PBPs are the major resistance determinants in the pneumococcus. The low-affinity variants of PBPs are the results of recombination of the genes coding for these proteins with genes of other species, such as viridans streptococci. Previous studies have suggested that PBP1a, − 2x, and − 2b are generally recognized as the major PBPs associated with the activities of penicillins and some cephalosporins [4, 12]. In our study, changes found in PBP1A, PBP2b and PBP2X are globally similar to those previously reported [6, 13, 14].
The maximal level of resistance of PNSP responsible of invasive pneumococcal disease reported in Casablanca, is relatively low with the maximal MICs = 2 mg/L compared to MICs of PNSP in many countries where MICs ≥ 8 mg/L were reported [14, 15]. Our investigation of the PBP1a, − 2x, and − 2b amino acid sequences of 30 clinical pneumococci demonstrates that the degree of diversity within these amino acid sequences correlates with increasing resistance to β-lactam antibiotics. Analysis of PBP1a, − 2x, and − 2b penicillin-binding motifs revealed the absence of substitution in or close to the active site of all motif analyzed in PSSP strains in this study. These findings differed from previously studies. Indeed Nagai et al. are found Thr445 → Ala substitutions close to SNNT motif in PBP2b gene and Leu546 → Val substitutions close to the LKSG motif in PBP2x gene in PSSP strains . Granger et al. are also found Thr445 → Ala substitutions in the SNNT motif in PBP2b in one PSSP strain in Canada . It is not clear how some PSSP isolates can harbor these two mutations without becoming non-susceptible to penicillin. PSSP analyzed in this study are probably associated with a limited number of clones according to the RFLP profiles of the three pbp genes.
Analysis of PBP1a and PBP2b motifs revealed the absence of substitution in or close to the active site of conserved KTG and SVVK motifs. These findings are in agreement with results from other studies, suggesting that these motifs are not involved in the development of penicillin resistance [17, 18].
Interestingly, PISP isolates with amoxicillin MICs ≥ 0.125 mg/L and MICs ≥ 0.25 mg/L harbored amino acid substitutions close to PBP1a conserved motifs STMK (Thr371 → Ala) and SRNVP (Pro432 → Thr), respectively. This result suggests that alteration in conserved motif of PBP1a may be occurred among S. pneumoniae with low level resistance to penicillin and amoxicillin. The diversity of the pattern of amino acid motifs in the PNSP as well as pbp2b and pbp2x genes suggests these isolates have emerged independently as previously described .
For PRSP strains, they shared a similar pattern of amino acid motifs but had different genotypes of the three pbp genes. All of these isolates harbored the same amino acid substitutions close to PBP1a, PBPB2b and PBP2x conserved motifs. Similar results were published by Zhou et al. in China . In addition, one strain had Ala618 → Gly substitutions close to KTGTA motif in the PLP2B. These changes are identical to those previously reported [6, 13]. However, we reported in this study, amino acid alteration among PNSP with low-levels of MICs (2 mg/L). In several study, amino acid alteration, especially for PBP1a, is reported for high-level penicillin resistance MICs > 4 mg/L [16, 18, 19]. Our explanation for this difference is the origin of our isolates. Indeed, non-invasive pneumococcal isolates frequently have a higher prevalence and high-levels of antimicrobial non-susceptibility, compared to invasive isolates, but they can share the same amino acid alterations.
Moreover, we found that all PNSP strains had generally some co-resistances associated with other antibiotics families especially to cotrimoxazole, tetracycline and erythromycin as reported elsewhere .
PCR–RFLP analysis of the pbp1a, pbp2b and pbp2x genes yielded six, eleven and ten distinct fingerprint patterns, respectively. Genotype 5/2/7 was found most frequently among PSSP isolates and there was a single genotype for plp1a and pbp2x. In contrast, genotype of PNSP strains, showed several composite pattern profiles for the three resistance genes. Variations in the RFLP patterns demonstrate the highly variable nature of the pbp genes, suggesting a high frequency recombination or point mutations that they undergoes over the time .