Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid.

Abstract

Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24-79.86; ermB: 80.88-82.56; mef: 74.85-76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S. pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.