| Objective:To evaluate the performance of gene-,protein-and polysaccharide-based techniques in Streptococcus pneumoniae(the pneumococcus)identification.To investigate the serotype and molecular epidemiology in invasive S.pneumoniae isolates in China.To analyze the in vitro antimicrobial susceptibility and variations of penicillin binding proteins of S.pneumoniae.To compare the virulence of the major serotypes of invasive S.pneumoniae isolates in China.Methods:A total of 181 viridans group streptococci(VGS)from Peking Union Medical College Hospital and 300 invasive S.pneumoniae isolates from 27 teaching hospitals in China were collected during 2010-2015.Using 16S rRNA gene and gyrB gene sequencing as a gold standard,the performance of two MALDI-TOF MS instruments in the identification of 181 VGS clinical isolates was studied.A range of pneumococcal strains with different capsular types were studied for DBA/HPA binding with flow cytometry and super resolution fluorescence microscopy.Genetic experiments constructing gene knock-outs and knock-ins using Janus Cassette and pPEP1 plasmid were used to study the role of phosphorylcholine esterase(Pce)in DBA/HPA binding.Serotypes and sequence types(STs)were determined by Quellung reaction and multilocus sequence typing(MLST)of the 300 invasive S.pneumoniae isolates.The multiplexed PCR and cpsB gene-based sequencing were evaluated in serotyping.Standard broth microdilution was performed to investigate the in vitro activity of 20 drugs against invasive S.pneumoniae isolates.Amino acid variations in penicillin binding proteins(PBP)1a,2b and 2x were analyzed to study the relationship of penicillin resistance phenotype and genotype.In vivo sepsis model constructed in mice was used to evaluate the virulence of major prevalent serotypes.The expression of six non-capsule related virulence genes ply,lytA,nanA,psaA,pspA and HylA were determined by real-time quantitative PCR.Results:The Bruker Biotyper system had a tendency to misidentify S.mitis/S.oralis/S.pseudopneumoniae as S.pneumoniae while the Vitek MS IVD demonstrated a better resolution for pneumococci and non-pneumococci despite the inability to distinguish between S.mitis/S,oralis.Based on MALDI-TOF analysis and selected 16S rRNA gene plus gyrB genes sequencing,we designed a practical VGS identification algorithm.DBA and HPA bind only to the teichoic acid of pneumococcal isolates with a functional Pce enzyme.A newly-discovered Pce variant(allele B)is seen only in serotype 11A and 11 E strains functional in vivo but nonfunctional when assayed in vitro.Bloodstream infection especially children under 5 years old predominated in all invasive S.pneumoniae infections,which accounted for around 50%of isolates.Forty serotypes and one non-typeable strain were identified in 300 isolates,among which the coverage of PCV7,PCV10,PCV13 and PPV23 were 42.3%,45.3%,73.3%and 79.3%,respectively.The major prevalent serotypes detected were 23F,19A,19F,3,14,6A and 6B.In terms of accuracy and simplicity,the cpsB gene sequencing-based serotyping had an advantage over the multiplexed PCR method and is thus suitable for most laboratories.MLST analysis identified 123 STs among which 33 STs were discoveredin this study.ST320,ST81,ST271 and ST876 were the most common STs.Major clonal complexes(CC)correlated well with serotypes,CC320→19A,CC271→19F,CC81/CC880→23F,CC876→14,CC3173→ 6A and CC505→3.All the isolates were susceptible to ertapenem,levofloxacin,moxifloxacin,linezolid and vancomycin but had a resistance rate higher than 90%to azithromycin,clarithromycin,erythromycin,clindamycin,chloramphenicol and tetracycline.A decreasing trend of resistance rates was seen in all the antimicrobial agents except for imipenem during 2010-2015.Serotype 23F,19A and 19F were the major resistant serotypes and ST271,ST320 and ST81 were the major resistant STs while serotype 3,ST180 and ST505 were susceptible to most antimicrobials.Strains within the vaccine coverage showed a higher resistance rate to most of the antimicrobials than those beyond the vaccine coverage.However,an increasing resistance trend was seen in strains beyond the vaccine coverage against some of the antimicrobials from 2010-2015.Strains isolated from cerebrospinal fluid had a high resistance rate to penicillin while no penicillin-resistant strains were found in strains from non-cerebrospinal fluid specimens.Variations in PBPs were highly diverse and was associated with minimum inhibitory concentrations(MIC)of penicillin instead of specimen types and breakpoints.Strains with penicillin MIC<0.25 μg/ml had no mutations in PBP active sites with one exception,in which a T451A mutation was found in the active site of PBP 2b in a serotype 6A strain.Strains with penicillin MIC≥0.5 μg/ml had different mutations in PBP active sites.Mutations in PBP1 a active sites were seen in all strains with higher penicillin MICs(MIC>1 μg/ml),accompanied by PBP2b and PBP2x mutations.It’s worth noting that strains isolated from non-cerebrospinal fluid specimens had different mutations in PBP active sites even through they were considered as penicillin-susceptible S.pneumoniae(PSSP).A total of 105 point mutations in PBP 1a,111 point mutations and two insertion mutations in PBP2b and 98 point mutations in PBP2x,among which the two insertion mutations YIW and YTW were reported in two PSSP isolated from blood for the first time.In vivo mice experiment revealed that serotype 3 was highly virulent in sepsis model while serotype 23F,19A,19F and 14 were unable to cause sepsis in healthy mice.There was a difference in the virulence among different strains of serogroup 6,even in the same serotype.There was no obvious correlation between the expression of ply,lytA,nanA,psaA,pspA and HylA in strains of serogroup 6 with different virulence levels as determined by the animal experiment.Conclusion:The Vitek MS IVD system had a better accuracy in SP identification than Bruker Biotyper.DBA and HPA can bind to pneumococcal isolates with different serotypes and thus had a potential in rapid identification of SP.Pce played a critical role in DBA/HPA binding to pneumococci and the newly discovered pce allele B was only found in serotype 11A and 1 1E strain,which can be used for serotype prediction.The prevalent serotypes and STs dominating in invasive S.pneumoniae isolates in China were serotype 23F,19A,19F,3,14,6A and 6B and ST320,ST81,ST271 and ST876.Despite a low vaccination rate,capsular switch phenomenon was observed in our study,possibly due to the antibiotic selection pressure.Mutation in PBP1a active sites was indispensable in strains with higher penicillin MICs(MIC>1 μg/ml),accompanied by PBP2b and PBP2x mutations.Among the prevalent serotypes,serotype 3 is of high virulence but low resistance while serotypes 23F,19A,19F,3 and 14 were avirulent but highly resistant.There was a variation in the virulence levels among different strains within serotype 6A,6B and 6C.Our study on one hand has provided an important referential basis for routine laboratories to improve SP diagnostic ability,to learn about the serotype and ST distribution,antimicrobial resistance and virulence status of major serotypes of invasive S.pneumoniae in China;on the other hand,it has great significance in guiding the use or development of vaccines in China with the epidemiological data basis. |
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