A study published in the journal PLUS ONE could one day help health professionals determine whether bacteria of the species Streptococcus pneumoniae which causes meningitis – an inflammation of the membranes that surround the brain and spinal cord – are resistant to antibiotics.
This type of analysis is not an easy task when using the conventional method. The bacteria must be isolated from a patient sample and analyzed while they are still alive, which is difficult because the microorganisms are sensitive and usually do not survive the journey to the laboratory.
A highly feasible new method has been developed in Brazil by researchers at the Santo André branch of the Adolfo Lutz Institute (IAL), the central epidemiological surveillance laboratory of São Paulo state. Between 2014 and 2020, they analyzed 873 samples of cerebrospinal fluid from patients with suspected streptococcal meningitis at health clinics in six cities in the state – Diadema, Mauá, Santo André, São Bernardo do Campo, São Caetano do Sul and Ribeirão Pires. Cerebrospinal fluid is produced by tissue that lines the ventricles of the brain. It flows into and around the brain and spinal cord to cushion them from damage and provide nutrients.
As part of the lab’s routine, the researchers analyzed the samples using real-time PCR, the gold standard for diagnosing infectious diseases, including COVID-19. The technique amplifies a specific gene or gene sequence from the target microorganism, if present in the sample, so that it can be more easily identified. In this case, S. pneumoniae (pneumococci) were detected in 149 samples.
They then reanalyzed the samples that tested positive for pneumococcus to detect the three genes associated with antibiotic resistance, again using real-time PCR, but this time with SYBR Green, a dye that binds to DNA and emits a fluorescent signal which is picked up by the equipment.
To find out which classes of antibiotics the bacteria resisted – penicillin, lincosamides or macrolides – they used the dissociation curve technique. “This technique involves raising the temperature of the samples degree by degree, which separates the dye from the DNA as the twin strands of the double helix that make up the genetic material amplified in the PCR machine gradually unwind. We measured the melting temperature [ Tm ], which is when half of the structure is still intertwined and the rest is separated. This temperature varies depending on the amplified gene, so it can be used to identify the gene that has been amplified and thus the antibiotic to which the bacteria are resistant,” said Ivan Campos principal investigator for the study.
After performing all these procedures, the researchers compared the results with the results obtained by the conventional method used to analyze resistance to antibiotics, where live bacteria are observed while in contact with each drug to see if they are able to reproduce. This conventional test was performed on 25 samples, which were the only ones that contained viable pneumococci for the procedure. The results were similar, confirming the potential of the new technique.
“We found that 51% of the analyzed samples received by the IAL between 2014 and 2020 were sensitive to antibiotics. This is positive, which means that these patients must have had a good prognosis. On the other hand, 17% resistant to different drugs, which is very dangerous because in these cases it is more difficult to treat the disease and other classes of antibiotics must be tried,” said Campos.
Moreover, S. pneumoniae is able to change its genetic makeup as it multiplies, so that new copies have the genes associated with drug resistance. “We therefore concluded that the test we developed can be used to study the resistance profile of pneumococcus, even in the absence of the isolated strains, as proven for our region,” she said.
The study was supported by FAPESP via two projects (17/03022-6 and 18/22718-4. The results obtained will be useful both for epidemiological monitoring and for improving the treatment of patients in the future.
About the São Paulo Research Foundation (FAPESP)
The São Paulo Research Foundation (FAPESP) is a public institution with the mission of supporting scientific research in all fields of knowledge by awarding grants, scholarships and grants to investigators associated with higher education and research institutions in the State of São Paulo, Brazil. FAPESP is aware that the very best research can only be carried out by working with the best researchers internationally. Therefore, it has established partnerships with funding agencies, higher education, private companies and research organizations in other countries known for the quality of their research and has encouraged researchers funded by its grants to further develop their international collaboration. You can learn more about FAPESP at www.FAPESP.br/da and visit the FAPESP news agency at www.agencia.FAPESP.br/en to keep up to date with the latest scientific breakthroughs that FAPESP helps achieve through its many programs, awards and research centers. You can also subscribe to the news agency FAPESP at http://agencia.FAPESP.br/subscribe
Article title
Multiplex real-time PCR using SYBR Green: Nonspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid
Article Publication date
June 14-2022
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