Study On Resistance And Molecular Characteristics Of Hospital-associated Streptococcus Pneumoniae From Children Under 5 Years Old In Liuzhou

ObjectiveTo understand antibiotic resistance of hospital-associated Streptococcus pneumoniae?S.pneumoniae?isolated from children under 5 years old and drug-resistant spectrum,and grasp the serotype of S.pneumoniae,MLST?Multilocus sequence typing?analysis and the virulence genes,and further explore the resistance phenotypes,resistant and viral genes characteristics among different molecular typing isolates,which may provide important data support for pneumococcal vaccine related clinical research,and also provide evidence for prevention and control of S.pneumoniae related diseases.MethodsThis study was a hospital-based retrospective study.The clinical samples were collected from children under 5 years old in Liuzhou,China.S.pneumoniae was identified and confirmed by traditional biochemical tests.All S.pneumoniae isolates were tested for antimicrobial susceptibility by E-test method,molecular characteristics including resistance genes,virulence genes?capsule structure and pilus structure virulence genes?and serotypes by PCR method.Multilocus Sequence Typing?MLST?was used to identify the Sequence Type?ST?of the strains.The Clonal Complex?CC?was identified by eBURST analysis.Categorical variables were compared by Pearson chi-squared??²?test or Fisher exact test.Minimum spanning tree and correspondence analysis were used to reveal the potential correlation between serotypes and STs.Multifactor Dimensionality Reduction?MDR?was applied to explore the differences and interactions of molecular characteristics between pneumococcal conjugate vaccines?PCVs?covering serotypes strains and without non-PCV covering serotypes strains.Results1.Basic characteristics of the study subjects:A total of 537 children with S.pneumoniae infection were included.The ages of 537 children ranged from 0 to 60 months,with median age(P₂₅-P₇₅)as 12.0?7.2²7.0?months.Of the 537 children,527?98.1%?were diagnosed as a non-invasive disease?including 419 pneumonia,48 bronchitis,37otitis media and 23 upper respiratory infections?and 10?1.9%?were diagnosed as an invasive disease?including eight bacteremia and two pleural inflammation?.2.Antibiotic susceptibility:All SP isolates were susceptible to vancomycin in this study.Most of the S.pneumoniae isolates were susceptible to penicillin G?98.3%?or levofloxacin?98.7%?,but resistant to clindamycin?94.4%?,erythromycin?93.8%?,tetracycline?86.4%?,trimethoprim-sulfamethoxazole?SXT??80.3%?and azithromycin?77.5%?.83.8%isolates were co-resistant to erythromycin and tetracycline.erm?B??92.6%?and mef?A/E??56.4%?were the primary erythromycin-resistant genes and there were 54.0%isolates carried two erythromycin-resistant genes simultaneously.tet?M??95.5%?and tet?O??2.8%?were the primary tetracycline-resistant genes found among isolates.Notably,there were 489?91.1%?isolates co-carrying tet?M?and erm?B?,299?55.7%?isolates co-carrying tet?M?and mef?A/E?,and 286?53.3%?isolates co-carrying tet?M?,erm?B?and mef?A/E?.3.Molecular characterization:Among the 537 pneumoniae isolates,514 isolates?95.7%?were properly serotyped,and a total of 16 serotypes were identified in this study.The predominant serotype was 19F,followed by serotypes 6,19A,23F,14.The overall coverage rate for PCV7 was73.7%,for PCV10 was 74.3%,and for PCV13 was 85.8%.There were significant differences in PCV13?P=0.002?and PCV10?P=0.045?among three age groups,but no significant differences between pneumonia and non-pneumonia groups?P>0.05?.The predominant ST was ST271?33.0%?,followed by ST320?11.5%?,ST81?4.3%?,ST3173?4.3%?,and ST876?3.4%?.As to virulence genes,the dominant virulence genes were lytA?95.7%?,ply?91.8%?and pspA?67.0%?,followed by the pilus genes?rlrA:34.1%;sipA:44.9%?.4.Analysis of resistance and toxin characteristics of different molecular types of streptococcus pneumonia:The resistance and virulence characteristics was significantly different between different serotype streptococcus pneumoniae,showing that serotype 19type isolates?including 19Fand 19A?are easier to resistant to cefotaxime,tetracycline and trimethoprim-sulfamethoxazole,and mainly carrys mef?A/E?,rlrA and sipA genes.The results of MDR analysis showed that the sipA rate of the serotype strain covered by PCVs was significantly higher than that of the strains covered with non-PCVs?for PCV7:OR=2.39,95%CI=1.55-3.70;for PCV10:OR=1.87,95%CI:1.22-2.86;for PCV13:OR=18.33,95%CI=6.56-51.25?.There are also some significant differences of resistance and virulence characteristics between different type ST isolates,mainly showing that ST271 and ST320strains are easier to resistant to cefotaxime,tetracycline and SXT,and main carry mef?A/E?,rlrA and sipA genes.5.Relationship between STs and serotypes:ST271/19F?32.6%?and ST320/19A?7.3%?were the predominant isolates in this study and we found there were relations between serotypes and STs which could be proved by correspondence analysis?c2=2761.13,P<0.001?,showing that serotype 6A/B was associated with ST386,ST90,ST902,ST3173,ST1661 and ST9789;serotype 23F was associated with ST872,ST81,ST242 and ST1437,and serotype 14 was associated with ST876.6.The main phenotypic and molecular characteristics of STs/serotype strains analysis:There were statistically significant associations between STs/serotypes and resistance to cefotaxime?P<0.001?,tetracycline?P<0.001?,SXT?P<0.001?.Although no significant relations were observed for phenotype resistance to erythromycin,there was a significant association between STs/serotypes and mef?A/E??P<0.001?.As to virulence genes,there were significant associations between STs/serotypes and rlrA?P<0.001?or sipA?P<0.001?.These results revealed that ST271/19F and ST320/19A were associated with carrying rlrA and sipA genes.Conclusion1.Streptococcus pneumoniae strains are mainly resistant to macrolides and clindamycin.92.2%of streptococcus pneumoniae isolates are multiple drug resistant strains,and the main resistance pattern is simultaneously nonsusceptible to macrolides,erythromycin,clindamycin,tetracycline and SXT.The main resistance genes is erm?B?,mef?A/E?and tet?M?.These results reflected that the resistance of hospital-associated streptococcus pneumoniae isolated from children in Liuzhou of China is serious,and long-term dynamic monitoring is needed for the resistance level of streptococcus pneumoniae.2.The main serotypes of streptococcus pneumoniae isolates are 19F,6B,19A,23F and 14.The serotype coverage rate of PCV13 was high in 85.8%,indicating that Liuzhou needs to introduce and apply PCV13 vaccine in children.3.MDR analysis results indicated that the sipA gene was associated with the PCVs serotypes,which provided a new idea for the new vaccine developed by bacterial pilues structure combined with PVC serotypes under the confirmation by large-scope researches.4.This study finds that there are correlations between the serotypes and ST types of streptococcus pneumoniae,and the main popular clones were ST271/19F and ST320/19A,and there were significant differences of resistance and virulence characteristics among different types of strains.This study found a correlation between the serotype and ST of streptococcus pneumoniae,and the main popular clones were ST271/19F and ST320/19A.This study also finds that ST271/19F and ST320/19A S.pneumoniae clones with resistance to cefotaxime,tetracycline and SXT are mainly carrying of mef?A/E?,rlrA and sipA genes,suggesting the value of monitoring multiple characteristics to detect new epidemiologic trends and provide implications for the formulation of multivalent pneumococcal vaccines.