Pneumococcal isolates and characteristics of patients
A total of 287 pneumococcal isolates were identified from sputum and blood cultures in this study. Three isolates did not fulfill the microbiologic diagnostic criteria and were excluded. 204 isolates subsequently re-grew on culture, and antimicrobial susceptibility testing was performed. Real-time PCR of lytA confirmed that they were pneumococcal isolates. These 204 isolates from 200 adult pneumonia patients which were considered causative pathogens for pneumonia, and their data were processed for analysis (Selection flow is shown in Additional Figure 1). The characteristics of the patients are shown in Table 1. The majority of patients (n=145, 72.5%) were ≥65 years old with a median age of 72.5 years. 159 (79.5%) cases were community-acquired pneumonia (CAP) and mostly mild pneumonia with CURB-65 ≤2. 127 (63.5%) patients were admitted to the hospital, and the mortality rate was 3.5%. Only 55 (27.5%) patients were confirmed to have received PPSV23, and 32 (16.0%) had a course of antimicrobials prior to the enrollment.
Distribution of serotypes and molecular epidemiology
Figure 1 shows the STs, CCs, and serotypes of the isolates. We detected 41 CCs, 62 STs and 2 singletons (STs 7801 and 13216). CC/ST 180 of serotype 3 was the most frequently observed strain (n=45, 22%), followed by CC/ST 236 of serotype 19F (n=12, 6%) and CC/ST 99 of serotype 11A (n=12, 6%). We identified 10 new STs (STs 13216, 13217, 13218, 13219, 13220, 13221, 13302, 13303, 13304, and 13389). In total, 109 (53.4%) isolates belonged to PMEN-type CCs, and 144 (70.6%) isolates were PPSV23-type serotypes.
Antimicrobial susceptibility
The susceptibility patterns for b-lactam antimicrobials, the number of nonsusceptible antimicrobial classes other than b-lactams, and the number of MDNS strains in relation to CCs and PMEN strains are shown in Table 2. Susceptibility for antimicrobials other than b-lactams are also shown in Additional Table 1. In total, 40 (19.6%) isolates (from 39 patients) were categorized as bNS strains. The most common molecular type among bNS strains was CC 236 (n=9, 22.5%), followed by CC63 (n=6, 15%), CC242 (n=5, 12.5%), and CC558 (n=5, 12.5%). In total, 30 (75%) bNS strains were PMEN clones. The remaining 10 (25%) bNS strains, which had 6 CCs and one singleton, have not previously been categorized as PMEN-type CCs. These included CC3111, which was isolated from 3 patients, and CC2756, which was isolated from 2 patients. Similarly, 121 (59.3%) isolates from 119 cases (33 CCs out of the total 41 CCs, 80.5%) were categorized as MDNS strains. Of those, 62.8% (n=76) of MDNS strains were PMEN related. MDNS strains were found among CC180 (n=36/50, 72% of total isolates in the CCs), CC236 (n=11/12, 91.7%), CC63 (n=7/8, 87.5%), CC2755 (n=7/8, 87.5%), and CC242 (n=7/7, 100%) (Table 2). A total of 83 (68.6%) of MDNS strains, in particular CC180 and CC2755, met the MDNS criteria without being bNS strains, whereas almost all bNS strains were MDNS strains.
Comparison of bNS and bS isolates
Figure 2 (A and B) compares the CC distribution by serotype among bS and bNS strains. Out of 40 bNS strains, 22 (55%) were PPSV23 types (serotypes 6B, 11A, 14, 19A, 19F, and 23F), and the remainder belonged to PPSV23 nonvaccine types (serotypes 6A, 15A, 23A, and 35B). We found that bNS strains were significantly associated with serotypes 19F, 15A, and 35B (Additional Table 2, P<0.05, respectively) when compared to bS pneumococcal strains.
Serotype 19F strains were mainly CC236 and serotype 15A of CC63, while serotype 35B contained multiple CC strains. Among bNS strains, CC63 and CC558 were exclusively present in the nonvaccine serotype 15A and 35B, respectively (Figure 2B). Among PMEN-related bNS strains (n=30, 75%), 6 (20%) had different serotypes from those of the original PMEN strains. It was noted that the serotype had shifted from vaccine type to nonvaccine type in four of those six PMEN strains: from CC242/serotype 23F to 23A (n=2) and from CC81/serotype 23F to 6A (n=1) and 15A (n=1) (Figure 1).
There was no significant difference in demographic and clinical characteristics of patients with b-lactam-susceptible (bS) pneumococcal strains compared with patients with bNS pneumococcal strains, except that patients with bNS strains tended to be associated with bronchial asthma (Additional Table 2).