S. Pneumoniae is one of the common pathogens in CAP that leading cause of mortality in children under 5 years old. To prevent pneumococcal-related diseases and reduce the economic burden of that, many developed countries have introduced PCVs targeting serotypes of S. Pneumoniae into National Immunization Program. However, PCVs targeting capsular polysaccharide locus cannot overcome replacement serotype or non-capsular serotypes among S. Pneumoniae. Virulence factors, as good antigen targets, can effectively supplement the failure of PCVs. Therefore, we conducted this retrospective study to investigate molecular characteristics of STs, serotypes and virulence factors of S. Pneumoniae isolated from children under 5 years with CAP in Chengdu, so as to grasp epidemiological surveillance data and guide for vaccine vaccination and development.
Large, accurate, long-term serotype distribution monitoring of S. Pneumoniae isolates contributes to better multivalent vaccine development and guides for vaccination. In this study, The prevalent serotype was 19F, followed by 6B, 19A, 6A, 23F, which resembled other cities in China or Asian countries, but their rank order varied by region[15–17]. However, the prevailing serotypes were 19A, 7F, 3 and 1 in Europe and that was 14, 1 and 5 in America[18, 19]. Exception for region factor, S. Pneumoniae serotype distribution was limited by vaccine usage, age and so on. For example, the prevalent serotypes were 19F, 4, 6, 18 in the pre-PCV7 period in Russian children, and that serotypes were 4, 6, 18 after the introduction of PCV7[20], indicating a substantial reduction of 19F by vaccination. It also can be seen that different multivalent vaccines can only prevent infection with certain serotypes of S. pneumoniae. In this study, The overall coverage rates of PCV7, 10 among all isolates were 47.32%, 48.1%, respectively. PCV10 seemed not better than PCV7 because 1, 5, 7F were rarely detected in our study. When taking considering into age factor, we found that the coverage rate of PCV13 among isolates from children aged < 1 year was as high as 84.2%, which surpassed that of PCV7 and PCV10 among isolates from all children with no vaccination. We strongly suggest that it would be best to vaccinate with PCV13 for infants in the high coverage areas of serotypes, which can release the pressure caused by most vaccine-covered pneumococcal diseases.
Compared to pulsed-field gel electrophoresis (PFGE) technology, MLST is a simple, effective, comparable method to describe the molecular characterization of microorganisms. It has been applied in many aspects, such as epidemiological investigation, molecular evolution of bacteria[21]. In this study, CC271 was the most common group of STs, including the prevalent ST271 and ST320. A few previous research have suggested that there is a good correlation between STs and serotypes. For example, ST320 19A was reported as the prevalent clone in Latin American[22], while ST271 19F was prevalent in China[23]. By using minimum spanning tree analysis, we found that ST271 19F and ST320 19A originated from Taiwan19F-14 were common clones in this study. Additionally, there may exist also consistency between some STs and serotypes due to the limited sample size in this study, such as ST505 with serotype 3, ST3173 with serotype 6B, ST876 with serotype 14, which was similar to the previous studies reported in China[8, 24]. Interestingly, one ST may contain diverse serotypes sometimes. In this study, ST271 included 6A and 1 besides the absolutely prevailing 19F. The results by Tocheva et al[25] also showed that ST199 contained serotypes 19A, 14, 15 and ST162 contained 19F, 9V. Taken together, these results of studies indicated there was the evolution of serotypes switching among STs in order to adapt vaccine or antimicrobial pressure[26].
It is established that virulence factors, as important components of bacteria, can be alternative vaccine antigens. Recent report proved that conserved proteins that have been proposed for candidate vaccines should possess favorable immunogenicity and immunoreactivity of antigen, such as pspA, inhibiting the activation of the C3 complement protein and elevating the colonization ability of S. Pneumoniae [27]. Besides the above-mentioned conditions, an additional requirement for selecting virulence factors as candidate vaccines is widespread distribution in the population. In this study, all isolates carried lytA and most of the isolates carried ply, psaA, nanA, pavA and piaA, which was similar to other cities of China[14]. Based on our results, we suggested that these virulence factors including lytA, ply, pasA, nanA, pavA, piaA might be candidate vaccines in the future. However, the carrier rate of pspA was significantly lower than that of other virulence factors, which might be not suitable for vaccine candidate. Currently, little is known about the relationship between ST/CC and virulence factor. A few previous studies reported that CC271 carried more virulence factors than non-CC271[28, 29]. In this study, the positive rate of nanA in the CC271 was higher than that in the non-CC271. Recent research has revealed that pneumococcal neuraminidase A encoded by nanA gene can synergize with that of influenza A virus strengthening cytotoxicity[30]. These results together indicated CC271 harboring more nanA might exhibit much pathogenicity. Additionally, the most common virulence pattern was lytA-ply-psaA-nanA-pavA-piaA mainly distributed in serotype 19F and 19A in this study, while lytA-ply-psaA-pspA-nanA-pavA-piaA pattern in serotype 6B. It can be seen that the distribution of virulence factors might be associated with serotypes. Virulence factor pspA consisted of two families (family 1 and family 2), whose distribution was different from region. It was reported that the carriage rate of family 2 was higher than that of family 1 in Japan[31]. Although pspA of 19F and 6B belong to family 1, their distribution is also distinct. Our results indicated that serotype 6B isolates were more aggressive than 19F isolates. Furthermore, purified protein-vaccine from the homologous family 2 cannot against infection caused by S. pneumoniae carrying family 1[32, 33]. Therefore, we considered that supplemental vaccine strategy that PCVs coupled with certain virulence factors maybe reduce the burden of pneumococcal disease based on the distribution of STs, serotype and virulence factors[34].
To the best of our knowledge, this is the first study on potential relationships between molecular characteristic of S. pneumoniae from children with CAP under 5 years in Chengdu via minimum spanning tree and correspondence analysis, while several limitations warrant attention in this study. Firstly, the sample size of pneumococcus needs to be further expanded to a more comprehensive assessment of molecular epidemiology of S. pneumoniae. Secondly, some serotypes have not been identified so far, which might need other methods to identify. Ultimately, The results only represent the data of the last two years when the time span of specimens collection was between January 2019 to January 2021 in this article. More high-quality and long-term surveillance on STs, serotypes, virulence factors needs to be carried out in the future.
In summary, our findings indicated that CC271 was the predominant epidemic clone, especially ST271 19F and ST320 19A. Moreover, there were strong relationships between STs, serotypes, virulence factors, which indicated the formulation of vaccine strategies needs to carefully consider molecular epidemiological factors.