The Role Of Streptococcus Pneumoniae In Children With Acute Respiratory Tract Infection And Its β-lactams Resistant Related Genes

PartⅠThe clinical feature and Antimicrobial Resistance in children with acute respiratory tract infection caused by Streptococcus pneumoniaeObject To understand the prevalence,drug resistance and clinical features of children with acute respiratory tract infection(ARI) caused by Streptococcus pneumoniae in Soochow area.Methods From Jan.2006 to Dec.2008,4865 patients with ARI who were admitted to hospital were chosen and their nasopharyngeal secretions were obtained and cultured.K-B disc diffusion for 9 antibiotics susceptibility were performed for these clinical isolates.4β-lactam antibiotics MICs were determined by E-test.Results Among 4865 patients,509 cases were isolated streptococcus pneumonia, the rate was 10.5%.Children between three and five years old were likely infected by SP, compared with other age groups.12.3%and 11.8%of SP were detected in winter and spring,separately,which were higher than other seasons.The disease caused by SP is mainly bronchopneumoniae(86.2%),with the manifestation of cough(96.9%), fever(60.3%),wheezing(49.3%),rales(81.6%) and abnormal X-ray(90.4%).Single SP infection were 149 cases,the coinfection of SP with mycoplasma and syncytial virus were common,the rate was 28.5%and 17.5%separately.The resistance of SP to Erythromycin, Clindamycin and Tetracycline were fairly high,which were all above 90%.The resistance to Chlormycetin,Ofloxacin and Rifampicine were low.All strains were sensitive to Vancomycin.91.1%of SP were intermediate to Penicillin.Resistant rate to Cefuroxime, Ceftriaxone and Cefotaxime was 87.3%,32.9%,10.1%respectively.The rate of multidrug resistant SP was 93.7%,the most common phenotype(resistant) was Erythromycin + SMZ/TMP + Clindamycin + Tetracycline + Cefuroxime(29.1%).Conclusions(1)SP was the most susceptible bacteria pathogen of children's ARI in Soochow area.(2)SP prevailed predominantly in the spring and winter time.(3)SP infection was mainly associated with lower respiratory infections.(4) The sensitivity of SP to Erythromycin,Clindamycin and Tetracycline were fairly low.Majority of SP were Penicillin and Ceftriaxone intermdiatate.Cefotaxime resistant was relatively low.As the MIC of PG raised up,there is a tendency to be more resistant to other antibiotics,includingβ-lactam and non-β-lactam agents.The drug resistance of SP is severe in Soochow area, clinical management of SP infections should be regulate.PARTⅡThe research on related genes ofβ-lactams resistant Streptococcus pneumoniaeObject To analysis the mutations in the related genes(penicillin-binding protein, PBP1a,-2b,-2x) ofβ-lactams resistant streptococcus pneumoniae,and determine the relationship between minimal inhibitory concentrations(MICs) and mutations of PBP1a, -2b and -2x.Methods 59 strains of S.pneumoniae were isolated from clinical samples between April and October in 2008 at Soochow Children's hospital.The MICs of Penicillin G(PG), Cefuroxime(XM),Ceftriaxone(TX) and Cefotaxime(CT) were determined by the E-test method.Chromosomal DNA encoding the Pencillin-binding domains(PBDs) of PBP1a, -2b,and -2x were amplified by nest-polymerase chain reaction followed by DNA sequencing.Nucleotide and deduced amino acid sequences of PBP1a,-2b,and -2x were aligned and compared with those of the respective reference SP R6 strain.Results According to the CLSI criteria,8 isolates(13.5%) were classified as PSSP, 46 isolates(78.0%) as PISP and 5 isolates(8.5%) as PRSP.The PBP1a gene was not successfully amplified from two strains(sp1,sp62) with the set of R6 primers.The most prominent active-site mutations,which have also been associated previously with resistance,were Thr371→Ala/Ser in PBP1a,Glu481→Gly followed by Thr451→Ala in PBP2b,and Thr338→Ala in PBP2x.Meanwhile,some fairly uncommon mutations were also detected in our research,such as Ser337→Thr,Gln552→Glu(which increase the MIC of cephalosporin by three folds) in PBP2x,Gly472→Leu,Ser474→Asn,Gly488→Ala in PBP2b,and Ser351→Ala,Ile358→Thr in PBP1a.Some mutations are closely associated with the MICs ofβ-lactam antibiotics.26 strains(45.6%) harboring the mutaions of Thr338→Ala,Met339→Phe,Met400→Thr and Arg384→Gly in PBP2x,whose MICs of cephalosporin increase dramatically,with each≥5.0μg/ml.The affiliated mutations of Ala624→Gly and 570QLQPT→AIDTK of PBP2b in 29 strains(49.2%) increase the average MICs of cephalosporin by three folds,and the average MIC of PG reach to 1.0μg/ml.The mutation phenotype of PBP1a was very simple,with substitutions of Thr371→Ala/Ser,Pro432→Thr and 574TSQF577→NTGY around three conserved motif, theβ-lactam antibiotics' MICs of which can be raised up by combination of Ser351→Ala and Ile358→Thr in PBP1a,reach to intermediate or resistant level.Parts of strains have the same amino acid substitutions with different MICs ofβ-lactams.Conclusions(1) The mutations of PBP1a,-2b and -2x are associated with antibiotics resistance of S.pneumoniae to theβ-lactams.(2)With the augment of the mutations around conserved motif,the MICs ofβ-lactams increased.(3) There are differences on the active-site mutations in PBPs between clinical SP and laboratory strains.(4)The differentiation among parts of strains couldn't be explained by the amino acid substitutions of PBP1a,-2b and -2x completely,other antibiotic-resistant mechanisms such as mutations of PBP1b,-2a or non-PBPs may works.