Quantitation of mecA Transcription in Oxacillin-Resistant Staphylococcus aureus Clinical Isolates

Abstract

The transcription of mecA, the gene required for oxacillin resistance in staphylococci, was quantified in a collection of 65 geographically and genetically diverse clinical and 8 defined laboratory Staphylococcus aureus isolates. mecA transcription was measured by real-time reverse transcription-PCR, confirmed by Northern blot analysis, and correlated with the presence and DNA sequence of the two mecA repressors, mecI and blaI. Isolates were first examined that contained mecI and/or blaI with wild-type sequence. BlaI provided significantly more repression of mecA transcription than did MecI, unrelated to blaI genetic location. Both together repressed mecA better than either one alone. In clinical isolates containing only wild-type mecI, mecA transcription repression was 10-to 25-fold less effective than that seen in previously studied constructs derived from strain N315. There was a difference in the mecI ribosomal binding site (RBS) between the clinical isolates (GGAA) and N315 (GGAG). The GGAA RBS was associated with 5.5-to 7.3-fold less mecA repression than GGAG in isogenic constructs. The values generated for wild-type repressors were compared to those in 26 isolates containing mecI mutations. mecA transcription appeared to be repressed only by BlaI in isolates with mecI nonsense and frameshift mutations. In contrast, mecI repression seemed to be partially or fully retained in many of the isolates with mecI and one isolate with blaI missense mutations, providing structure-function correlates with the site and type of mutation. We conclude that mecA repressor activity is highly variable in clinical S. aureus isolates due to mecI mutations, RBS polymorphisms, and unidentified genomic adaptations.Two repressors, BlaI and/or MecI, regulate transcription of mecA, the gene required for oxacillin resistance in staphylococci (8,15). mecA encodes a penicillin binding protein (PBP2a) that is poorly bound by ␤-lactam antibiotics and can perform essential functions of cell wall construction when ␤-lactams inactivate the cell's normal penicillin binding protein complement (3,14). Two signal transducers, BlaR1 and MecR1, regulate repressor activity (20). MecR1 and BlaR1 are transmembrane ␤-lactam sensory transducers, having a surface-exposed ␤-lactam binding domain and a cytoplasmic zinc peptidase motif. Signal transduction is thought to occur following interaction of a ␤-lactam antibiotic with the binding domain, transmitting a signal through four membrane-spanning segments. This induces conformational change in the cytoplasmic metalloprotease domain causing autoproteolysis. Proteolytic cleavage of the inducer is followed by cleavage of the repressor, leading to derepression of target genes. The genes for signal transducers and repressors are contained in two-gene operons (blaR1-blaI and mecR1-mecI) that are divergently transcribed from their regulated genes (blaZ and mecA, respectively) (8, 13, 15). The repressors bind to specific palindromic sequences that overlap divergent promoters for mecA and mecR1 (8,18). A helix-...

Comparison of Tests To Detect Oxacillin Resistance in Staphylococcus intermedius , Staphylococcus schleiferi , and Staphylococcus aureus Isolates from Canine Hosts

“…Real-time mecA PCR was performed with previously described oligonucleotide primers and a mecA dye-quencher probe (24). Standard PCR mixtures and thermocycler profiles were employed (12,26).…”

Microarray Transcription Analysis of Clinical Staphylococcus aureus Isolates Resistant to Vancomycin

“…gyrA and 16S rRNA were amplified as control and standard, respectively. Amplification and detection of specific products was performed with an ABI Prism 7700 sequence detection system (PE Applied Biosystems) as previously described (11). The primer sequences used are available at http: //www.tigr.org/microarray/Vanco_Paper/.…”

Bodas de plata de la reacción en cadena de la polimerasa (PCR)

“…La PCRtr, se ha usado para la identificación de mutaciones puntuales asociadas con resistencias a fármacos antimicrobianos (62). En sólo una hora se puede determinar la presencia en heces de Enterococos resistentes a vancomicina (18), facilitando el control de la transmisión de este patógeno.…”

“…La PCRtr dúplex o multiplex ha sido una gran herramienta para el diagnostico acertado en estas enfermedades, diferenciando en un mismo tubo agentes etiológicos diferentes como en el caso de Chlamydophila pneumoniae, Mycoplasma pneumoniae y Legionella pneumophila (69,70) Otra aplicación en investigación básica con células bacterianas, ha sido la cuantificación de ARN y la valuación de su expresión. Este es el caso del estudio de transcriptos del gen mecA en Staphylococcus aureus meticilin resistente, en donde se correlaciona la presencia del gen, con su expresión y el efecto de sus genes represores e inductores sobre esta (62). A manera de cierre, la PCRtr continuará evolucionando, al ser una herramienta versátil, que permite su uso en gran cantidad de áreas de investigación básica y aplicada.…”