New technique helps find out whether bacteria that causes meningitis are resistant to antibiotics

New technique helps find out whether bacteria that causes meningitis are resistant to antibiotics

Image: Ivana Campos/Disclosure

Text: Thais Szego, from Agência FAPESP

A study published in the journal PLOS ONE may, in the future, help health professionals to determine whether bacteria of the species Streptococcus pneumoniae that cause meningitis – inflammation in the membranes that surround the brain and spinal cord – are resistant to some type of antibiotic.

Until then, this was not an easy task. It required isolating the microorganism in the patient’s sample and analyzing it while it is still alive – something difficult, since the bacteria are sensitive and, in most cases, cannot withstand the trip to the laboratory.

To get around the problem, researchers from the Adolfo Lutz Institute – Regional Laboratory of Santo André (SP) – developed a new way of carrying out this analysis. Between 2014 and 2020, the group received 873 samples of cerebrospinal fluid (cerebrospinal fluid) from people suspected of having the disease at health centers in six cities in São Paulo (Diadema, Mauá, Santo André, São Bernardo do Campo, São Caetano do Sul and Ribeirão Pires). ) for the diagnosis of bacterial meningitis. This substance is a sterile fluid present in the cavities of the brain and spinal cord that acts as a shock absorber, protecting these structures from trauma.

As part of the laboratory routine, the scientists subjected the samples to a real-time PCR assay (commonly used in the diagnosis of infectious diseases, including COVID-19), which amplifies a specific gene of the target microorganism in vitro, if it is present in the sample, allowing it to be identified more easily. It was possible to identify the presence of Streptococcus pneumoniae, or pneumococcus, in 149 samples.

With these positive samples, another real-time PCR assay was performed, this time using a dye called SYBR Green. The substance binds to DNA and emits a fluorescence signal that is captured by the equipment to detect one of the three genes studied, simultaneously, which are related to antibiotic resistance.

In order to determine which classes of antibiotics the bacteria were resistant to – penicillin, lincosamides or macrolides – the group used a technique known as the dissociation curve.

“In this method, we increase the temperature of the samples degree by degree, which causes the dye to detach from the DNA, as the double helix that forms the genetic material amplified in PCR is gradually separating. Then we analyze the so-called melting temperature [Tm], which is when half of the structure is joined and half is separated, which varies according to the amplified gene. Depending on how many degrees this happened, we were able to differentiate which gene was amplified, therefore, which antibiotic this bacterium is resistant to”, explains biologist Ivana Campos, responsible for the study.

Validation

After carrying out all these processes, the researchers compared their results with those obtained by the method traditionally used to assess antibiotic resistance – which consists of placing live microorganisms in contact with each of the drugs and observing whether they are able to proliferate in the presence of the antibiotic. substance. This conventional test was only performed with 25 samples, since only they showed viable pneumococci for the process. The results were similar, which confirmed the potential of the new technique developed.

“We were able to verify that 51% of the samples evaluated, received between 2014 and 2020, were sensitive to antibiotics, which is a good sign, since these patients must have had a good prognosis”, says Campos. “On the other hand, 17% were resistant to several drugs, which is very dangerous, because in these cases it is more difficult to carry out the treatment and we need to adopt antibiotics of other classes”, he explains.

The researcher also points out that S. pneumoniae has a great ability to exchange genetic material, that is, it is easy to pass these genes that confer antimicrobial resistance to other bacteria, making them resistant as well.

“Therefore, we concluded that with the assay we developed, it is possible to study the pneumococcal resistance profile even in the absence of isolated strains, as evidenced for our region.”

The work was supported by FAPESP through two projects (17/03022-6 and 18/22718-4) and the results obtained by the specialists can be useful both for epidemiological surveillance actions and to improve the treatment of patients in the future.

The article Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid can be read at: https://journals.plos.org/plosone/article?id=10.1371/journal. pone.0269895.

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