Streptococcus pneumoniae (S. pneumoniae) is an important pathogenic bacterium in children with respiratory tract infections. Infants and young children are likely the primary reservoirs of this agent, with cross-sectional point rates of nasopharyngeal carriage ranging from 27% in developed to 85% in developing countries.1 In a global study of the epidemiology and etiology of childhood pneumonia in 2010, S. pneumoniae was found in 18.2% of severe cases and in 32.7% of the subgroup of the children who eventually died of the disease.2 The development and spread of resistance to commonly used antibiotics, such as penicillin, macrolide, cephalosporin and co-trimoxazole, in these bacteria have already been a global problem that underlines the urgent need for vaccines that can effectively control pneumococcal diseases. The implementation of pneumococcal conjugate vaccines (PCVs) into national immunization programs has significantly reduced the carriage rates and incidence of this bacteria; moreover, it restricts the circulation of drug-resistant strains.1,3–7
Seven-valent PCV (PCV7) was licensed in China in 2008, but it has not been introduced into the national program of immunization. The immunization rates of this vaccine are low, only 10% children had received PCV7 in 2011,8 primarily because of its expense (860 RMB/dose, approximate 138.5 $/dose). A previous study on outpatient children younger than 5 years with respiratory tract infections showed that the license of PCV7 did not prevent the spread of vaccine serotypes in China because of the low immunization rate.9 Many studies have confirmed that some pneumococcal serotypes, especially vaccine serotypes, were related to more severe invasive pneumococcal infections (ie, serotypes 19A and 19F).5,6 The pathogenetic conditions of inpatients are severe. The distribution of serotypes and the sequence types (STs) of S. pneumoniae isolated from inpatients were investigated for potential changes after the license of PCV7.
This study collected clinical S. pneumoniae isolates, including both invasive and noninvasive, from inpatients younger than 14 years from March 2013 to February 2014. The serotype distribution, coverage rates of PCVs, antimicrobial resistances and multilocus sequence typing (MLST) were evaluated.
MATERIALS AND METHODS
This study cohort was composed of children younger than 14 years at Beijing Children’s Hospital from March 2013 to February 2014. The patients were children admitted to the Infectious Diseases Department, the Respiratory Diseases Department or the Intensive Care Unit. This hospital is a National Children’s Medical Center, and it has more than 3 million outpatient visit and 70,000 hospitalized every year.
Only 1 isolate was included in this study when 2 or more isolates were cultured from 1 patient. The invasive isolates were defined as S. pneumoniae strains isolated from normal sterile specimens, such as blood, cerebrospinal fluid and pleural effusion. The noninvasive isolates were defined as S. pneumoniae strains isolated from nonsterile specimens, such as hypopharyngeal aspirates, bronchoalveolar lavage and nasopharyngeal swabs. All the isolates were cultured in the clinical laboratory following a similar procedure as in the following citation. The isolates were transported to the Microbial and Immunology Laboratory for serotyping, antimicrobial susceptibility tests and MLST.
All isolates were identified based on their typical colony morphology, Gram staining, an optochin sensitivity test (Oxoid Company, Britain, United Kingdom) and an Omni serum assay (Statens Serum Institute, Copenhagen, Denmark). All isolates were stored at −80°C in freezing tubes for further study.
The serogroups were tested using the Quellung reaction with Pneumotest kits, and the serotypes were tested with factor antisera (Statens Serum Institute, Copenhagen, Denmark). The interpretation of the serotyping depended on the capsular swelling under phase-contrast microscopy with an oil immersion lens (magnification, 100×), as described in the literature.10 The coverage rates of PCV7, PCV10 and PCV13 were estimated by calculating the percentage of isolates that expressed the serotypes included in the vaccines.
Antimicrobial Susceptibility Testing
For all isolates, the minimum inhibitory concentrations (MICs) were determined against penicillin, erythromycin, amoxicillin–clavulanic acid, cefaclor, cefuroxime, ceftriaxone, levofloxacin, linezolid, vancomycin and imipenem using E-test strips (BIOMERIEUX, France), and the antimicrobial susceptibilities to chloramphenicol, tetracycline and trimethoprim–sulfamethoxazole were determined using the Kirby–Bauer disk diffusion (OXOID Company). The isolates were cultured on trypticase soy agar (Oxoid Company) plates supplemented with 5% sheep blood at 35°C under a 5% CO2 atmosphere for 20–24 hours. Suspend the grown bacteria in normal saline and adjust the suspension to a 0.5 McFarland standard. Sterile cotton swabs were dipped into the bacterial suspension and were uniformly spread onto Mueller–Hinton agar (Oxoid Company) plates supplemented with 5% sheep blood. The plates were then incubated at 35°C under a 5% CO2 atmosphere for 20–24 hours. The 2012 criteria of the Clinical and Laboratory Standards Institute for MICs were applied to classify isolates as susceptible, intermediate or resistant.11 The S. pneumoniae American Type Culture Collection 49619 (ATCC 49619) was used as the quality-control strain and included in each set of tests to ensure the accuracy of the results. Multidrug resistant S. pneumoniae (MDRSP) were defined as a resistance to 3 or more classes of antibiotics tested in this study.
Multilocus Sequence Typing
The housekeeping genes aroE, gdh, gki, recP, spi, xpt and ddl were amplified via polymerase chain reaction.12 The sequences of each of the 7 loci were compared with those of all known alleles at the loci, as well as with the STs in the database of the pneumococcal MLST website (http://pubmlst.org/spneumoniae/). New allelic numbers or new ST numbers were assigned by the curator of the pneumococcal MLST website. The eBURST v3 software (http://eburst.mlst.net/) was used to investigate the relationships between the isolates and assign a clonal complex (CC) based on the stringent group definition of 6 of 7 shared alleles. STs that shared 6 identical alleles of the 7 MLST loci with another ST in the group were subdivided into 1 group as a CC.
The parent(s) or legal guardian(s) of each participant signed a written informed consent document before enrolment and before any procedure was performed. This study was reviewed and approved by the Ethics Committee of Beijing Children’s Hospital Affiliated to Capital Medical University. Ethical problems were not encountered in this study.
The serotype distribution, coverage rates of PCVs, antimicrobial resistance and MLSTs were analyzed. The distribution of the international antibiotic STs identified by the Pneumococcal Molecular Epidemiology Network was also analyzed.13 All the data were analyzed by WHONET 5.6 as recommended by the World Health Organization. The χ2 test and Fisher exact test for accurate probabilities were used for comparing proportions via SPSS 17.0 (SPSS Inc., Cary, NC). A 2-tailed cut-off of P <0.05 was considered as a significant difference, but for the comparison on coverage rates of PCV7 and PCV13, a 1-tailed cut-off of P <0.05 was used.
Throughout the study period, 187 S. pneumoniae isolates, including 21 invasive pneumococcus specimens and 166 noninvasive pneumococcus specimens, were collected from inpatients. These isolates were cultured from hypopharyngeal aspirates (n = 107), bronchoalveolar lavage (n = 56), nasopharyngeal swabs (n = 1), ear discharge (n = 2), cerebrospinal fluid (n = 3), pleural effusion (n = 4) and blood (n = 14). Three age groups were divided: younger than 2 years (n = 119), 2–5 years (n = 34), and older than 5 years (n = 34).
Serotype Distribution and Vaccine Coverage
The serotype distribution and PCVs coverage rates of the 187 S. pneumoniae isolates are shown in Table 1. A total of 21 serotypes were identified. The most common serotypes were 19F (31.5%), 19A (19.8%), 23F (11.2%), 6A (9.1%), 14 (9.1%) and 15B (5.9%), accounting for 86.6% of all isolates. The most common serotype in each age group was the serotype 19F. Serotype 19A was more common in 0-to-2-year-old children (23.5%, 28/119), and serotype 14 was more frequent in 2-to-5-year-old children (20%, 7/34). The total coverage rates of PCV7, PCV10 and PCV13 were 56.2% (105/187), 56.7% (106/187) and 86.1% (161/187), respectively. The coverage rate of PCV7 was higher in 2-to-5-year-old children than in other age groups; however, this difference was not significant (χ2 = 1.58; P = 0.21). The coverage rates of PCV13 in the 3 age groups did not significantly different from each other (χ2 = 1.87; P = 0.39). The coverage rates of PCVs in invasive pneumococci were higher than those in noninvasive pneumococci, although this difference was not significant (PCV7: χ2 = 0.32, P = 0.57; PCV10: χ2 = 0.86, P = 0.35; PCV13: P = 0.32).
Antimicrobial Susceptibility Testing
The susceptibility and MICs of the 187 S. pneumoniae isolates to 13 antibiotics are shown in Table 2. The total nonsusceptibility (intermediate and resistant) rates of penicillin were 8.0%, 91.5% and 91.4%, as defined by the nonmeningitis parenteral breakpoint [intermediate (4 mg/L), resistant (≥8 mg/L)], oral nonmeningitis breakpoint [intermediate (0.12–1 mg/L), resistant (≥2 mg/L)] and parenteral meningitis breakpoint [susceptible (≤0.06 mg/L), resistant (≥0.12 mg/L)]. The nonsusceptibility rates to cefuroxime, cefaclor, tetracycline and trimethoprim–sulfamethoxazole were also high. All but one of the isolates were highly resistant to erythromycin. Measured with regard to the nonmeningitis parenteral breakpoint, the rate of penicillin nonsusceptibility was significantly higher in invasive pneumococcus strains (χ2 = 13.54; P = 0.00); however, measured with regard to the oral nonmeningitis and parenteral meningitis breakpoints, no between-group difference was observed. The nonsusceptibility rates of the other antibiotic agents were slightly (but not significantly) higher in invasive pneumococci.
The antibiotic resistance patterns of the pneumococcal isolates are shown in Table 3. Approximately 93.5% (174/187) of the isolates were MDRSP. The most prevalent antibiotic resistance pattern was macrolides/β-lactams/tetracyclines/sulfonamides.
Multilocus Sequence Typing
The MLSTs of the 187 S. pneumoniae strains according to the eBURST analyses are shown in Figure 1. A total of 58 STs were detected in the 187 S. pneumoniae isolates, in which 16 were newly assigned via the MLST database: 9765 and 9779 to 9792. The 5 most predominant STs for all pneumococci were ST271 (24.1%, 45/187), ST320 (18.2%, 34/187), ST81 (7.5%, 14/187), ST876 (7%, 13/187) and ST3397 (5.3%, 10/187), which were primarily related to serotypes 19F, 19A, 23F, 14 and 15B, respectively. The eBURST analysis showed 8 CCs and 30 singletons. CC271 was the most frequent CC, with a proportion of 49.2% (92/187), followed by CC81 (8.6%, 16/187), CC876 (7%, 13/187) and CC3397 (11, 5.9%). In addition, CC271 was the most prevalent CC in both invasive isolates (57.1%, 12/21) and noninvasive isolates (48.2%, 80/166).
The relationships among MLSTs and antibiotic nonsusceptibility were analyzed. Based on the nonmeningitis parenteral breakpoint [susceptible (≤2 mg/L), intermediate (4 mg/L), resistant (≥8 mg/L)], all the ST81 and ST3397 pneumococci were susceptible to penicillin. Based on the oral breakpoint [susceptible (≤0.06), intermediate (0.12–1 mg/L), resistant (≥2 mg/L)], ST271, ST320, ST81, ST876 and ST3397 pneumococci were not susceptible to penicillin. ST271, ST320, ST81 and ST3397 pneumococci were nonsusceptible to cefuroxime and cefaclor. The nonsusceptibilities of ST271, ST320 and ST81 to imipenem were clearly higher than those of the other STs (P < 0.05). All but one of the ST271 pneumococci were resistant to erythromycin. The nonsusceptibility rates of tetracycline and trimethoprim–sulfamethoxazole were high for all STs except ST876.
This study described the serotype distribution, antimicrobial resistance and molecular characteristics of S. pneumoniae isolated from hospitalized children in Beijing. This study only identified 21 (11.2%) invasive pneumococcal strains; however, this number was higher than a previous study, which collected 171 invasive pneumococcal isolates from 11 hospitals during 2006–2008 (only 5.2 isolates from each hospital every year on average).14
The monitoring of pneumococcal serotype distributions is an important epidemiological investigation that can provide the reliable baseline information required for S. pneumoniae vaccine preparation and government immunization strategies. The present data showed that the serotypes 19F, 19A, 23F, 6A, 14 and 15B were the most common types in inpatient isolates. Types 6A and 15B were not found as frequently as in previous inpatient studies.14–16 Compared with research from the 1980s, serotypes 5, 6, 1, 19, 2, 14 and 3 were more common,17 the serotype distribution showed a difference. Serotypes 19F and 19A were most common, which have relationships with severe pneumococcal diseases,5,6 that might be because of their high-level antibiotic resistance. In a previous study of serotype 19F regarding S. pneumoniae, the nonsusceptibility rates to cefaclor and cefuroxime increased significantly from 14.2% in 1997–1998 to more than 80% in 2010.18 A recent study in the US also found that serotype 19A was the major MDRSP (38.5%).19
The coverage rates of PCV7, PCV10 and PCV13 were 56.2%, 56.7% and 86.1%, respectively, and these rates are slightly higher than those reported in research from 2006–2008 conducted before the use of PCV7 among the inpatients of the same hospital (ie, 46.0%, 47.6% and 74.6%).15 PCV7 was made available in China in the private sector in 2008. The immunization rates of this vaccine are low primarily because of its expense (860 RMB/dose, approximate 138.5 $/dose), the low immunization rate limited the effectiveness of PCV7. The coverage rate of PCV13 is significantly higher than that of PCV7, suggesting that PCV13 prevents pneumococcal diseases effectively in Beijing. PCVs were prepared to prevent invasive pneumococcal diseases or severe pneumococcal diseases, and if these vaccines (especially PCV13) were universally used in Chinese immunizations, then the effect on the rates of vaccine serotypes would be obvious.
There was a high-level antibiotic resistance in this study. Because our epidemiological survey of noninvasive and carriage pneumococcal isolates sought to evaluate the characteristics of clinical infectious isolates, the susceptibility rates to penicillin of the isolates were analyzed according to different interpretation breakpoints. The penicillin nonsusceptibility rate was 8% based on the nonmeningitis parenteral breakpoint. However, this rate increased to 91.5% when based on the oral penicillin breakpoint, which is significantly higher than developed countries (24.4% in Europe,20 38.9–42.7% in the US21). The nonsusceptibility rates to penicillin are increasing, and this finding coincides with the global trend.22–26
Recently, the nonsusceptibility rates of S. pneumoniae to other β-lactam antibiotics have also increased, which is associated with the increasing use of cephalosporins at tertiary hospitals.27 In this study, the rates of nonsusceptibility to cefuroxime and cefaclor were 89.9% and 90.3%, respectively, which were higher than those reported in our previous study (ie, 79.3% and 81.6).16 The nonsusceptibility rates to tetracycline and trimethoprim–sulfamethoxazole were also high. This high-level antibiotic resistance is most likely because of the excessive use of antibiotics in general clinical practice.27 Pneumococcal immunization has decreased the number of antimicrobial-resistant infections.19,20 Nevertheless, some of the PCV7-specific and PCV13-specific serotypes exhibited antimicrobial resistance or multidrug resistance. Therefore, the judicious use of antimicrobials remains pivotal in curtailing the emergence and spread of antimicrobial resistance within pneumococcal strains.
Presently, more than 10,000 STs are listed in the MLST database, which emphasizes the variety and complexity of pneumococci at the gene level. The 5 predominant STs for all pneumococci were ST271, ST320, ST81, ST876 and ST3397, which match the results of a previous study.16 In West Africa, the relatively common STs among invasive strains are ST4012, ST3404, ST289 and ST618.28 ST156, ST180 and ST218 are common in Brazil.29 Together, those research indicates that the pneumococcal ST distribution is disparate across different countries and regions. Besides, PCVs might limit the spread of vaccine-related STs. Research from England and Wales found that STs commonly associated with PCV7 declined after PCV7 implementation. Of the PCV7 STs that cause meningitis, the 5 most common clones in decreasing order were ST162, ST9, ST113, ST191 and ST124.30 In this study, CC271 was the most prevalent CC, with a proportion of 49.2%, indicating that CC spread was the major cause of pneumococcal infection in hospitalized children. It also emphasizes the notion that universal immunization is important for preventing pneumococcal diseases.
The analysis of the relationships between STs and antibiotic resistance indicate that ST271, ST320, ST81 and ST3397 were the predominant resistant STs. These STs might lead to the high-level antibiotic resistant of pneumococci. In this study, CC271 was the most frequent CC, including 9 STs such as ST271, ST320, ST236 and others. The serotypes of stains included in CC271 were serotype 19A and 19F, except for 1 strain of serotype 15C. ST320 was associated with international antibiotic-resistant strains Taiwan19F-14 (defined by the Pneumococcal Epidemiology Network).12 That is, CC271 was the main resistant clone in this study. This finding matched with the previous studies.16,27 Many studies confirmed that CC271 was associated with multidrug resistance and carriage of the ermB and mefA genes, contributed to the increased prevalence of antimicrobial resistance.31–33 ST81 (Spain23F-1, 8.6%, 16/187) and ST90 (Spain6B-2, 1.0%, 2/187), which were also listed as internationally spread resistant STs by Pneumococcal Epidemiology Network, were found in this study at lower frequencies. The above results indicated that the prevalence of antibiotic-resistant CCs might be the major reason for the spread of resistant S. pneumoniae.
Notably, the differences between the invasive pneumococcus strains and the noninvasive pneumococcus strains in PCV coverage, antibiotic resistance and MLSTs were not significant. This finding might indicate that the monitoring of noninvasive pneumococci in routine hospital work is important given that the isolated rates of invasive pneumococci were low.
This study has several limitations. It was a single-center research study, and thus, it might not represent the overall epidemic trends in China. Future longitudinal and multicenter surveillance of S. pneumoniae serotype distributions, antimicrobial susceptibility and molecular epidemiology is needed to confirm the present results and evaluate the influence of universal immunization. The number of invasive isolates was limited in this study. This number might increase when antibiotic prescriptions are well controlled, and the inspection rate of the invasive samples improves in the future.
The National Natural Science Foundation of China (Grant No. 81371853), the Capital Health Research special development (Grant No. 2014-2-1141) and the National Clinical Centre Project of the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2013BAI09B11) financially supported this study. We thank the Infectious Diseases Department, the Respiratory Diseases Department, the Intensive Care Unit and the Clinical Laboratory of Beijing Children’s Hospital for their contribution to this study.
Authors’ Contribution: All of the authors had access to the full dataset (including the statistical reports and tables) and take responsibility for the integrity of the data and the accuracy of the data analysis. Y.K.H., Y.Y.H., L.S., X.B.P., D.F., L.G., W.Q. and S.K.L. conceived and designed the study. L.S., X.B.P., D.F., L.G., W.Q., S.W., T.J.J. and Y.Y. collected the data and designed the analysis. Y.K.H., Y.Y.H. and L.S. interpreted the data. Y.K.H. and L.S. wrote the first draft of the paper. Y.K.H. and Y.Y.H. reviewed and approved the final report.
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