1 Laboratoire d'immunogénétique, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CHUM), Montréal, Canada; 2 Département de microbiologie-immunologie, Hôpital Notre-Dame du CHUM, 1560 Sherbrooke Est, Montréal, Québec, Canada H2L 4M1; 3 Département de microbiologie, Hôpital Saint-Luc du CHUM, Montréal, Canada; 4 Département de microbiologie, Hôpital Maisonneuve-Rosemont, Montréal, Canada
Received 16 November 2004; returned 22 January 2005; revised 10 March 2005; accepted 11 March 2005
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Abstract |
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Methods: The pbp2x genomic region encompassing codons 178703, which includes the entire region of the transpeptidase domain, was sequenced and compared for 52 clinical isolates comprising 20 penicillin-susceptible (PSSP), 20 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci.
Results: The degree of diversity within PBP2x correlated with increased resistance to ß-lactam antibiotics. There were an average of 5.0 ± 1.8 mutations in PSSP, 37.9 ± 4.4 in PISP, and 63.0 ± 2.0 in PRSP isolates when compared with the control penicillin-susceptible strain R6. At least six distinct amino acid profiles were identified among PISP strains isolated in Quebec. In contrast, all PRSP isolates shared a similar pattern of altered amino acids compared with the sequence from susceptible strains.
Conclusions: These data will be useful in future studies to monitor the genetic changes associated with the emergence and spread of ß-lactam resistance in Quebec.
Keywords: penicillin-binding proteins , ß-lactams , pneumococci , serotype , penicillin resistance , cefotaxime resistance
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Introduction |
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ß-Lactam antibiotics exert their biological effects by interacting with a unique class of proteins, the penicillin-binding proteins (PBPs). PBPs are membrane enzymes that catalyse the polymerization and transpeptidation of glycan strands, during the assembly of the bacterial cell wall.1 ß-Lactam resistance in clinical pneumococci is mediated by altered PBPs, specifically PBP1a, PBP2x and PBP2b.24
PBP2x of S. pneumoniae, the first target to be modified under antibiotic pressure, is the most thoroughly studied PBP.57 The crystal structure of PBP2x revealed that it comprised three-domains: a central 350-residue domain (amino acid positions 266 to 616), which corresponds to the transpeptidase domain, and two flanking domains of unknown function.8 The active site of transpeptidase activity is formed by three conserved amino acid motifs, STMK (including the active-site serine), SSN and KSG. Mutations, within or in positions flanking these motifs, have been observed in resistant laboratory mutants and clinical isolates.5,914 PBP2x of penicillin non-susceptible pneumococci (NSSP) may harbour several dozen amino acid mutations outside these motifs compared with homologous sequences from penicillin-susceptible S. pneumoniae (PSSP).9,15 Some of these amino acid substitutions have been shown to be common to penicillin-resistant pneumococci (PRSP) isolated from different countries.1618 Only a few of those changes are likely to be linked to the resistance phenotype.1720
In Canada, data from the 19972002 national surveillance study demonstrated that the prevalence of S. pneumoniae with reduced susceptibility to penicillin did not significantly change over the 5 year period and ranged from 21.2% in 1997 to 24% in 2002.2123 However, the proportion of isolates with high-level penicillin resistance (MIC > 2 mg/L) increased dramatically from 2.4% in 1999 to 13.8% in 2002, and the proportion of multidrug-resistant S. pneumoniae isolates increased from 2.7% to 8.8% over the 5 year period.23 The prevalence of PRSP varied considerably by geographical location in Canada, and was higher in Ontario and Quebec (central Canadian provinces) than the eastern provinces.22,23
Although several studies have described the genetic profile of the pbp2x gene in pneumococci from different countries, only one study was done on very few clinical isolates from Canada.13 Given that the proportion of PRSP with high-level resistance has considerably increased in Canada since 1999, and Quebec is part of this emerging trend, we investigated the nature of the PBP2x amino acid motifs in 52 clinical pneumococci isolated across the Quebec province.
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Materials and methods |
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Fifty-two clinical isolates of pneumococci [20 penicillin-susceptible, 20 penicillin-intermediate (PISP) and 12 penicillin-resistant strains, for which the penicillin MICs were 0.06 mg/L, > 0.06 and < 2 mg/L, and
2 mg/L, respectively] were chosen randomly to represent a range of MICs and a cross-Quebec geographical distribution, for molecular characterization. Pneumococci were obtained from the bacteria bank of the Reseau EQUERE (Etude QUEbecoise des pathogenes REspiratoires) at Maisonneuve-Rosemont Hospital (Montreal, Canada) and from St-Luc Hospital (Montreal, Canada). The R6 strain of S. pneumoniae (MIC of penicillin
0.016 mg/L) and the laboratory strain ATCC 49619 (MIC of penicillin between 0.125 and 1 mg/L) were used as the penicillin-susceptible and -intermediate reference strains, respectively. The clinical and demographic parameters of the S. pneumoniae isolates analysed in this study are listed in Table 1. Bacteria were cultured at 37°C on agar supplemented with 5% sheep blood (Quelab, Montreal, Canada) in a 5% CO2 atmosphere. Capsular typing was based on the Quellung technique. Bacterial DNA was extracted from overnight growth cultures by boiling pneumococcal cells in chelex (SigmaAldrich, Oakville, Canada) for 15 min. The supernatant was then recovered by spinning the specimen for 10 min at 15 000 rpm. The supernatant was kept at 4°C until used for PCR.
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For each clinical strain, penicillin and cefuroxime susceptibility testing was performed by broth microdilution according to NCCLS guidelines.24 Interpretative criteria used were those published in the NCCLS M100-S12 document.25 For all NSSP isolates, penicillin and cefotaxime MICs were also determined in triplicate using Etest strips (AB Biodisk, Solna, Sweden), as recommended by the manufacturer. MICs were measured independently by two observers and the mean was calculated for each bacterial strain tested in triplicate. MIC values for both methods appear in Table 1.
Amplification of the pbp2x gene and nucleotide sequencing
The coding region of the transpeptidase domain of pbp2x genes of all strains of S. pneumoniae, listed in Table 1, was amplified from bacterial DNA by PCR. Primer pairs used for amplification of the pbp2x gene were designed from the DNA sequence of the R6 strain (GenBank accession no. X16367): pbp2x-F, 5'-721GGGATTACCTATGCCAATATGAT743-3'; and pbp2x-R, 5'-2463AGCTGTGTTAGC(G/A/C)CGAACATCTTG-2440-3'. Amplification reactions contained 200 mg of bacterial DNA, 0.25 mM of each dNTP (Pharmacia, Piscataway, NJ, USA), 1 x PCR buffer (Applied Biosystems, Foster City, CA, USA), 2.5 units of Taq DNA polymerase (Applied Biosystems) and 15 pmol of primers (Invitrogen Canada Inc., Burlington, Canada) in a total volume of 50 µL. PCR cycling conditions included a 5 min initial denaturation step at 95°C, followed by 35 cycles of 1 min at 95°C, 75 s at 62°C and 75 s at 72°C, and a final extension step at 72°C for 4 min. Amplified fragments of 1742 nucleotides were purified into Multiscreen96 PCR plates, using vacuum filtration with the cleaning kit (Millipore, Bedford, MA, USA).
The nucleotide sequences of purified PCR products were determined by direct sequencing using BigDye terminator cycle sequencing reactions (Applied Biosystems). The reaction products were run in an automated DNA sequencing ABI PRISM 3100 capillary sequencer (Applied Biosystems). All PCR products were sequenced in both directions.
Sequence analysis
Nucleotide and derived amino acid sequence data were compared with those of the pbp2x R6 reference strain (X16367). The analysis was done by Lasergene software (Version 5, DNAstar Inc., Madison, WI, USA), using the ClustalW alignment. Comparison of our sequences with all other sequences was performed using the Genetics Computer Group (GCG) package. Numbers of amino acid mutations are presented as means ± SEM.
Nucleotide sequence accession numbers
The nucleotides sequences determined in this study were submitted to the GenBank nucleotide sequence database under accession numbers AY950507 to AY950558.
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Results |
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The diversity of amino acids in PBP2x (codons 178703) was determined for each clinical isolate. In total, we observed 110 amino acid changes compared with the R6 PBP2x control sequence (Figure 1). Of those, 105 amino acid substitutions were found in NSSP and occurred mostly (67%) within the transpeptidase domain (residues 266616). Mutations at positions in or close to each of three conserved motifs (STMK, SSN and KSG) are indicated in bold (Figure 1).
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Finally, we compared the pbp2x gene sequence of the 52 clinical isolates analysed in this study with other published sequences in the Genetics Computer Group (GCG) database and identified five novel amino acid sequence variants in our samples. The amino acid substitutions Asn185Thr, Ser252
Ala and Lys505
Glu were observed in the PISP isolates 14736, 14622/14625 and 2259, respectively. Leu429
Cys was found in the PSSP isolate 14756 and Asp643
Ala was identified in PRSP strain 14893. These new mutations are not frequent and are probably the result of point mutations.
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Discussion |
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We identified the presence of at least six distinct amino acid profiles among PISP strains isolated in Quebec. The acquisition and spread of ß-lactam resistance in pneumococci is a complex process, involving clonal diffusion, horizontal DNA transfer, and point mutations. The diversity of the pattern of amino acid motifs in the PISP pbp2x gene suggests that PISP isolates have emerged independently in Quebec, on more than one occasion. In contrast, all PRSP strains shared a similar pattern of amino acid motifs and are probably associated with a limited number of resistant clones.
Our analysis of PBP2x penicillin-binding motifs showed the presence of substitutions at the amino acid Thr338 in most NSSP strains (Table 1). However, one PSSP isolate (14900) contained this mutation while it was absent in three PISP (2076, 14896 and 14763). The Thr338Ala/Ser/Pro mutation, adjacent to the active-site serine Ser337 in the first conserved motif (STMK), was identified as an important determinant of penicillin resistance.27,28
Although the Thr338 residue is not directly in contact with the antibiotic,8,29
the Thr338
Ala substitution results in the absence of a hydroxyl group at this position, which drastically lowers the acylation efficiency of PBP2x for ß-lactams.29
The Thr338 substitution is frequently found in clinical isolates with ß-lactam resistance4,9,13,18,28
but it is sometimes observed in PSSP isolates.9,13,18
However, it is not clear how some PSSP isolates can harbour this substitution without becoming resistant. It is possible that other compensatory structural alterations help to stabilize the primary active-site mutation of PBP2x. The His394
Leu mutation just before the second conserved motif (SSN) was found only in one PISP isolate (14763) not bearing the Thr338
Ala substitution in the STMK motif (Table 1). In fact, the His394
Leu substitution has never been observed in association with the Thr338
Ala mutation in NSSP isolates.12,13,18,28
On the other hand, the Met339Phe substitution, also in the first conserved motif, was found in only one PRSP isolate (14760). Site-directed mutagenesis demonstrated that this substitution decreases the acylation efficiency 3- to 10-fold, depending on the sequence context and the ß-lactam antibiotic.10
In clinical S. pneumoniae, the PBP2x Met339
Phe substitution has been found in strains with penicillin MICs
4 mg/L12,20,30
and cefotaxime MICs
2 mg/L2,9,18,20,31
isolated in France, USA and Japan. In our case, penicillin and cefotaxime MICs for the 14760 isolates were 2 mg/L for penicillin and 1.5 mg/L for cefotaxime. This substitution was not observed in the previous study of Canadian pneumococci strains.13
This strain was isolated in January 2002 and may represent the recently increasing incidence of highly resistant pneumococci in Quebec.22,23
This observation is interesting and will be useful to monitor the emergence and spread of isolates with high-level penicillin resistance in Quebec and other parts of Canada.
The amino acid substitution Leu546Val preceding the third conserved motif (KSG), was observed in 18 clinical isolates; and penicillin MICs for these strains were generally the highest (
1 mg/L), except for two strains (2259 and 14681) whose MICs were 0.25 and 0.5 mg/L, respectively (Table 1). The Leu546
Val substitution is associated with high-level ß-lactam resistance in clinical pneumococci.9,12,13,20,28
In our study, all strains presenting cefuroxime MICs
2 mg/L and cefotaxime MICs > 0.5 mg/L (Table 1) harboured the Leu546
Val substitution. However, some strains susceptible to these cephalosporins contained this mutation. Although the Leu546
Val substitution is not sufficient by itself to cause high-level ß-lactam resistance, it seems to play an important role in the development of resistance.
The Thr550Ala mutation just after the KSG motif was observed in only one of our clinical isolates. Interestingly, this mutation was found in an isolate (14758) with a penicillin MIC of
0.06 mg/L and a cefuroxime MIC of 0.5 mg/L. It has been clearly shown by site-directed mutagenesis that this mutation in PBP2x is involved in low-level resistance to cephalosporins.5,7
However, several studies of clinical S. pneumoniae found no or very few resistant strains with a Thr550
Ala substitution.12,13,18
Our results are in agreement with the observed difference between clinical and laboratory PBP2x mutants. In fact, resistance in laboratory mutants does not always correlate with the definition of resistance for clinical isolates in terms of their MICs. Furthermore, one amino acid change may contribute to a decreased susceptibility selectable in the laboratory without conferring high resistance levels.
Substitutions outside penicillin-binding motifs that may alter the conformational change of active-site structure, might also contribute to resistance. Amino acid substitution Gln552Glu was previously associated with cephalosporin resistance in laboratory mutants.2,6,11,19,32
Although this substitution was observed in four clinical pneumococci isolated in Quebec (14770, 14771, 2076 and 14896), none was resistant to either cefuroxime or cefotaxime (Table 1). These results confirm previous studies reporting that this particular mutation is associated with ß-lactam resistance of laboratory mutant strains rather than clinical S. pneumoniae isolates.17,18,30
Analysis of amino acid sequences of PBP2x revealed other prominent substitutions that occurred among most NSSP isolates. In fact, 97% (31/32) of NSSP isolates contained both Arg254Gln and Met256
Val substitutions. Our search in different databases and among PBP2x published sequences revealed that this double substitution in the C-terminal domain is present in most NSSP clinical isolates19,20,28,30
and ß-lactam-resistant Streptococcus mitis B6.15
However, like other mutations previously described to be linked to resistance, this double substitution was also found in two PSSP isolates. The prevalence of these double substitutions (97%) is higher than the Thr338 substitution (94%) usually used to predict resistance. The amino acid motif Q254V256 (Figure 1) could represent a new target for the development of PCR-based predictive diagnosis of ß-lactam resistance in S. pneumoniae. Further studies of mutagenesis in this area are still necessary to confirm if these preponderant amino acid alterations are directly associated with penicillin resistance development.
This study constitutes the first investigation of pbp2x gene alterations in clinical S. pneumoniae isolates throughout the province of Quebec. Based on the deduced amino acid sequence, we distinguished six distinct motif profiles among the PISP that will play an important role in tracking the genetic changes to chart the emergence, spread and evolution of ß-lactam and notably penicillin resistance in this part of Canada.
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Acknowledgements |
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