Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

Abstract

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.The selective pressure resulting from the extensive use of antibiotics over the last 50 years has led to the emergence of bacterial resistance and to the dissemination of resistance genes among pathogenic microorganisms (2,17,18). The progressive emergence and rapid dissemination of antibiotic resistance in staphylococci and its association with the use and consumption of antibiotics constitute a major health concern and have been considered a global crisis (7,11,16,32). Staphylococci are ubiquitous microorganisms present in the respiratory tract and on the skin of a high percentage of adults. However, several population groups are at serious risk of suffering pathogenic staphylococcal infections. Within the genus Staphylococcus, S. aureus is the causal agent of most staphylococcal infections and is associated with serious communityacquired and nosocomial diseases. Serious complications occur because of multiple-antibiotic-resistant S. aureus. The introduction of new antibiotics in the fight against staphylococcal infections has stimulated a remarkable case of bacterial evolution in the face of changing selective pressures. Thus, the use of a new drug has always been followed by the prompt appearance of new staphylococcal resistance.The first semisynthetic penicillin, namely, methicillin, was introduced in 1959 to overcome the problems that arose from the increasing prevalence of penicillinase-producing S. aureus resistant to penicillin (15). During the 1980s, methicillin-resistant S. aureus (MRSA) started to constitute a widespread human health concern (7). Methicillin resistance in S. aureus is primarily mediated by the overproduction of PBP2a, an additional altered penicillin-binding protein with low affinity for beta-lactam antibiotics. The mecA gene, the structural determinant encoding PBP2a, is therefore considered a useful molecular marker of putative methicillin resistance in S. aureus. MRSA is one of the most important pathogens that caus...

Melipona mondury produces a geopropolis with antioxidant, antibacterial and antiproliferative activities

“…This strain was isolated from raw human milk (LC2). Phenotypic and genotypic tests, identification of the nuc gene (Brakstad et al 1992), detection of the mecA gene (Perez-Hoth et al 2001), and chromosomal cassette typing mec (ssmec) (Boye et al 2007) were conducted to identify the strains. The isolated LC2 was positive for the nuc gene and mecA as well as ssmec, and thus typed as IV, which characterized it as a CA-MRSA strain.…”

Correlation Between the Genotype Detection Determined by Rt- PCR Assays and the Phenotypic Detection Determined by Antibiotic Susceptibility Patterns of Methicillin-Resistant Staphylococcus Aureus Clinicalisolates in Algeria.

“…In recent years, several studies have demonstrated the high capacity of molecular methods such as Pulsed-field gel electrophoresis (PFGE), RT-PCR and PCR-based methods to increase the rapidity and accuracy of resistance testing [1,6,14,15,16,21,22].…”

“…These methods detect only phenotypic expression of methicillin resistance without the presence of the mecA gene [17]. Since phenotypic methods are not discriminating enough and are highly dependent on growth conditions, the use of genetic approach, such as DNA hybridization and PCR, is essential to stop the spread of S. aureus methicillin-resistant and to increase the rapidity and accuracy of their identification and their antibiotics resistance patterns [6].…”

“…The detection of MRSA has become an important tool in clinical diagnosis for the management and treatment of patients [6,17]. Routine methods used in microbiology laboratories for identification of methicillin resistance in S. aureus are based on antibiotic susceptibility disk diffusion agar and dilution [14,17].…”

Methicillin-resistant Staphylococcus aureus (MRSA)

“…The mecA gene which encodes PBP2a is considered a useful molecular marker of putative methicillin resistance in S. aureus. S. aureus strains have a tendency to accumulate additional resistance determinants, resulting in the formation of multiple-antibiotic resistant MRSA strains which are creating therapeutic problems and limiting the choice of therapeutic options [12].…”