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Antimicrobial Agents and Chemotherapy, December 2001, p. 3625-3628, Vol. 45, No. 12
Unité des Neisseria, Centre National de
Référence des Méningocoques, Institut Pasteur,
75724 Paris Cedex 15, France
Received 8 May 2001/Returned for modification 16 August
2001/Accepted 12 September 2001
We developed a nonculture method to predict the susceptibility of
Neisseria meningitidis to penicillin G. The
penA gene was amplified and submitted to restriction
fragment length polymorphism analysis. This approach was first
validated with a collection of 75 meningococcal strains of known
phenotypes. It was next successfully applied to 29 clinical samples.
Treatment of meningococcal
infections is a medical emergency and requires a rapid diagnosis by the
isolation of bacteria from cerebrospinal fluid (CSF), blood, or other
body fluids (synovial, pericardial, etc.). However, the isolation of
Neisseria meningitidis by culture is frequently hindered by
early antibiotic treatment, which is highly recommended whenever
meningococcal infection is suspected (6, 21). Several
methods for nonculture diagnosis of N. meningitidis have
been recently reported. These methods rapidly (1 to 2 h) identify
N. meningitidis by amplifying one target gene, such as that
encoding 16S rRNA (12, 14), IS1106 (17), porB (29), dhpS
(13), ctrA (9), or crgA
(25). The serogroup, which reflects the capsule
immunospecificity, is subsequently predicted by PCR amplification of
serogroup-specific allele of the siaD gene for sialic
acid-containing serogroups (B, C, Y, and W135) and by PCR amplification
of the mynB gene for the mannoseamine-containing serogroup A
(3, 4, 8, 19, 24, 25). These nonculture methods provide
essential data for assessing the etiological diagnosis of the disease,
but they do not provide information about antibiotic susceptibility. Penicillin G is still effective for the treatment of meningococcal infections. However, meningococcal strains with reduced susceptibility to penicillin (Peni), for which MICs range
from 0.125 to 1 µg/ml (11), have been increasingly
reported in several countries (18). Penicillin-resistant strains (Penr) for which MICs are >1 µg/ml are
now emerging (Table 1). This phenotype is
thought to be due to a reduction in the affinity of penicillin-binding
protein 2 (PBP2), encoded by an altered penA gene, for
penicillin (16, 22). Mosaic structures in penA result from horizontal DNA exchange by transformation between commensal
Neisseria species and N. meningitidis (5,
22, 23). We have previously used restriction fragment length
polymorphism (RFLP) and DNA sequencing to show that
penicillin-susceptible strains (Pens) of
different geographic origins, antigenic formulas, and genetic lineages
have the same penA allele, whereas
Peni strains have a variety of different
penA alleles (1). Moreover, penA
sequences from Peni strains are highly divergent,
particularly in the transpeptidase-encoding region (nucleotides 718 to
1743). This approach was shown to be suitable for the analysis of
genetic relatedness between different penA alleles. The aim
of the present study was to develop a nonculture method to predict the
susceptibility of N. meningitidis to penicillin G.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3625-3628.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nonculture Prediction of Neisseria
meningitidis Susceptibility to Penicillin
ABSTRACT
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TABLE 1.
Distribution of the 75 N. meningitidis strains
tested in this study according to PCR-RFLP and susceptibility
to penicillin (MICs)
DNA sequence alignment revealed conserved regions in the transpeptidase-encoding region of the penA gene of both Pens and Peni strains (1). Two oligonucleotides were designed on the basis of these conserved sequences: AA-1 (5'-ATCGAACAGGCGACGATGTC-3'; nucleotides 1237 to 1256) and 99-2 (5'-GATTAAGACGGTGTTTTGACGG-3'; nucleotides 1728 to 1748). They amplify a 500-bp fragment of the 3' end of the penA gene. These oligonucleotides were first tested on a collection of 75 meningococcal strains for which there were different MICs of penicillin G (39 Pens and 36 Peni strains).
Penicillin G susceptibility was tested by the agar dilution method on G
medium with G supplement (Sanofi Diagnostic Pasteur, Marnes La
Coquette, France). The strains were tested with inocula of
108 CFU/ml on plates containing penicillin G at
the following concentrations: 0.06, 0.125, 0.25, 0.5, and 0.75 µg/ml.
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 on G medium (Etest; AB-Biodisk,
Solna, Sweden). 
-Lactamase activity was detected with a
Cefinaseä disk (BioMérieux, Marcy l'Etoile, France).
PCR was performed as previously described (1, 25).
Amplification products of the expected size (500 bp) were obtained for
all of the strains (Fig. 1A). After
digestion with TaqI and separation on a 4% agarose gel, six
different profiles were obtained (Fig. 1B). An arbitrary number was
assigned to each profile (patterns 1 to 6). The results of PCR-RFLP
from 75 Pens and Peni
N. meningitidis strains are shown in the Table 1. On the
basis of the RFLPs, two classes of strains were identified. (i) All Pens strains for which the MIC was <0.125
µg/ml had the same pattern (RFLP1). (ii) Peni
strains for which the MIC was 0.125 µg/ml had altered patterns (RFLP2
to -6) compared to Pens strains. However, for
five strains for which the MIC was 0.125 µg/ml, we found RFLP1 as for
Pens strains. MICs for these strains were
controlled by Etest and the agar dilution method. These strains could
have been misclassified as Peni strains, as we
have previously suggested for such strains for which the MIC of
penicillin G is at the breakpoint level (1). The fact that
these strains are susceptible to amoxicillin supports this
interpretation (Table 1). This observation underlines the technical
difficulties in determining meningococcal susceptibility to penicillin
by MIC tests. Indeed, 14 European Reference Laboratories using
identical methods, media, and strains reported differences in MIC
determinations. Only molecular approaches showed complete agreement in
detection of Peni meningococcal strains
(2). The PCR-RFLP results with the penA gene
were highly consistent with susceptibility to penicillin. No
correlation was observed between a particular RFLP pattern and MICs in
Peni strains. Indeed, the MICs for strains with
the same RFLP could be different (Table 1). Molecular approaches are
therefore able to overcome technical problems of MIC determination and
to detect meningococcal strains with reduced susceptibility to
penicillin G, regardless of the MIC for them. Treatment could be
immediately adapted.
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Our PCR-RFLP assay was subsequently used to test 29 clinical samples
from various biological fluids obtained from different patients with
suspected meningococcal infection (Table
2). Samples were treated as previously
described (25), and PCR was performed with 15 µl of
each sample. PCR was first used to amplify the crgA gene as
recently described (25) and all of the samples were found
to contain meningococcal DNA. Culture methods also found that 10 of the
samples were positive. This allowed the MICs of penicillin G for them
to be determined (Table 2). We next amplified the penA gene.
Amplification products of the expected size (500 bp) were obtained for
all 29 samples. Analysis of restriction patterns after digestion with
TaqI showed that 20 samples had the same RFLP as the
Pens strains (RFLP1), whereas 9 samples had
different RFLPs, as observed for Peni strains. A
complete correlation was observed between the PCR-RFLP results for
biological samples, the PCR-RFLP results from bacterial DNA extracts,
and the MIC of penicillin G for the corresponding strains (Table 2).
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Molecular detection of genes associated with bacterial resistance to
antibiotics has been developed for several species. Methicillin resistance in Staphylococcus aureus and coagulase-negative
staphylococci results from the synthesis of a novel PBP encoded by the
mecA gene. The PCR-based detection of the mecA
gene is becoming the standard method for the detection of methicillin
resistance (27, 28). Resistance to vancomycin encoded by
the van genes (vanA, vanB,
vanC, vanD, vanE, and vanG)
is now widespread in enterococci. Therefore, amplification assays for
the detection of the van genes have been developed (7,
20). -Lactamase-producing Haemophilus influenzae
can be detected by PCR with specific primers for the blaTEM and
blaROB -lactamase genes
(26).
Early antibiotic treatment is the major element in the immediate management of meningococcal infections. The use of rapid and specific methods of nonculture diagnosis and the development of a molecular approach for the prediction of meningococcal susceptibility to penicillin are needed because of the increasing number of culture-negative cases. The use of PCR to detect reduced susceptibility to penicillin was recently reported with seven different PCRs with the penA gene (15). A strain was considered to be Peni if it failed to produce at least one PCR product. However, this approach was not tested in clinical samples. Our approach only uses one PCR product that is produced in all Pens and Peni strains and is easily and directly applicable for clinical samples. Our data clearly suggest that the detection of an altered penA allele is always correlated with reduced susceptibility to penicillin. Moreover, the combination of this molecular approach with the phenotypic antibiotic susceptibility tests to penicillin may enable us to clearly classify strains as Pens or Peni and to overcome the difficulties encountered for strains for which the MIC of penicillin G is close to the breakpoint level of 0.125 µg/ml (1).
The acquisition of -lactamases seems to be rare in N. meningitidis, and resistance to penicillin G is now evolving by
alteration of PBP2. Penr strains (MIC >1
µg/ml) may be expected in the near future, analogous to
Streptococcus pneumoniae and as suggested by the recent
characterization in our laboratory of meningococcal strains for which
the MICs were 1 and 1.5 µg/ml (Table 1). The threshold of 1 µg/ml
corresponds to the therapeutic concentration in the CSF obtained during
treatment with penicillin G (10). The emergence of
meningococcal strains for which the MIC was >1 µg/ml may provoke
treatment failure. The development of a reliable molecular approach to
surveillance is clinically relevant and is expected to anticipate and
enhance detection of the resistant strains in order to administer an
adequate antibiotic treatment. Moreover, this nonculture detection of
meningococcal strains with altered susceptibility to penicillin G
provides information that otherwise is inaccessible in culture-negative
cases of meningococcal infections. It also complements our approach
combining nonculture diagnosis and serogroup prediction of
meningococcal infections (25).
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ACKNOWLEDGMENTS |
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We thank Magaly Ducos for help with MIC determination.
This work was supported by the Institut Pasteur. A. Antignac was supported by a fellowship from the Caisse Nationale d'Assurance Maladie.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité des Neisseria, Centre National de Référence des Méningocoques, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 44 (0) 1 45 68 84 38. Fax: 44 (0) 1 45 68 83 38. E-mail: mktaha{at}pasteur.fr.
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Antimicrobial Agents and Chemotherapy, December 2001, p. 3625-3628, Vol. 45, No. 12
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.12.3625-3628.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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