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Antimicrobial Agents and Chemotherapy, March 2006, p. 887-892, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.887-892.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Interlaboratory Comparison of PCR-Based Methods for Detection of Penicillin G Susceptibility in Neisseria meningitidis

Muhamed-Kheir Taha,^1* Maria Leticia Zarantonelli,¹ Arianna Neri,² Rocío Enriquez,³ Julio A. Vázquez,³ and Paola Stefanelli²

Neisseria Unit and the French National Reference Center for Meningococci, Institut Pasteur, Paris, France,¹ Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanita, Rome, Italy,² Reference Laboratory for Neisserias, National Center for Microbiology, National Institute of Health Carlos III, Majadahonda, Madrid, Spain³

Received 15 June 2005/ Returned for modification 21 August 2005/ Accepted 11 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
We carried out a study for the nonculture detection of susceptibility of Neisseria meningitis to penicillin G in three laboratories of the European Monitoring Group on Meningococci (EMGM). Thirteen clinical samples (cerebrospinal fluids) and corresponding bacterial isolates from 13 cases of invasive meningococcal infection were distributed to the three laboratories. The MICs of penicillin G were determined for the isolates. Each laboratory used an "in-house" PCR-based method to determine alterations to the penA gene, which is associated with a reduced susceptibility to penicillin G. Nucleotide sequences from the 3' end of the penA gene were also determined. We observed a good correlation between genotyping of penA and the phenotypic determination (MIC) of susceptibility to penicillin G. The results obtained by the three methods for penA in the samples correlated very well with those obtained in bacterial isolates and with sequence data. The kappa coefficient that was used to estimate the level of agreement between genotypic results varied between 0.65 and 1, indicating a good agreement. This suggests that genotyping can predict susceptibility of N. meningitidis to penicillin G. These data strongly suggest that genotyping of penA should be used to determine meningococcal susceptibility to penicillin G in culture-negative cases. Although the nucleotide sequence of penA may be the gold standard in genotyping of penA, the less expensive PCR-based approach reported in this study may be quicker when a large number of isolates and clinical samples need to be tested.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture-negative cases are frequently found in suspected meningococcal infections, particularly after early antibiotic treatment (9). Molecular methods have been developed for the nonculture diagnosis (identification and genogrouping) of Neisseria meningitidis, which reduces the time needed for detection and characterization of N. meningitidis in culture-negative clinical samples (25). The immediate management of invasive meningococcal infections requires rapid diagnosis and prompt and adequate antibiotic therapy. The first choice antibiotics for treating invasive meningococcal infections are beta-lactams. However, an increasing number of meningococcal isolates are showing reduced susceptibility to penicillin G. These isolates are known as Pen^I and are defined as having an MIC between 0.094 mg/liter and 2 mg/liter. The biological significance of this phenotype remains to be determined for therapy by penicillin G. The emergence of meningococcal strains with an MIC of >1 mg/liter can cause treatment to fail because this threshold corresponds to the therapeutic concentration obtained in the cerebrospinal fluid (CSF) during treatment with penicillin G (13). Pen^I isolates account for 33%, 27.2%, and 37% of the total meningococcal isolates in France, Italy (2004), and Spain, respectively (4, 24, 28). However, a recent interlaboratory study showed that participating laboratories were able to detect the Pen^I isolates in 18.2% to 100% of cases (29). Moreover, there is no information available on antibiotic susceptibility for culture-negative cases. Therefore, a consensus molecular method is needed for the reliable prediction of meningococcal susceptibility to antibiotics. Three penicillin-binding proteins (PBP1, PBP2, and PBP3) can be detected in N. meningitidis, as also reported for the closely related species Neisseria gonorrhoeae (6). Genes encoding additional PBP have also been found in the complete genome sequence of two strains of N. meningitidis (17, 27). The reduced susceptibility of N. meningitidis to penicillin G involves alterations to penicillin-binding protein 2 (PBP2) (21). In all Pen^I isolates, between five and eight positions in the C-terminal part of PBP2 (amino acids 427 to 581) are modified (3). These modifications are directly linked to a reduced susceptibility of N. meningitidis to penicillin G and can be revealed by sequencing penA (3). This study reports three rapid methods to detect penA polymorphisms. The first method amplifies the 3' part of the penA gene and then uses restriction fragment length polymorphism analysis to reveal alterations to penA and thus predict decreased susceptibility to penicillin G (2). The second method uses a real-time PCR assay to detect one of the alterations to the penA gene, which has been chosen as a marker of penA modifications: a different melting temperature between Pen^I and Pen^S isolates (22). The third method uses a differential PCR, in which oligonucleotides were designed to obtain a positive PCR only when penA is not altered, thus indicating an isolate susceptible to penicillin G. These methods were applied to meningococcal isolates for the rapid screening of the penicillin-intermediate genotype (2, 22, 24). Moreover, these methods can be directly applied to clinical samples, such as cerebrospinal fluid and blood, and allow a rapid and easily interpretable detection of Pen^I isolates without requiring culturing. The aim of this study was to establish gold standards for the molecular detection of alterations to penA. We used clinical samples from patients with well-known clinical histories and the corresponding cultured and characterized isolates to compare the three approaches as molecular methods for predicting susceptibility of N. meningitidis to penicillin G.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, samples, and conventional bacteriology. Three laboratories (named L1 to L3), which are members of the European Monitoring Group on Meningococci (EMGM), participated in this study. Biological samples (n = 13, named PenNet01 to PenNet13) were obtained from the CSF of 13 different patients admitted to several hospitals with a clinical diagnosis of meningitis. Samples were cultured, and bacteria were isolated using standard methods. The MIC of penicillin G was determined using the E test on Mueller Hinton agar supplement with 5% sheep blood, as previously described. Laboratories 3 and 1 used the breakpoints of 0.094 mg/liter or 0.125 mg/liter to define Pen^I isolates, respectively (29). Serogroup was determined by bacterial agglutination with serogroup-specific immune sera (Bio-Rad). Serotypes and serosubtypes were determined as previously described (1, 15, 26).

Sample preparation for PCRs. Samples were freeze-thawed once, heated at 100°C for 3 min, and then centrifuged for 5 min at 10,000 x g to obtain the supernatant. Laboratory 1 carried out nonculture detection of N. meningitidis in clinical samples using PCR amplification of the crgA gene, and genogrouping of N. meningitidis was carried out using PCR amplification of serogroup-specific genes (25). Laboratory 1 sent 200 µl of each sample at room temperature to the participating laboratories. Each laboratory carried out one of the rapid techniques for detecting alterations to penA.

RFLP of penA. Laboratory 1 (L1) used restriction fragment length polymorphism (RFLP) of penA. PCR was carried out using two oligonucleotides, penA-1F and penA-1R, to amplify a 511-bp fragment of the 3' end of the penA gene. PCR was carried out as previously described (2, 5). Amplification products were digested with TaqI and separated on a 3% agarose gel. Pen^S isolates had the same profiles, whereas Pen^I isolates showed different profiles. The restriction profiles corresponding to Pen^S and Pen^I isolates were as previously reported (2).

Real-time PCR and oligonucleotide thermal analysis. Laboratory 2 (L2) used real-time PCR and oligonucleotide thermal analysis. Primers, fluorescent resonance energy transfer probes, and PCR parameters were as previously described (22), and primers and probes are listed in Table 1. The real-time PCR mixture contained either 1 µl of purified chromosomal DNA (100 ng) extracted from the cultured isolates (22) or 10 µl of boiled CSF added directly to the mixture with no additional purification step.

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TABLE 1. Oligonucleotides used in this study

A total of 1 µl (10 pmol) of each primer, 2 µl (2 pmol) of FL and LC640 probes, 2.5 µl MgCl₂ (final concentration, 4 mM), 2 µl of Fast-start master hybridization probe reaction mixture (Roche Diagnostic, Mannheim, Germany), and PCR-grade sterile water was used in a final volume of 20 µl. PCR included an initial denaturation step of 10 min at 95°C, followed by 40 cycles of denaturation for 10 s at 95°C, annealing for 10 s at 48°C, polymerization for 10 s at 72°C, and detection of the fluorescence for 15 s at 38°C. This last detection step was added to each PCR cycle to increase the red fluorescence levels for the quantitative PCR analysis. The temperature transition rate was 20°C/s for all segments.

Fluorescence was measured in channel 2 at 640 nm. Data were analyzed with LightCycler software, version 5.32, according to the manufacturer's instructions (Roche Diagnostics). The susceptible reference strain, one Pen^I strain with a defined penicillin MIC, and a negative control (sterile water instead of the DNA template) were included for reproducibility of the results and to check contamination.

Single-PCR-based method to predict penicillin susceptibility in N. meningitidis strains and in clinical samples. Laboratory 3 (L3) used a single-PCR-based method to predict penicillin susceptibility in N. meningitidis strains and in clinical samples. Two oligonucleotides, RT-3(F) and RT-4(R), were designed based on the polymorphic sites that discriminate Pen^S/Pen^I strains. In both oligonucleotides, the 3' end corresponds to one of the polymorphic sites. The oligonucleotides target a complementary sequence of the penA gene in penicillin-susceptible strains. They amplify the region from nucleotides 1526 to 1714 inclusive, which corresponds to amino acids 509 to 572 in Pen^S strains. Therefore, if the amplification is successful, this implies penicillin susceptibility, whereas no amplification implies penicillin resistance. Amplification products were analyzed on a 2% agarose gel.

DNA sequencing of penA and MLST. Laboratory 1 also partially sequenced penA. The penA gene was amplified using two oligonucleotides having adaptors that corresponded to the universal forward and reverse sequences that were added to the 5' end upstream and downstream from oligonucleotides, respectively (Table 1). After amplification, the universal forward and reverse oligonucleotides were used for sequencing. Multilocus sequence typing (MLST) was used to genotype the isolates. The polymorphism of seven chromosomal genes in N. meningitidis that encode housekeeping enzymes was determined. The analysis was performed on the DNA sequence of approximately 450 bp from PCR products corresponding to these genes. The combination of the seven corresponding alleles of these genes defines the sequence type (ST) of a given strain. Oligonucleotides used for MLST were as previously described (15, 26).

Statistical methods. We used the kappa coefficient (K) to estimate the level of agreement between results from the PCR-based detection of penA polymorphisms (10). This test determines whether agreement between results exceeds chance levels. We compared the results from each laboratory with the results obtained from sequencing of the cultured bacteria. K was calculated using K = Po-Pe/1 – Pe, where Po is the observed agreement and Pe is the agreement obtained from a random guess. A K value of 0.60 or more was considered a good level of agreement.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of isolates and clinical samples. The bacterial isolates from the 13 samples belonged to four major serogroups (6 to serogroup B, 4 to serogroup C, 2 to serogroup W135, and 1 to serogroup Y). We also found different phenotypes (serotypes and serosubtypes) (Table 2). Two laboratories (L1 and L3) determined the MIC of penicillin G using the E test. L1 applied an MIC breakpoint of 0.125, whereas L3 applied 0.094 as the penicillin MIC breakpoint. Accordingly, the isolates were classified as Pen^S and Pen^I by the two labs. We observed a good correlation between the results from the two laboratories, except for isolate LNP21615, which was classified as Pen^I by L1 and as Pen^S by L3 (Table 2). Moreover, the MICs obtained by L1 were always higher than those found by L3 (Table 2).

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TABLE 2. Phenotype and genotype of N. meningitidis isolates

MLST typing revealed that the isolates belonged to several genetic lineages, including major clonal complexes such as the ST-41/44/lineage III and ST-269 clonal complexes for isolates of serogroup B and the ST-11/ET-37 clonal complex for serogroup C isolates (Table 2).

The nonculture detection of meningococcal DNA using crgA and ctrA was positive in all 13 clinical samples. The results of serogrouping by conventional agglutination of isolates were identical to the results predicted by PCR (genogrouping) (data not shown). The results obtained for conventional and molecular characterization suggest that the panel tested corresponded to different isolates and may be suitable for analyzing penA polymorphisms.

Molecular detection of alterations to penA. The three participating laboratories successfully amplified penA from all cultured isolates. PBP2 sequences (amino acids 427 to 581) that were predicted from the DNA sequences of the PCR products agreed with the MIC-based phenotypic classification (Tables 2 and 3). Eight of the 13 sequences were identical and corresponded to susceptible isolates (Table 3; Fig. 1). These PBP2 sequences (PBP2s) were also identical to those previously reported for susceptible strains (5). Five PBP2 sequences differed from PBP2 by 14 or 17 substitutions, showing between 89% and 91% identity with PBP2, and corresponded to Pen^I isolates (Fig. 1 and Tables 2 and 3). Three different sequences were observed among these five isolates, with three isolates having identical sequences (LNP17244, LNP21316, and LNP21338) with 14 substitutions. The other two isolates (LNP21321 and LNP21332) were different, with 17 and 14 substitutions, respectively (Fig. 1). All of the five altered PBP2 sequences showed modifications in the polymorphic positions previously reported to be altered in all Pen^I strains (3). When the three laboratories tested the polymorphism of penA using rapid PCR approaches (see Materials and Methods), they all successfully detected the altered penA alleles in the Pen^I isolates (Table 3). Similar results were also obtained when directly amplifying the clinical samples. However, two laboratories could not amplify penA from the PenNet03 sample despite detecting meningococcal DNA by PCR amplification of crgA and ctrA. We obtained a good correlation between laboratories, except for the samples PenNet09 and PenNet13, which L3 typed as susceptible while L1 and L2 detected altered penA genes in them (Table 3). However, an altered penA gene was found in the cultured isolates by all three laboratories, in accordance with DNA sequencing (Table 3 and Fig. 1).

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TABLE 3. penA typing in cultured isolates and clinical samples (CSF)

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FIG. 1. The partial sequences of the C-terminal part of PBP2 (amino acids 427 to 581) from the sequences of penA genes from isolates of N. meningitidis tested in this study. Ditto marks indicate identical residues. Polymorphic residues are indicated by a one-letter code. The positions that are modified in all PenI isolates are indicated by asterisks. The KTG motif is indicated in a box. The isolate number is indicated on the left.

Performance of nonculture detection of penicillin G susceptibility. We estimated the performance of the three rapid approaches using kappa statistics (see Materials and Methods) by calculating the K coefficient, using the results from each laboratory and the sequencing data as references. As expected, the agreement was perfect, with a K value of 1 (maximum agreement), for the results obtained with the cultured isolates by the three laboratories. The results from nonculture rapid approaches in detecting alterations of penA compared to sequencing data gave a K value of 1 for L1 and L2, whereas L3 had a K value of 0.65.

The sensitivity and specificity of methods for detecting the Pen^I isolates used by L1 and L2 were both 100%, whereas they were 100% and 80%, respectively, for L3.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The phenotypic determination of susceptibility to penicillin G by the E test is still difficult due to differences in the critical values used by different laboratories (29). Moreover, our data indicate that differences in MICs for the same set of isolates may be observed even when using the same medium. This could be due to differences in the sources or batches of medium and/or sheep blood. Nonculture PCR-based methods are increasingly being used for diagnosis of meningococcal infection. Although current nonculture diagnosis approaches allow the identification and genogrouping of N. meningitidis, they cannot predict meningococcal susceptibility to antibiotics. The global approach to immediate management of invasive meningococcal infections requires information on antibiotic susceptibility to provide adequate treatment for patients and prophylaxis for contacts. This study is the first attempt to find a rapid test for penicillin G resistance for wide clinical application.

The major antibiotics currently used in treatment and prophylaxis are beta-lactams, quinolones, chloramphenicol, and rifampin. Several studies have shown that alterations to penA are directly linked to reduced meningococcal susceptibility to penicillin G (3, 18, 21) and that no isolate with a sequence characteristic of susceptible strains has ever had the same MIC as Pen^I strains. Mutations in the rpoB gene encoding the beta subunit of the RNA polymerase are directly linked to high levels of meningococcal resistance to rifampin (8, 16, 23). Meningococcal resistance to quinolones can also be inferred from mutations in the gyrA, parC, and mtr genes (11, 19). Resistance to chloramphenicol can be inferred from the catP gene (12, 20), and resistance to sulfonamide can be inferred from mutation in folP gene (7, 14). Therefore, molecular methods have been developed to detect alterations in these genes.

The enhanced surveillance of invasive meningococcal infections worldwide requires standardized methods. The EMGM represents a good forum for conducting interlaboratory comparisons of PCR-based methods. An interlaboratory study of PCR methods of identification and genogrouping of N. meningitidis in laboratories of the EMGM has already been carried out (25). The current study is a part of our effort in the EMGM to provide a basis for detecting standardized protocols for molecular identification and characterization of N. meningitidis.

Our sequencing data of the 3' part of the penA gene clearly confirmed the direct correlation between alterations in this region and the Pen^I phenotype. The detection of those positions that are always modified in Pen^I strains is a powerful tool for identifying Pen^I strains. The three rapid PCR-based methods reported in this study were in total agreement with each other and completely correlated with sequences from cultured bacteria. We observed the same level of agreement with clinical samples and the nonculture characterization of the penA gene using PCR-based RFLP of penA (laboratory L1) and real-time PCR and oligonucleotide thermal analysis (laboratory L2). The discrepancy with a different PCR method (laboratory L3) that we observed for the PenNet09 and PenNet13 samples suggests that recommended PCR-based methods for penA typing should give a positive PCR regardless of the phenotype of the isolates. It is most likely that this discrepancy is related to the method used by laboratory 3.

The molecular detection of penA alterations should not be used in first-line detection of N. meningitidis. Nonculture detection and genogrouping have been previously reported, and their sensitivity and specificity have also been estimated (25). Subsequent analysis of the products of PCR amplification of the penA gene can be used to predict susceptibility to penicillin G. As a nonculture method on clinical samples, amplification of penA may be less sensitive than amplification of crgA and ctrA for detecting meningococcal DNA, as suggested by the failure of PCR for one clinical sample (PenNet03). However, transport and/or storage conditions of samples may also be responsible for this failure. It may be prudent to ship processed samples frozen. Moreover, the performance of the amplification may be improved by designing different primers. Standardization of PCR-based methods might also benefit from a collection of seeded sterile CSF samples with known concentrations of N. meningitidis organisms.

Nonculture assays for diagnosis of meningococcal disease should be used together with conventional methods, in particular when culturing fails to isolate bacteria. Nonculture assays should not replace culturing, which should always be carried out, as cultured bacteria are still an invaluable source of information on meningococcal pathogenesis.

 
This work was supported by European Union Contract no. QLK2-CT-2001-01436.

We thank a number of reviewers who improved the quality of the manuscript with their comments and criticisms.

 
* Corresponding author. Mailing address: Neisseria Unit and the French National Reference Center for Meningococci, Institut Pasteur, 28 Rue du Dr Roux, Paris, France. Phone: 33 1 44 38 95 90. Fax: 33 1 40 61 30 34. E-mail: mktaha{at}pasteur.fr.

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Antimicrobial Agents and Chemotherapy, March 2006, p. 887-892, Vol. 50, No. 3
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.3.887-892.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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