, nucleotide sequences for each target DNA were obtained from the National Centre for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov/) and used for multiple alignments in the clustalW suite via the European Bioinformatics Institute (www.ebi.ac.uk/tools/clustalW).…”