Genotypic and phenotypic characteristics of Streptococcus pneumoniae from community-acquired pneumonia patients and healthy asymptomatic participants in Sichuan province, China

Community-acquired pneumonia (CAP) is an infection of lung parenchyma that occurs in persons outside the hospital, which has high rates of morbidity and mortality, especially in children and elderly [1, 2]. Among the pathogens, Streptococcus pneumoniae (S. pneumoniae) is considered as a major cause of CAP [3]. Since the first detection of antibiotic-resistant S. pneumoniae in 1977, multidrug-resistant (MDR) S. pneumoniae has appeared in various countries [4‐6]. Additionally, previous study has stated that the multidrug resistance of S. pneumoniae in China was the highest among the 11 Asian countries [7], indicating the urgent needs for controlling antimicrobial resistance.

It is well known that analysis of the molecular characteristics exerts a key role in clinical treatment. To date, over 90 serotypes of S. pneumoniae were identified [8]. The diversity is based on variation in structure of the repeating units of capsular polysaccharide [9]. Additionally, capsular polysaccharides as virulence factors, are the basis of S. pneumoniae vaccines, including the 7-valent pneumococcal conjugate vaccine (PCV7), the 10-valent PCV (PCV10), and the 13-valent PCV (PCV13) [10]. Although PCV7 vaccine is introduced in 2008, the immunization rate of PCV7 was less than 10% because it had not been included into National Immunization Program in China. Especially among migrant children, they are not largely vaccinated [11]. In 2017, PCV13 vaccine is available in big cities of China, but this vaccine is given only on own expense. Additionally, a previous study has stated that serotype distribution varies in Asian countries, and non-PCV serotypes have emerged as well [12]. Therefore, monitoring local serotypes can prevent the occurrence of pneumococcal disease and provide the guidance for developing new pneumococcal vaccines.

Streptococcus pneumoniae normally colonizes the upper respiratory tract of humans, and previous studies have shown the prevalence of S. pneumoniae carriage of 16.6% for healthy children in Shanghai, and nearly 70.0% for healthy children in Xinjiang, China [13, 14]. In the pathogenesis of pneumococcal disease, nasopharyngeal colonization is the necessary first step [15], suggesting that exploration of S. pneumoniae characteristics in asymptomatic carriers is warranted. Although epidemiology studies of S. pneumoniae from patients with CAP and IPD have been conducted in China [16, 17], limited data are available for the genotypic characteristics of S. pneumoniae from asymptomatic participants and CAP patients.

The current study was the first to evaluate the serotypes distribution, virulence/pili genes, and antibiotic susceptibility of S. pneumoniae from healthy asymptomatic participants and CAP patients in China. We found that the most frequent serotypes were 3 in CAP carriers and asymptomatic carriers. Additionally, 4 serotypes exclusively associated with CAP carriers, and 7 with asymptomatic carriers were observed. Over half of the isolates belonged to PCV13 serotypes and were resistant to ERY, CLI, SXT, and TET. The MDR rate was up to 90.2%. The dominant STs were ST271, ST320, and ST3397, respectively. The most interesting findings were significant association between the rlrA and sipA genes and the source of S. pneumoniae.

It is well known that the distribution of S. pneumoniae serotypes changes over age, geographic region, time, and diseases [27, 28]. In our study, the most frequent serotypes in CAP carriers group were 3, 19F, 19A, which were similar to other recent studies in China [16, 29], however different with serotypes in Italy [30]. Additionally, the major serotypes from asymptomatic carriers were 3, 6, and 19F in our study, whereas were 19F, 6B, and 6C in Brazil [31], indicating that serotypes is closely related to geographic region. NT isolates are deemed to be less virulent, due to lacking the polysaccharide capsule [32]. Correspondingly, previous study has analyzed the distribution of NT isolates between the asymptomatic carriers and IPDs, and found NT isolates were only from asymptomatic carriers [31]. Similarly, NT isolates were only detected from asymptomatic carriers in our study as well. On the other side, we found that serotypes 3 and 19F were frequently found in both asymptomatic carriers and CAP carriers. Indeed, these serotypes are common in asymptomatic carriers and CAP carriers worldwide [16, 33‐35].

PCVs can effectively reduce the burden of pneumococcal diseases and improve the population immunity [36]. Previous studies have revealed that incidence of vaccine-covered serotypes was significantly decreased after vaccination with PCVs. In parallel, occurrence of non-PCVs serotypes was increased over time [37, 38]. In our study, the overall coverage rate of PCV13 was 66.3%, which was conformed to previous study reported the 50–68% coverage of PCV13 in mainland China [39]. Additionally, we also found that the major serotypes in CAP carriers and asymptomatic carriers were PCV13 serotypes. Those results indicated that PCV13 may be useful to prevent against S. pneumoniae. However, several studies have concluded the different points regarding the PCV13 efficacy for serotype 3 in different regions. For example, Morre et al. [40] have reported that PCV13 presents high effectiveness against pneumococcal disease in USA. On the contrary, Andrews et al. [41] have stated that PCV13 is effective for most serotypes except for serotype 3 in UK. Additionally, a similar study in Hong Kong, China has found that the efficacy of PCV13 against pneumococcal disease caused by serotype 3 is low [42]. Therefore, whether the introduction of PCV13 against pneumococcal disease caused by serotype 3 is effective needs to be monitored, because serotype 3 was the most common in both asymptomatic carriers and CAP carriers.

The increase of antibiotic resistance enables the S. pneumoniae to be a worrying threat for public health. Previous studies have demonstrated that overuse of antibiotic contributes to the increase of antibiotic resistance [43, 44]. Although many measures have been taken to regulate the use of antibiotics in hospitals, antibiotics use is also widespread in China. A previous report has shown that duration of bacterial carriage is closely associated with prevalence of resistance [45]. In our study, higher resistance rates to CEF, TET and PEN in CAP carriers compared with asymptomatic carriers were shown. This may be related to the differences in serotype distribution and duration of bacterial carriage. Additionally, a few pneumococcal isolates in asymptomatic carriers and CAP carriers groups were susceptible to other antibiotics, such as CHL, CEF, CRO, MER, CFR, MOX, LEV, VAN, RIF, AMPC/CVA, and LIN, which brought hope to the treatment of drug-resistant S. pneumoniae. Therefore, rational use of antibiotics may be appropriate measure to control the spread of antibiotic-resistant strains.

The most common STs in this study were ST271 and ST320, which was similar to the findings of other reports in other regions of China [46, 47]. The predominant CC was CC271 in our study, which it was found in both CAP carriers and asymptomatic carriers. Additionally, CC271 was also closely associated with the carriage of rlrA and sipA. Consistent with our study, previous study has revealed that the PI-1 and PI-2 encoded by rlrA and sipA genes are present in isolates of CC271 [48]. In the current study, the carriage rates of rlrA and sipA genes from CAP carriers were higher than those of asymptomatic carriers, indicating that rlrA and sipA may be closely associated with high virulence. Additionally, our study showed that the genes including ply, lytA, pspA, cbpA, cbpG and pavA were widely conserved, suggesting that these may be candidates for developing vaccine in future. Moreover, we found the rlrA was present in serotypes 19F, 3, 19A, which has also been reported in previous study [26]. However, it has been reported that sipA gene is present in over half of pneumococcal isolates and commonly detected in serotypes 19F, 11A and 7A/F [26]. In this study, the most dominant serotype carrying sipA was 19F, followed by 3 and 19A. Taken together, rlrA and sipA may be important for colonization and pathogenicity of pneumococcal isolates.

The limitations of this study should be noted. First, our surveillance was not a population-based study but rather an actively participating surveillance, which led to the small numbers of isolates collected. All study populations have not been vaccinated with PCVs, and the changes after introduction of PCVs in this population remain unclear. Last, serogroup 6 was not typed, due to the limited laboratory equipment and reagents.

The current study showed the serotype distribution and antimicrobial resistance of pneumococcal isolates from CAP carriers and asymptomatic carriers in Sichuan, China. In addition, high prevalence of MDR and high coverage of PCV13 emphasized the importance of rational use of antibiotics and the necessity to expand the immunization with PCV13. Importantly, some virulence/pili genes were conserved, which may supply new guidance for pneumococcal vaccines in the future.