Population biology of 225 serogroup 6 Streptococcus pneumoniae isolates collected in China

Background In this study, we defined the population biology of serogroup 6 Streptococcus pneumoniae collected in China and their antibiotic resistance profiles. Methods The serotypes of 225 S. pneumoniae strains isolated between 1997 and 2011 were identified with the Quellung reaction and serotype-specific PCR. All isolated pneumococci were tested for their sensitivity to 11 kinds of antibiotics with the E-test method or disc diffusion. The sequence types (STs) were analyzed with multilocus sequence typing (MLST). Results The frequencies of serotypes and subtypes 6A, 6B-I, 6B-II, 6C, and 6D among the 225 isolates were 46.7% (105/225), 19.6% (44/225), 25.8% (58/225), 6.2% (14/225), and 1.8% (4/225), respectively. Serotype 6E was not found in the serotype 6A isolates, and neither serotype 6F nor 6G was identified in any isolate. MLST analysis revealed 58 STs. The most common STs were ST982 (23.1%), ST90 (14.7%), ST4542 (7.6%), and ST2912 (4.9%). The rates of clonal complex 90 (CC90) and CC386 among the oral-penicillin-nonsusceptible isolates decreased over the years, whereas the rates of CC855 and CC3173 increased. The four CCs had similar penicillin MIC distributions, with a maximum MIC of 2 μg/ml. Conclusions This study identified the serotypes/subtypes and CCs/STs of group 6 S. pneumoniae present in China. No salient antibiotic-resistant clones were isolated among the serogroup 6 S. pneumoniae. Electronic supplementary material The online version of this article (doi:10.1186/1471-2334-14-467) contains supplementary material, which is available to authorized users.


Background
Streptococcus pneumoniae remains the leading cause of community-acquired respiratory infections, and is one of the most important human pathogens causing severe invasive infections [1]. So far, more than 90 pneumococcal serotypes, classified into 46 groups, have been identified. Serogroup 6 is one of the most common causes of invasive pneumococcal disease in unvaccinated populations. Serogroup 6 traditionally comprises two serotypes, 6A and 6B. Two new serotypes, 6C and 6D, previously identified as serotype 6A and 6B, respectively, by the Quellung reaction were identified as two distinct serotypes using a multibead assay with monoclonal antibodies [2,3]. A factor antiserum, 6d, was then developed against these two new serotypes. Therefore, these two serotypes can now be identified with the traditional Quellung test [4,5]. Two subtypes of 6B, designated 6B-I and 6B-II, can be distinguished by polymerase chain reaction (PCR) [6]. Another study in Korea demonstrated that some serogroup 6 isolates encode distinctive cps (capsular) loci, differing from those of most other serogroup 6 isolates. These were designated the "6X" group, and include both traditional serotype 6A and 6B isolates [7]. Based on the differences in their capsular gene sequences, serotype "6E" was assigned to this group in a subsequent study [8]. In a German epidemiological survey, two serogroup 6 isolates could not be assigned to a serotype using factor sera [9]. The latest report by Oliver et al. [10] showed that the two isolates were assigned to new serotypes, "6F" and "6G".
That study showed that a single codon change in the cps gene, encoding a glycosyl transferase, altered the substrate specificity, caused the biosynthesis of heterogeneous capsule repeating units, and resulted in a new hybrid capsule type. Antigenic formulae and specific factor antisera against serotypes "6E", "6F", and "6G" have not yet been developed, but are necessary for their traditional serotype classification [11]. Because the new serotypes have not been confirmed with the traditional Quellung test, they are designated with quotation marks in the present text.
We did not want to investigate the effects of the vaccine PCV7, because its immunization coverage is too low (<1%) to reflect its effectiveness. We set out to identify any new serotypes (serotypes "6E", "6F", and "6G") in serogroup 6 S. pneumoniae in China and to report the population biology of this group. In our previous study [12], we examined the genetic structures of only serotypes 6C and 6D, without the most frequent serotypes 6A and 6B. In the present study, we report the STs of all the serogroup 6 isolates and an association between the STs and antibiotic resistance. We intended to use identical isolates in these two studies, but some isolates from our previous study were dead when we went to include them in the present study. Therefore, the present study includes some new isolates collected in 2008-2011 from Shenzhen and Beijing. There were differences in the antibiotic resistance in different clones. For completeness, some published data or similar information from our previous paper have been included in the present results, but these are not included in the analysis or discussion.

Pneumococcal isolates
A total of 225 serogroup 6 S. pneumoniae strains isolated from children in 1997-2011 were included in the study. The details of these S. pneumoniae collections have been published previously [13][14][15][16][17][18]. The isolates were cultured from nasopharyngeal specimens, except those collected from Beijing in 2006-2008 and from Shenzhen in 2009-2011, which were cultured from hypopharyngeal aspirate specimens. All isolates were identified as serotypes 6A, 6B, 6C, and 6D with a capsule Quellung test using a pneumotest kit and specific factor antisera (Statens Serum Institut, Copenhagen, Denmark). Typing was performed with phase-contrast microscopy, according to previously reported procedures [19].
A parent and/or legal guardian of each participant signed a written informed consent document before enrollment and before any study procedure was performed. This study was viewed and approved by the Ethics Committee of Beijing Children's Hospital Affiliated to Capital Medical University. No ethical problems existed in this study.

Antimicrobial susceptibility
The minimum inhibitory concentrations (MICs) of all isolates were determined for penicillin, amoxicillin-clavulanic acid, ceftriaxone, cefuroxime, erythromycin, imipenem, levofloxacin, and vancomycin using E-test strips (AB Biodisk, Solna, Sweden), and their susceptibility to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol was assessed with disc diffusion tests (Oxoid). The use of the penicillin parenteral (nonmeningitis) breakpoints for susceptible, intermediate, and resistant rates to compare penicillin susceptibilities in this study blurred the differences between the serotypes [22]. Therefore, the oral penicillin V breakpoints were also used to analyze penicillin susceptibility. Streptococcus pneumoniae ATCC49619 was used as the reference strain in the susceptibility tests. Isolates were considered multidrug resistant if they were not susceptible to three or more classes of antimicrobials.

Molecular epidemiology of serogroup 6 strains
All strains were characterized with multilocus sequence typing (MLST). Bacterial chromosomal DNA was extracted as described above. The PCR fragments of the seven MLST genes (aroE, gdh, gki, recP, spi, xpt, and ddl) were amplified from the chromosomal DNA. The products were sent to BGI Company (Beijing, China) for sequencing on both strands. The STs were determined by comparing the allelic profiles with the recognized STs at the MLST website (http://spneumoniae.mlst.net). The new alleles and allelic profiles identified in the present study have been submitted to the MLST database for name assignment. The eBURST v3 software (available at http://www.mlst.net) was used to estimate the relationships among the isolates and to assign strains to a clonal complex (CC) using the stringent group definition of six of seven shared alleles [23].

Statistical analysis
The serotyping and antimicrobial resistance data were analyzed with the WHONET 5.3 software, as recommended by the World Health Organization (http://www. who.int/drugresistance/whonetsoftware/en/). The χ 2 test, performed with the SPSS software v. 13.0 (SPSS Inc. USA), was used for statistical comparisons. A two-tailed cut-off of P < 0.05 was deemed to indicate statistical significance.
The analysis of antibiotic resistance revealed similar results to those in our previous report [12], which are shown in the tables with no further description.
The distribution of the CCs/STs of the serogroup 6 S. pneumoniae isolates according to serotype/subtype is shown in Table 1 and Figure 1. Each serotype/subtype had a main CC/ST that included the majority of the isolates in the serotype/subtype. For instance, the proportion of ST982 in the serotype 6A isolates (44.8%) was The CC/ST distributions of the serogroup 6 S. pneumoniae changed significantly during the study period. The common CCs/STs, such as ST982, CC90, CC4542, and CC4536, mainly occurred in 1997-2005, but these isolates decreased or even disappeared in later years. Two CCs among the common CCs/STs, CC2754 and CC386, were mainly found in 2006-2010, with some fluctuations. No CC or ST showed a clearly increasing trend, except the group involving the infrequent "other" CCs/STs ("others" in Table 1). Considerable isolates identified in this study belonged to the infrequent "other" CCs/STs (46/225), especially in 2009-2011 (10/26).

Associations between STs/CCs and antimicrobial resistance
The numbers and percentages of isolates not susceptible to oral penicillin and other antibiotics are summarized according to CCs/STs in Tables 2 and 3. Notably, all CC90 isolates were not susceptible to oral penicillin, with high MIC 50 and MIC 90 values, whereas other common CCs/STs were usually susceptible, except that some intermediately susceptible strains (40.0%) were present in CC386. More than half the infrequent "other" CC/ST isolates were not susceptible to oral penicillin. High rates of nonsusceptibility to cefuroxime were found in CC90 (92.7%) and the infrequent "other" CCs/STs (45.6%). The differences in some other antimicrobial resistances between the CCs/STs are shown in Table 3.
The distributions of serotypes/subtypes and CCs/STs in the oral-penicillin-nonsusceptible strains (OPNSP) were analyzed further, as shown in Table 4. Although 6B-II was the main subtype in the total OPNSP strains, its proportion decreased from 82.1% in 2000-2002 to 41.7% in 2009-2011 (χ 2 = 5.6, P < 0.05). The proportion of serotype 6A strains increased significantly from 10.3% to 50.0% during the same period (χ 2 = 6.8, P < 0.01). The constitution of CCs/STs among the OPNSP also changed. The proportions of CC90 and CC386 that were identified as subtype 6B-II decreased. However, the proportions of CC855 (including six serotype 6A isolates) increased, as did the proportions of CC3173 (including six serotype 6A isolates and two subtype 6B-I isolates). The distribution of penicillin MICs between the CCs/STs did not differ, except that the MICs of the CC386 isolates reached 0.125 μg/ml.

Discussion
Recently, Ko et al. [8] identified a distinct group among serotypes 6A and 6B S. pneumoniae isolates, designated serotype "6E". Based on sequence data, the serotype "6E" isolates from traditional serotype 6B were the same as those from subtype 6B-II in the present study, which is exactly consistent with subtype 6B-III reported by Elberst et al. [24]. Subtype 6B-II was isolated frequently in the present study. However, no serotype "6E" isolate was identified in traditional serotype 6A with PCR.
As-yet-unconfirmed serotype "6E" has only been identified based on its capsular gene sequence [8] because no new serotype including both traditional serotypes 6A and 6B can be constructed within the serological typing system [11]. If differences in gene sequences indicate new serotypes, two new serotypes must be individually recognized within the traditional serotypes 6A and 6B.
All the common CCs/STs included more than one serotype or subtype, except CC90 and CC386, which included subtype 6B-II isolates only. One subtype 6B-II isolate belonged to CC4542, which otherwise included only serotype 6A and subtype 6B-I isolates. However, this isolate had a unique ST, ST8618. Two classes of cps sequences have been reported for serogroup 6 isolates.
The class 2 cps sequences are limited to a few serotype 6B isolates [25], which are in fact subtype 6B-II. The present data show that our subtype 6B-II isolates displayed genetic backgrounds different from those of other serotypes/subtypes.
Our previous studies have suggested that a clonal shift occurred in serotype 19F and 23F isolates between 1997 and 2010. In the serotype 19F S. pneumoniae isolates, the spread of CC271 with high β-lactam-antibiotic resistance may be driven by the selective pressure applied with the overuse of antibiotics, whereas the spread of susceptible CC983 is limited. CC271 became the main population of serotype 19F S. pneumoniae after 2003 [26]. In the serotype 23F S. pneumoniae isolates, ST81 (with high β-lactam-antibiotic resistance) took the place of ST342 and became the absolutely predominant population in 2010 [27]. Both CC271 and ST81 are also important international epidemic clones [26,27]. We noted that selection by antibiotic abuse caused an increase in subtype 6B-II isolates with significantly higher β-lactamantibiotic resistance than that of other serogroup 6 S. pneumoniae isolates in 1997-2008 [12]. The present data show that most subtype 6B-II isolates belong to CC90. ST90 is one of the globally epidemic resistant  clones, Spain 6B -2 (http://web1.sph.emory.edu/PMEN/index. html). CC90 isolates were the main components of the OPNSP in our study. However, isolates belonging to this clone decreased in 2009-2011, and its substitutes, CC855 and CC3173, did not show higher β-lactam-antibiotic resistance. Therefore, this replacement cannot be explained by antibiotic overuse and remains undefined at present. Most of the CC855 and CC3173 isolates were identified as serotype 6A, which is not covered by the 7-valent pneumococcal conjugated vaccine (PCV7). The proportion of serotype 6A isolates in the OPNSP increased significantly (Table 4). This replacement may be attributable to immunization with PCV7, which was used after September 2008 in China. However, this rationale is not convincing because the vaccine immunization coverage was so low (less than 1%) that the spread of serotypes 19F and 23F was not restricted at all [26,27].
The MIC 90 and maximum MIC of penicillin in serogroup 6 S. pneumoniae isolates remained at 1 μg/ml and 2 μg/ml, respectively, from 2000 to 2010 in China (Table 2), whereas the MIC 90 of serotypes 19 F and 23 F increased to 2 μg/ml and the maximum value was 8 μg/ml, and even reached 256 μg/ml [28,29]. The present data also show the absence of salient resistant clones in serogroup 6 S. pneumoniae. They all may be under antibiotic selective pressure. Therefore, the CC/ST components of this group have not changed regularly, like serotypes 19 F and 23 F. This speculation corresponds to the decreasing frequency of serogroup 6 among all S. pneumoniae isolates over time [12]. The population biology of this serogroup should be monitored continuously in the future.

Conclusions
In summary, this study clarifies the serotypes/subtypes and CCs/STs of group 6 S. pneumoniae present in China. Serotype "6E" was not identified in the traditional 6A isolates, and neither serotype "6 F" nor "6G" was assigned in our collection. Although the subtype 6B-II isolates were the main constituents of the OPNSP, their place was taken by serotype 6A isolates over time. The genetic constitution of the group 6 S. pneumoniae and its changes showed distinct regional characters. Unlike the serotype 19 F and 23 F isolates, no salient resistant clones were identified in serogroup 6 S. pneumoniae. Further long-term surveys of serogroup 6 S. pneumoniae are required to monitor the clonal prevalence and antimicrobial resistance in this important human pathogen.