a Unité des Neisseria and Centre National de Référence des Méningocoques, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris cedex 15, France; b National Reference Laboratory for Meningococcal Infections, NIPH, Prague, Czech Republic; c National Meningococcal Reference Laboratory, 196 Alexandras Avenue, 11521 Athens, Greece
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Abstract |
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Introduction |
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PBP2, a 60 kDa protein, is one enzymic target of ß-lactam antibiotics. By analogy with Escherichia coli, PBP2 of N. meningitidis is probably involved in the biosynthesis of peptidoglycan, particularly in the transpeptidation reaction required for cross-linking peptidoglycan.11 Penicillin binds covalently to PBPs, thereby inhibiting the transpeptidase activity of these proteins.11 Modifications in PBP2 that may reduce its affinity for penicillin result from changes in penA caused by horizontal DNA transfer to pathogenic Neisseria spp. from commensal ones.12 This DNA exchange probably occurs by transformation.13 penA has been reported to be polymorphic in meningococcal penI strains,14 but to demonstrate a direct role for penA polymorphism in reduced susceptibility to penicillin, the genetic relationships between meningococcal strains should be analysed. As N. meningitidis is highly diverse, it should be demonstrated that altered penA is related to the penI phenotype but not to a random distribution of different penA alleles in different genetic lineages.
Classical serological typing (serogroup:serotype:serosubtype) of meningococcal strains is no longer adequate for this purpose. Such typing is based on the immunological specificity of surface structures such as the capsule and outer membrane porins (PorA and PorB).15,16 These antigenic structures are subject to strong selection by the host immune response. They are therefore highly variable and are not good markers of genetic relatedness among meningococcal strains. Genetic typing methods have been developed and shown to be more reliable for the characterization of meningococcal strains. Multilocus enzyme electrophoresis (MLEE),17 multilocus sequence typing (MLST)18 and multilocus DNA fingerprinting (MLDF)19 are now generally used to study population genetics and for epidemiological characterization of meningococcal strains.
The aim of this study was to analyse penA polymorphisms in a genetically well-defined collection of meningococcal strains obtained from three national reference centres and to assess the relationship between these polymorphisms and the reduced susceptibility of these strains to penicillin.
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Materials and methods |
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The meningococcal strains used in this study and their geographical origins are listed in Table I. The CAP1 strain was derived from strain LNP 8013, as described previously.20 It harbours pilA::aph3' and is resistant to kanamycin. Bacteria were grown at 37°C under 5% CO2 on G medium with G supplement (Sanofi Diagnostic Pasteur, Marnes La Coquette, France) and on GCB medium (Difco, Detroit, MI, USA) with Kellogg supplements.21 Penicillin G susceptibility was tested by the agar dilution method with G medium. The strains were tested using inocula of 108 cfu/mL on plates containing penicillin G at 0.06, 0.125, 0.250, 0.500 and 0.750 mg/L. The MIC was defined as the lowest concentration of penicillin G that inhibited visible growth after 18 h of incubation in 5% CO2 at 37°C. Penicillin G susceptibility was also tested by the diffusion method (Etest) on G medium. When needed, kanamycin was added to the medium at 100 mg/L. ß-Lactamase activity was detected using Cefinase discs (bioMérieux, Marcy l'Etoile, France).
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Serological typing was performed as previously described.15,16
DNA fingerprinting
For genomic DNA extraction, bacteria were suspended in 500 µL of distilled water, subjected to one freezethaw cycle, heated at 100°C for 3 min and then centrifuged for 5 min at 10 000g. The pilA, pilD and penA genes were amplified by PCR from the supernatant using the oligonucleotide primers listed in Table II. PCR was performed as previously described.19,22 The amplicons obtained by PCR corresponding to the pilA (1.8 kb) and pilD (0.9 kb) genes were then digested with AluI, HpaII or TaqI. For penA amplicons (1.7 kb), the restriction enzymes HaeIII, HpaII and TaqI were used separately. An arbitrary number was assigned to each restriction endonuclease pattern and an allele was defined by three numbers corresponding to the restriction endonuclease patterns obtained for the three enzymes. Restriction digests were analysed by electrophoresis in 5% non-denaturing polyacrylamide gels prepared with 89 mM Trisborate and 2 mM EDTA (pH 8.0). Restriction profiles were analysed as described previously19 using the Taxotron package (PAD Grimont, Institut Pasteur, Paris, France).
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The penA gene was amplified by PCR using oligonucleotides 99-1 and 99-2 (Table II). The amplicons were purified on a Sepharose CL-6B column (Pharmacia, Uppsala, Sweden) and sequenced using oligonucleotides 99-16, 99-22, 99-24, 99-27, AA-1, AA-5 and AA-8 (Table II
) and a Sequenase PCR product sequencing kit (USBAmersham, Cleveland, OH, USA). Sequences were aligned using the Multiple Alignment Program (MAP).23
Transformation of N. meningitidis
Genomic DNA was prepared from three penI strains of N. meningitidis (W-39, TH-41 and LNP 16454) and used to transform a penicillin-susceptible (penS) strain, LNP 8013, as described previously.20 For penA amplicons to be used for transformation, PCR was performed with oligonucleotides 99-14 and 99-23. Oligonucleotide 99-23 contains the uptake sequence necessary for DNA transformation in Neisseria spp.24 Transformants were selected on GCB medium containing penicillin G at concentrations of 0.125, 0.25, 0.50 and 0.75 mg/L. For genetic transfer during bacterial growth (co-culture experiments), meningococcal strains CAP1 and LNP 16454 were suspended in GCB liquid medium supplemented with 5 mM MgCl2. The optical density at 600 nm was adjusted to 0.6. Suspensions of each strain were mixed together (1:1) and cultured at 37°C under 5% CO2 for 3 h. The mixture was then plated on selective GCB medium containing kanamycin 100 mg/L and penicillin G at 0.125, 0.25, 0.50 or 0.75 mg/L.
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Results |
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To analyse the structure of penA in N. meningitidis, we first set up a collection of 25 meningococcal strains corresponding to various MICs of penicillin G and to various geographical and anatomical sites of isolation (Table I). On the basis of MIC, these strains fell into two categories: penS strains (n = 13, MIC < 0.125 mg/L) and penI strains (n = 12, 1 mg/L
MIC
0.125 mg/L). Strains were isolated over a period of 10 years in several countries (Table I
). No ß-lactamase activity was detected in these strains using Cefinase discs. Serological typing showed that these strains also differed in antigenic formula (serogroup:serotype:serosubtype). All three major serogroups (A, B and C) were represented in our collection (Table I
).
Molecular characterization of meningococcal strains
Strains were then typed by MLDF, which involves amplification by PCR of several chromosomal loci and analysis of restriction fragment length polymorphisms (RFLPs). MLDF of the pilA and pilD genes has been shown to be reliable for characterizing strains and its resolution is as good as that of MLEE.19,22 Distance matrices for pilA and pilD were used to construct a dendrogram using the Taxotron package. Fifteen different groups were identified among the strains tested in this study (Figure 1). The distribution of penS and penI strains on this dendrogram did not depend on MIC. These results indicated that our collection was composed of different genetic lineages and that both types of strain (penS and penI) were present in several genetic groups of N. meningitidis. Several strains corresponded to major epidemic complexes as shown by MLEE analysis. For instance, strain LNP 10824 belongs to clone IV-1 which has been involved in large epidemics in Africa. Strains LNP 13302 and LNP 13408 belong to the ET-37 complex which is frequently encountered in Europe and North America.19,25
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It was necessary to have various genetic lineages represented among the meningoccocal strains in our collection for a thorough analysis of the genetic diversity of penA among these strains. The polymorphism of penA was analysed by investigating restriction endonuclease patterns for amplified penA following digestion with three enzymes, HaeIII, HpaII and TaqI. The entire open reading frame of penA was amplified and digested by these enzymes. Distance matrices were constructed and used to draw a dendrogram for the strains tested. All penS strains had identical restriction endonuclease patterns for the three enzymes (Figure 2). The penA allele harboured by these strains was named penA1. One susceptible strain (LNP 13129) harboured a slightly different allele (penA2), the HpaII profile of which differed from that of penA1 by only two bands. However, penA1 and penA2 clustered together on the dendrogram (Figure 1
). The penI strains showed a high degree of polymorphism for penA (Figure 2
): eight different penA alleles were observed among the 12 strains tested in this study (Figure 1
, Table I
). The pilA/pilD-based dendrogram and penA-based dendrogram cannot be directly superimposed (Figure 1
compares the two dendrograms). Several strains that clustered together on the basis of pilA/pilD polymorphism (LNP 16454, LNP 16467, LNP 16519, W-39 and W-88) harboured different penA alleles, penA5 (W-39 and W-88) and penA7 (LNP 16454, LNP 16467 and LNP 16519) (Figure 1
, Table I
). Two penI strains (LNP 16308 and 99/93) had the penA1 allele despite having an MIC of 0.190 and 0.125 mg/L, respectively (Figure 1
, Table I
).
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Correlation between reduced susceptibility to penicillin and alteration of the penA gene
We investigated the correlation between penA polymorphism and reduced susceptibility to penicillin by transferring penA alleles from three penI strains (W-39/penA5, TH41/penA6 and LNP 16454/penA7) to a susceptible strain (LNP 8013/penA1). Transformation was performed using genomic DNA or PCR-amplified penA from donor strains. Penicillin-resistant transformants were obtained at a high frequency (104/cfu). The penA alleles of these transformants (TR1TR6) were identical to the alleles in the donor strains (Table I). Transformants also had the pilA/pilD alleles of the recipient strain, LNP 8013 (pilA10 and pilD3) (Table I
), indicating that the altered penA allele had been acquired by the recipient strain. It is unlikely that, in these experiments, changes at another chromosomal locus in transformants were involved in the resistance of transformants to penicillin, as similar results were obtained with genomic DNA and PCR-amplified DNA. These results strongly suggest that the penI phenotype in our collection of strains is directly related to penA polymorphism. One transformant (TR2), which bore the pilA10 and pilD3 alleles (Table I
), had a new penA allele (penA10) different from those of both the donor strain (W-39/penA5) and the recipient strain (LNP 8013/penA1). However, penA10 differed from penA1 only in its TaqI profile by the presence of an additional TaqI site (profile 8, Table I
, Figure 2
). This transformant may have originated through a partial recombination between penA1 and penA5. However, it could also have resulted from a point mutation in penA1 of the recipient strain LNP 8013.
We investigated whether penA could be transferred from a penI strain to a susceptible strain during bacterial growth in a culture medium. A penicillin-susceptible, kanamycin-resistant strain, CAP1 (penA1), derived from strain LNP 8013 (Table I; see Material and methods),20 and a penI strain, LNP 16454 (penA7), were grown to midlogarithmic growth phase and mixed together. This coculture was performed to investigate how gene exchange could occur in vivo during mixed carriage. Colonies resistant to kanamycin and penicillin were obtained when the mixture was plated on medium containing these antibiotics, at a frequency of 105/cfu of the CAP1 recipient strain. These transformants (represented by TR7 in Table I
) harboured the penA7 allele (the allele of the donor strain, LNP 16454) but had the pilA/pilD alleles of the recipient strain CAP1 (pilA10 and pilD3), indicating that the altered penA allele (penA7) had been acquired by the recipient strain (Table I
).
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Discussion |
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All the penI strains tested here harboured an altered penA allele (and hence a modified PBP2) except for two strains which had the penA1 allele (LNP 16308 and 99/93). For these strains, another PBP or bacterial target may be involved. However, we cannot exclude the possibility that these two strains were misclassified, as their MICs were close to the cut-off point (Table I). Strain LNP 16308 (penA1, MIC 0.19 mg/L) was epidemiologically linked to strain LNP 16325 (penA1, MIC 0.04 mg/L). Both strains belonged to the same genetic group (Table I
, Figure 1
). Strain LNP 16308 (penI strain) was isolated from a 4 year old boy suffering from meningococcal infection. Strain LNP 16325 (penS) was isolated 10 days later from his sister, who also developed meningococcal infection. As the LNP 16325 strain was shown to be susceptible to penicillin and the two strains were epidemiologically linked and genetically identical, it seems most likely that both strains are susceptible to penicillin. This example clearly indicates the technical difficulties involved in interpreting a classical antibiogram.
Our study proposes a reliable and rapid molecular approach for analysing reduced susceptibility to penicillin in N. meningitidis. It overcomes problems such as differences in medium or inoculum size, which necessitate the careful standardization of antibiogram determination in different laboratories.
The penA gene seems to be stable, as all susceptible strains harboured the same penA allele (penA1 or the closely related penA2) regardless of genetic group. The similarity in the penA sequences of penS strains of N. gonorrhoeae (which is closely related to N. meningitidis) and penS strains of N. meningitidis also provides evidence of the stability of penA in pathogenic Neisseria before the era of antibiotics.27 These data suggest that penA was acquired before the two species separated. The widespread use of penicillin leads to selection pressure resulting in the emergence of penI strains; penA evolution under this pressure seems to be independent in N. meningitidis and N. gonorrhoeae. Indeed, whereas the acquisition of an extra aspartate residue (Asp345) is a common mechanism in N. gonorrhoeae for reducing the affinity of PBP2 for penicillin, no such extra aspartate residue was observed in the penI strains of N. meningitidis tested here.
The prevalence of penI strains increased in France from 4% of all meningococcal isolates in 1994 to 28% in 1998 (data not shown).28 The frequency of N. meningitidis with reduced susceptibility to penicillin is very low in the Czech Republic: of 655 strains isolated during 19911997, only four (0.6%) showed reduced susceptibility to penicillin.29 Our results indicate that the selection pressure acts directly on the evolution of penA and results in a high degree of penA polymorphism in strains with reduced susceptibility to penicillin. As the strains tested in this study were genetically diverse, the emergence of penI strains does not seem to be due to the expansion of one particular clone. Similar results were obtained with penI strains in Spain.30 A group of five strains (LNP 16454, LNP 16467, LNP 16519, W-39 and W-88 ), which clustered together on the basis of pilA and pilD, harboured two different penA alleles: penA5 (W-39 and W-88) and penA7 (LNP 16454, LNP 16467 and LNP 16519) (Figure 1, Table I
). These strains may originate from two closely related clones that acquired two different penA alleles.
The results regarding penA transfer by transformation and during co-culture are consistent with a model of penA evolution by transformation and homologous recombination rather than mutation. Indeed, N. meningitidis and N. gonorrhoeae are naturally competent for transformation and undergo autolysis throughout their growth phase, facilitating horizontal DNA exchange between these species and even with other species of the genus Neisseria.31 Transformation has been used previously to demonstrate inter-species recombination between the penA genes of N. meningitidis and commensal species during the emergence of penI strains of N. meningitidis.32
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Acknowledgments |
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Notes |
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References |
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Received 19 July 2000; returned 13 September 2000; revised 26 September 2000; accepted 19 October 2000