Novel technology helps detect whether bacteria

The test has been developed

Image: It can be used to study the resistance of pneumococci even in the absence of isolated strains (pneumococci observed under the microscope)
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Credit: Ivana Campos

study published in the magazine PLUS ONE It could one day help health workers determine whether the bacteria are Streptococcus pneumoniaewhich causes meningitis — inflammation of the membranes that line the brain and spinal cord — are resistant to antibiotics.

This type of analysis is not an easy task when using the traditional method. Bacteria must be isolated from a patient’s 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.

Researchers at the Santo André branch of the Adolfo Lutz Institute (IAL), the central laboratory for epidemiological surveillance in the state of São Paulo, have developed a new, highly feasible method in Brazil. Between 2014 and 2020, they analyzed 873 cerebrospinal fluid samples from patients with suspected streptococcal meningitis at health clinics in six cities in the state – Diadema, Mawa, Santo André, São Bernardo do Campo, São Caetano do Sul and Ribeirão Perez. . CSF is produced by the tissues that line the ventricles of the brain. It flows in and around the brain and spinal cord to protect them from injury and provide nutrients.

As part of the lab’s routine, scientists analyzed samples using real-time PCR, the gold standard for diagnosing infectious diseases, including COVID-19. This technique amplifies a specific genetic or genetic sequence of the target microorganism, if present in the sample, so that it can be more easily identified. In this case, pulmonary pulmonary (pneumococcus) in 149 samples.

They then reanalyzed samples that tested positive for pneumococcus for 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 that is captured by the equipment.

To find out what types of antibiotics bacteria were resistant to – penicillins, lincosamides or macrolides – they used the dissociation curve technique. “This technique entails raising the temperature of the samples one step at a time, making the dye separate from the DNA as the double strands in the double helix form the genetic material amplified in the PCR machine gradually. We measured the melting temperature [Tm], which is when half of the hull is still intertwined and the rest breaks off. This temperature varies depending on the amplified gene, so it can be used to determine which gene was amplified and then which antibiotic the bacteria are resistant to.” Ivana Camposthe principal investigator of the study.

After performing all these procedures, the researchers compared the results with the results obtained with the traditional method used to analyze antibiotic resistance, in which live bacteria are observed during 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 containing viable pneumococci for the procedure. The results were similar, confirming the potential of the new technique.

“We found that 51% of the samples analyzed, which the IAL received between 2014 and 2020, were sensitive to antibiotics. This is positive, which means that these patients should have had a good prognosis. On the other hand, 17% were resistant to various drugs, which is very dangerous because in these cases the disease is more difficult to treat and other classes of antibiotics must be tried.

Furthermore it, pulmonary pulmonary Able to alter its genetic makeup as it reproduces, so that new copies contain genes associated with drug resistance. “Therefore, we concluded that the test we developed can be used to study the resistance properties of pneumococci even in the absence of isolated strains, as demonstrated by our region,” she said.

The study was supported by FAPESP through two projects (17 / 03022-6 And the 18/22718-4. The results obtained will be useful both for epidemiological surveillance 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 whose mission is to support scientific research in all fields of knowledge by offering scholarships, fellowships and grants to researchers associated with institutions of higher education and research in the state of São Paulo, Brazil. FAPESP understands that only the best research can be done by working with the world’s best researchers. Therefore, it has established partnerships with funding agencies, higher education, private companies, and research organizations in other countries known for the quality of its research and encourages scholars funded by its grants to develop their international collaborations. You can learn more about FAPESP at www.fapesp.br/en And visit FAPESP news agency at www.agencia.fapesp.br/en To keep abreast of the latest scientific achievements that FAPESP helps to achieve through many programs, awards and research centers. You can also subscribe to FAPESP news agency at http://agencia.fapesp.br/subscribe


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