Avaliação de metodologia multiplex para pcr de identificação de genes de resistência a antibióticos em streptococcus agalactiae

Abstract

Streptococcus agalactiae is an opportunistic pathogen capable of causing invasive diseases in newborns, pregnant women and individuals with low immunity. Early-onset disease and late-onset disease that affect newborns are caused by the bacteria and account for over 300,000 cases annually globally and result in approximately 90,000 deaths. Intrapartum antibiotic prophylaxis is recommended for preventing these diseases, however, each year, resistance to antibiotics indicated in prophylaxis is growing in S. agalactiae. The polymerase chain reaction (PCR) is a technique that can be used to detect resistance genes in microorganisms. Its variation, multiplex PCR, allows the simultaneous amplification of multiple target genes in a single reaction. By using specific oligonucleotide primers for each gene of interest, multiplex PCR makes it possible to reduce detection time, as well as sample and reagent volumes. It also minimizes the risks of cross-contamination and consequently increases diagnosis reliability. This work aimed to evaluate the applicability of the multiplexed PCR methodology for identifying antibiotic resistance genes in S. agalactiae. The research selected sets of oligonucleotide primers that meet the requirements for a multiplex reaction and present specificity to the resistance genes erm(A/TR), erm(B), mef(A/E), tet(M) or tet(O). The applicability of the multiplexed methodology was then evaluated for each set of primers by making adjustments to their concentrations and annealing temperatures. The resulting amplification products were analyzed by agarose gel electrophoresis and visualized using a UV transilluminator. With the collected data, the sensitivity and specificity of the duplex reaction was compared with the monoplex reaction. For the first set composed of erm(B), mef(A/E) and tet(M) clear visualization of bands was obtained, with sensitivity estimated at 92.3% and specificity at 94.1% and excellent correlation of results between methodologies (kappa = 0.864 (P<0.001)). For the second set composed of erm(A/TR) and tet(O), clear visualization of bands was obtained with sensitivity and specificity estimated at 100% and ideal correlation of results between methodologies (kappa = 1.000 (P<0.001)). Thus, it was demonstrated that the multiplexed methodology can be used with confidence in places that carry out research on these genes