|Director : Jean-Michel ALONSO (firstname.lastname@example.org)|
Main research topics of the Neisseria unit, National Reference Center for the Meningococci, are in the field of the molecular epidemiology and pathogenesis of infections due to Neisseria meningitidis, including the molecular typing of clinical isolates from invasive infections (bacteremias and meningitis) and particularly the clones involved in epidemics in France and in Africa. Experimental research focuses on the characterization of virulence factors and regulatory genes involved in adhesion and invasion at structural and functional levels, in vivo, and the study of genetic alterations involved in resistance to beta-lactams.
1. Surveillance of meningococcal infections by the National Reference Center (J-M Alonso, M-K Taha et al.)
N. meningitidis is a major causative agent of meningitis (30% of bacterial meningitis) and of meningococcemias (septicemias) worldwide. In addition to neurologic sequels, purpura fulminans and fatal septic shock can complicate meningococcal infections. Meningococcal infections are potentially epidemic. Their world incidence varies, according to countries, from 1 to 10 per 100,000 inhabitants, with mortality rates reaching up to 10%. In France the incidence was stable at <1/100,000 for the past eleven years, but increased by >20% since 2000. Nevertheless, the cases remain sporadic and no epidemic has been recorded. The proportion of the various serogroups among the strains recovered from invasive infections is 55,7% for the prominent serogroup B (versus 66% in 2000), followed by an increasing proportion of the serogroup C (36,2 Vs 19,2% in 2000), the serogroup W135 (8%) and serogroup Y (3,4%). Forty-eight fatal cases of meningococcal infection have been recorded in France in 2001, mostly among children under 5 years and adults over 70.
2. Experimental research.
Our research activities are based on the constant surveillance of the actual status of meningococcal diseases, considering the evolution of the bacterial molecular determinants as well as the criteria of host susceptibility. They are conducted in collaboration with the correspondents of the Reference Center at the national and international levels. Our main goal is to improve the diagnostic and typing methods and to provide better knowledge on the pathogenesis of these diseases.
2.1. Molecular epidemiology of N.meningitidis.
2.1.1. Molecular diagnosis and typing.
Direct characterization of N.meningitidis from clinical samples (blood, cerebrospinal fluid, etc.) permits its determination by amplification of the regulatory gene crgA, followed by that of the genes siaD, encoding the capsule for serogroups B, C, Y/W135 or the gene mynB encoding the synthesis of capsular polysaccharides of serogroup A.
Molecular typing of N.meningitidis is essential for assessing an epidemiological link between cases.
Polymorphic chromosomal loci pilA, pilD, regF, igaA and crgA are amplified by PCR and further analyzed by multilocus DNA fingerprinting. Various alleles are then defined that are specific for each clonal group. This technique, fully correlates with the multilocus enzyme electrophoresis, and is completed by the pulsed-field electrophoresis and the multilocus sequence typing to characterize several geographic foci in France those confirm an endemic condition due to heterogeneous genotypes.
2.1.2. Detecting the involvement of N.meningitidis W135 during the 2001 epidemic in sub-Saharan Africa.
Burkina Faso and Niger were among the countries most severely affected by the meningitis epidemic in 2001, with 13039 cases, 1813 deaths, and 7906 cases, 595 deaths, respectively.
During a joint mission of the Association pour le Médecine Préventive and the Institut Pasteur, we collected a convenience sample of 57 cerebrospinal fluids (CSF) and one serum from 58 patients in Burkina Faso and 37 CSF and three sera from 40 patients in Niger. CSF and serum samples were tested by PCR for the presence of Streptococcus pneumoniae (Sp) DNA,for the presence of Nm DNAas well as for the presence of Haemophilus influenzae type b (Hib) DNA. Samples positive for Nm were further tested by PCR for capsule genes to predict the serogroups A, B, C, Y and W135. Of the 58 samples from Burkina Faso, 32 (55%) were positive for Nm, 4 for Sp and 22samples failed to give a detectable gene amplification for the three tested bacterial species. Of the 40 samples from Niger, 31 (78%) were positive for Nm, 3 for Sp, 2 for Hib and 4 samples failed to give a detectable gene amplification for the three tested bacterial species. Among the 32 Nm PCR positive samples from Burkina Faso, 8 corresponded to serogroup A, 12 to serogroup W135 and 2 to serogroup C.PCR failed to predict serogroup in 10 samples.PCR amplification of capsule genes of Nm led to the identification of 16serogroup A, 12 serogroup W135 and 1 serogroup C among the 31 Nm PCR positive samples from Niger, while 2 failed to determine the serogroup. Thus, among the Nm positive samples for which serogroup could be predicted, 38 % corresponded to serogroup W135, 38% corresponded to serogroup A, and 5% corresponded to serogroup C. In addition, 12 strains of N. meningitidis (Nm) had been isolated from other CSF samples (4 from Burkina Faso and 8 from Niger). No vaccination history was available for these cases. Among the four strains isolated in Burkina Faso, only one was A: 4: P1-9 while the 3 others were W135:2a: P1-2,5. Among the eightNm strains from Niger, seven had the antigenic formula A: 4: P1-9 and one, was W135:2a:P1-2,5. This field survey thus revealed the involvement of N. meningitidis serogroup W135 in equivalent proportion to that of serogroup A during an epidemic condition when the meningococcal A&C vaccination campaign had been started for several weeks. As in the case of the Hajj 2000 epidemic also due to serogroup W135, but with no obvious relationship, one can suspect an antigenic shift or the selection of an escape variant to the A&C vaccine.
2.2. The polymorphism of the gene penA and its relationship to diminished susceptibility to penicillin.
N.meningitidis strains with diminished susceptibility to penicillin (MIC*0.125mg/L) were detected since 1994 in France, reaching 30%. We have studied the polymorphism of the gene penA among 25 clinical strains, including 13 penicillin-susceptible strains (MIC<0.125mg/L) and 12 strains with diminished susceptibility (1 mg/L>MIC*0.125mg/L), of different geographical origin, antigenic formulas, and from different pathologic conditions. We observed that for different genotypes, according to restriction patterns of the genes pilA, pilD, crgA, regF and igaA, strains with MIC of penicillin <0.125mg/L (penS) harbored identical penA allele, whereas strains with MIC >0.125mg/L (penI) harbored altered sequences of penA. Transformation of penS strains into penI was obtained in vitro by using total DNA or PCR-amplified products of altered penA gene. Also mixed culture of both genotypes led to equivalent transformation, suggesting a direct correlation between alterations of penA and the expression of diminished susceptibility to penicillin. Detecting such gene alterations for penicillin susceptibility testing, can now directly be done without previous culture, whenever PCR diagnosis will be used.
2.3. Molecular genetic of virulence of N.meningitidis.
The meningococcal infectious process first involves bacterial adhesion to epithelial target cells. This is mostly due to the pili. The PilC1 protein plays a major role in the pili assembly and the adhesion to the cell. The gene pilC1 harbors a promoter region necessary for the induction of its expression when the bacteria adhere to the cell. A new transcriptional regulator has been identified, the gene crgA, of the lysR family, whose expression is induced by contact with the target cell along with that of pilC1. The gene crgA seems to regulate several other genes involved in adhesion, mostly in switching to intimate adhesion of N.meningitidis to the cell. These adhesion determinants also play an important role in the invasive process of the blood vessels by activating the TNFa in the endothelial cells in the presence of monocytes.
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
Jean-Michel ALONSO, IP
Muhamed-Kheir TAHA, IP
Mireille LARRIBE, Université Paris 7
Aude ANTIGNAC, PhD student, Paris 7.
Al Eddine DEGHMANE, PhD student, Paris 11.
Maria Leticia ZARANTONELLI, post-doctoral fellow, Argentina
Magaly DUCOS,Technicienne supérieure,IP.
René PIRES,Technicien supérieur,IP.
Annie GUIYOULE, Technicienne supérieure, IP.
Dario GIORGINI,Technicien supérieur,IP