Serotype distribution and antibiotic resistance of Streptococcus pneumoniae isolates from 17 Chinese cities from 2011 to 2016

This study was conducted at the Peking University People’s Hospital, a teaching hospital located in Beijing, China. A total of 881 S. pneumoniae isolates were collected from pediatric and adult patients with pneumococcal infections in 17 cities across China during the years 2011, 2012, 2013, and 2016. The study protocols were approved by Ethics Committee of the Pecking University People’s Hospital and all participants provided written informed consent prior to study commencement. For participants younger than 18 years of age, written informed consent was obtained from each participant’s parents or legal guardian. One isolate was collected from each patient. Duplicate isolates and patients colonized by bacteria but without any clinical evidence of infection were excluded. Isolates were obtained from sputum, blood, broncho-alveolar lavage fluid (BALF), cerebrospinal fluid (CSF), pharyngeal swabs, nasal swabs, and middle ear fluid. Sputum samples from children < 2 years old were collected by nasotracheal aspiration. S. pneumoniae isolates from sputums were included if they met the following criteria: less than 10 squamous epithelial cells and more than 25 leukocytes per low power field. Isolates were transported to Peking University People’s Hospital for antibiotic susceptibility testing and serotyping annually. Isolates were identified based on typical colony morphology, Gram staining, optochin sensitivity tests (Oxoid, Hampshire, UK), and Omni serum assays (Statens Serum Institut, Copenhagen, Denmark).

The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of the 881 S. pneumoniae isolates against 15 antibiotics (penicillin, amoxicillin/clavulanic acid, ceftriaxone, cefuroxime, cefaclor, vancomycin, erythromycin, azithromycin, clarithromycin, tetracycline, levofloxacin, moxifloxacin, trimethoprim/sulfamethoxazole, chloramphenicol and clindamycin) in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [8]. The CLSI 2013 criteria for MICs were applied to classify isolates as susceptible, intermediate, or resistant. The oral penicillin breakpoint was used to classify isolates as penicillin-susceptible (MIC ≤ 0.06 μg/ml), penicillin-intermediate (MIC between 0.12 and 1 μg/ml), or penicillin-resistant (MIC ≥ 2 μg/ml). For ceftriaxone, the non-meningitis breakpoint was used to classify isolates as susceptible (MIC ≤ 1 μg/ml) or resistant (MIC ≥ 4 μg/ml) [9]. S. pneumoniae ATCC 49619 was used as the quality control strain and was included in each set of tests to ensure accurate results. MICs were calculated as the MIC₅₀ and MIC₉₀ (MICs that inhibit 50% and 90% of the isolates, respectively).

Pneumococcal serotypes/groups were determined for the 881 isolates with Pneumotest-Latex kit (Statens Serum Institut, Copenhagen, Denmark) and type-specific antisera (Statens Serum Institut, Copenhagen, Denmark). The Pneumotest-Latex kit consisted of the 14 latex reagents pools A-I and P-Q. By testing all 14 pools and using the chessboard identification system, it was possible to identify the 23 vaccine serotypes to type/group level. Traditional quellung reaction with type-specific antisera was used for full serotyping of serogroup 19, 6 and 23. Isolates that reacted with the Pneumotest-Latex kit but did not belonged to serotypes or groups included in the PPV23 were classified as Non-vaccine types (NVT). The potential PCV7 and PCV13 vaccine coverage was estimated by calculating the percentage of isolates that expressed the serotypes included in the vaccines and related serotypes (7 for 7F, 9 for 9 V, and 18 for 18C).

Multilocus sequence typing (MLST) analysis was performed according to the S. pneumoniae MLST protocol [10]. Internal fragments of approximately 550–600 bp from the aroE, gdh, gki, recP, spi, xpt, and ddl genes were amplified by polymerase chain reaction using primers described previously [11]. Alleles and sequence types (STs) were assigned using software available at the Streptococcus pneumoniae MLST Database web page (http://​pubmlst.​org/​spneumoniae). Analysis of STs and assignment to clonal complexes were performed using all STs found in the online database using the eBURST program. STs were grouped into clonal complexes by their similarity to a central allelic profile. Visualization of phylogenetic results was performed using the PHYLOViZ online tool (http://​www.​phyloviz.​net/).

Data from the antibiotic susceptibility tests were analyzed using WHONET 5.6 software, Windows-based database software developed by the World Health Organization for the management and analysis of microbiological laboratory data with a special focus on the analysis of antimicrobial susceptibility test results. χ² and Fisher’s exact probability tests were performed using SPSS for Windows (version 18.0; SPSS, Chicago, IL, USA) to compare proportions. P values < 0.05 were considered statistically significant.

During the study period, a total of 881 non-duplicated isolates were collected from 23 teaching hospitals in 17 cities. The number of isolates varied with years and regions and it was detailed in the Additional file 1: Table S1. Of the 881 S. pneumoniae isolates, 131 were obtained from pediatric patients aged 0 to 2 years (≤ 2 years old), 45 were obtained from pediatric patients aged 2 to 5 years (> 2 but ≤ 5 years old), 43 were obtained from patients aged 5 to 18 years (> 5 but ≤ 18 years old), 408 were obtained from adults aged 18 to 65 years (> 18 but ≤ 65 years old), and 254 were obtained from patients > 65 years old. Sputum was the most common specimen source, accounting for 73.1% of samples (644 strains), followed by blood (56 strains), BALF (56 strains), pharyngeal swabs (22 strains) and CSF (13 strains) Table 1. Strains isolated from sterile sites, such as blood, CSF, and pleural fluid, were classified as IPD strains. The 17 cities were divided into three groups based on the per capita gross domestic product (GDP) of the province in which the city is located (China Statistical Yearbook 2016, http://​www.​stats.​gov.​cn/​tjsj/​ndsj/​2016/​indexeh.​htm). Cities in group 1 had a per capita GDP of more than 60,000 Chinese yuan (CNY) and included Tianjin, Beijing, Shanghai, Jiangsu, Zhejiang, Inner Mongolia, Guangdong, and Shandong. Cities in group 2 had a per capita GDP between 50,000 and 60,000 CNY and included Chongqing, Hubei, Jilin, Shaanxi, and Liaoning. Cities in group 3 had a per capita GDP of less than 50,000 CNY and included Ningxia, Hunan, Xinjiang, and Guangxi.

The in vitro activities of the tested antimicrobial agents against the 881 S. pneumoniae strains are shown in Table 2. Based on the MIC breakpoints of oral penicillin, the percentages of penicillin-resistant S. pneumoniae (PRSP), penicillin-intermediate S. pneumoniae (PISP), and penicillin-susceptible S. pneumoniae (PSSP) isolates were 51.6% (455/881), 12.3% (108/881), and 36.1% (318/881), respectively. The rates of resistance of S. pneumoniae to erythromycin, azithromycin, and clarithromycin were 95.2%, 96.9%, and 92.5%, respectively. Macrolides exhibited weak activity against all of the isolates. Of the 839 strains (95.2%) that were resistant to erythromycin, 776 (92.5% of all strains) were also resistant to clindamycin. S. pneumoniae susceptibilities to amoxicillin/clavulanic acid and ceftriaxone were comparable (using non-meningitis breakpoints), but the rate of resistance to amoxicillin/clavulanic acid was higher than to ceftriaxone (using non-meningitis breakpoints). Overall, the resistance of S. pneumoniae to penicillin (based on breakpoints of oral penicillin) increased from 48.8% in 2011 to 55.9% in 2016 (P = 0.390, Fig. 1). The proportion of PRSP isolates remained stable and there were no statistical differences in rates of PRSP from 2011 to 2016 (Table 3). During the same time period, the proportion of PSSP isolates fell from 39% in 2011 to 33.6% in 2016. The MIC₅₀ of penicillin was 1 μg/ml in 2011 and increased to 2 μg/ml in 2016, indicating a trend towards an increase in resistance to penicillin. Trends towards increases in resistance were also observed for other β-lactams and cephalosporins. However, fluoroquinolones maintained excellent activity against S. pneumoniae; the MIC₉₀ values for levofloxacin and moxifloxacin were 1 μg/ml and 0.25 μg/ml, respectively. There were 19 S. pneumoniae strains resistant to levofloxacin and the MIC values were between 8 and 32 μg/ml. All of the strains were susceptible to vancomycin and no vancomycin-resistant strains were found during the study period. The rates of resistance of penicillin non-susceptible S. pneumoniae (PNSP) to other antimicrobial agents were also significantly higher than those of PSSP. However, no statistical differences in resistance to azithromycin, clarithromycin, levofloxacin, or moxifloxacin were found between PSSP and PNSP strains. The proportion of PSSP isolates varied with patient age; PSSP was more common among older adults compared with children (42.0% vs 18.7%, P < 0.001). There were no significant differences in the proportion of PNSP isolates between IPD and non-IPD strains. The rate of PSSP isolated from patients in emergency units was higher than in other hospital wards (P = 0.041). Similarly, the rate of PNSP isolated from patients in intensive care units (ICUs) was higher than in other wards (P = 0.017, Table 3).