Final Activity Report Summary - MOL-MENATB (Molecular approach to the study of antibiotic susceptibility of Neisseria meningitidis and of its impact on pathogenesis of meningococcal infection)
Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and septicemia worldwide with a high case-fatality rate and the prompt management including antibiotic treatment of patients and chemoprophylaxis in contact persons is crucial. Our first aim was to develop molecular tools that allow detection and reliable characterisation of antibiotic resistance in N. meningitidis. We focused on antibiotics that are currently used in treatment (penicillin and beta lactamines) and chemoprophylaxis (rifampicin and ciprofloxacin). These tools are expected to be valuable as they can be also used as non-culture methods to overcome the increasing number of failure in culturing this bacterium.
Reduction of susceptibility to penicillin is increasing in N. meningitidis worldwide that is correlated with the alterations in penA gene encoding penicillin binding protein, PBP2. We developed an approach by sequencing of penA gene that allows directly to detect isolates with reduced susceptibility to penicillin. Indeed, data from 1551 Nm clinical isolates from 22 countries revealed the presence of 139 different penA alleles; among them 38 highly related alleles were clustered together and corresponded to the penicillin susceptible isolates. The other 101 penA alleles were highly diverse and corresponded to different genotypes/phenotypes. A new web site was created based on the data from this work (http://neisseria.org/nm/typing/penA).
We also showed that only isolates with rifampicin higher than 32 mg/L had mutations in the rpoB gene. Sequencing of rpoB gene also allows direct detection of resistance to rifampicin even in culture negative samples. We also focused on meningococcal resistance to ciprofloxacin, which is an alternative drug in chemoprophylaxis of Nm infections. During the study, the first invasive ciprofloxacin resistant isolates were detected in France. These 3 isolates had a mutation in the gyrA gene resulting in Thr91Ile substitution. Moreover, the characterisation of meningococcal isolates allowed us to suggest the lack of clonal expansion of Nm isolates resistant to rifampicin, ciprofloxacin or with reduced susceptibility to penicillin since there was a diversity of genotypes and/or gene mutations among resistant isolates. In conclusion, the results obtained in this part of the project argue for the use of penA, rpoB and gyrA genes sequencing to identify isolates with reduced susceptibility to penicillin, rifampicin and ciprofloxacin, respectively.
The second objective of the project was a pathophysiological analysis of the effects of reduced meningococcal susceptibility to penicillin. The Neisseria Unit at the Institut Pasteur has previously shown that peptidoglycan (PG) is altered in isolates with reduced susceptibility to penicillin. PG acts in signalling as a pathogen associated molecular pattern. Penicillin binding proteins (PBP) are involved in PG biosynthesis. We constructed isogenic mutants that were inactivated in genes encoding the PBPs that are suggested to act as carboxypeptidases (pbp3, pbp4 and pbp5). We analysed PG structure in these mutants by reverse-phase high pressure liquid chromatography (HPLC), matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS) and post source decay analysis (MALDI-PSD). The inactivation of these PBPs seems to result in a decrease in tri- and tetrapeptides and an increase in peptapetides containing muropeptides particularly in the mutant that lacks the three proteins (PBP3, PBP4 and PBP5). This mutant also showed reduced virulence and inflammatory response in the model of transgenic mice expressing the human transferrin (hTf+). Our data further underline the role of PG in the meningococcal pathogenesis.
Reduction of susceptibility to penicillin is increasing in N. meningitidis worldwide that is correlated with the alterations in penA gene encoding penicillin binding protein, PBP2. We developed an approach by sequencing of penA gene that allows directly to detect isolates with reduced susceptibility to penicillin. Indeed, data from 1551 Nm clinical isolates from 22 countries revealed the presence of 139 different penA alleles; among them 38 highly related alleles were clustered together and corresponded to the penicillin susceptible isolates. The other 101 penA alleles were highly diverse and corresponded to different genotypes/phenotypes. A new web site was created based on the data from this work (http://neisseria.org/nm/typing/penA).
We also showed that only isolates with rifampicin higher than 32 mg/L had mutations in the rpoB gene. Sequencing of rpoB gene also allows direct detection of resistance to rifampicin even in culture negative samples. We also focused on meningococcal resistance to ciprofloxacin, which is an alternative drug in chemoprophylaxis of Nm infections. During the study, the first invasive ciprofloxacin resistant isolates were detected in France. These 3 isolates had a mutation in the gyrA gene resulting in Thr91Ile substitution. Moreover, the characterisation of meningococcal isolates allowed us to suggest the lack of clonal expansion of Nm isolates resistant to rifampicin, ciprofloxacin or with reduced susceptibility to penicillin since there was a diversity of genotypes and/or gene mutations among resistant isolates. In conclusion, the results obtained in this part of the project argue for the use of penA, rpoB and gyrA genes sequencing to identify isolates with reduced susceptibility to penicillin, rifampicin and ciprofloxacin, respectively.
The second objective of the project was a pathophysiological analysis of the effects of reduced meningococcal susceptibility to penicillin. The Neisseria Unit at the Institut Pasteur has previously shown that peptidoglycan (PG) is altered in isolates with reduced susceptibility to penicillin. PG acts in signalling as a pathogen associated molecular pattern. Penicillin binding proteins (PBP) are involved in PG biosynthesis. We constructed isogenic mutants that were inactivated in genes encoding the PBPs that are suggested to act as carboxypeptidases (pbp3, pbp4 and pbp5). We analysed PG structure in these mutants by reverse-phase high pressure liquid chromatography (HPLC), matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS) and post source decay analysis (MALDI-PSD). The inactivation of these PBPs seems to result in a decrease in tri- and tetrapeptides and an increase in peptapetides containing muropeptides particularly in the mutant that lacks the three proteins (PBP3, PBP4 and PBP5). This mutant also showed reduced virulence and inflammatory response in the model of transgenic mice expressing the human transferrin (hTf+). Our data further underline the role of PG in the meningococcal pathogenesis.