Serotype Changes and Drug Resistance in Invasive Pneumococcal Diseases in Adults after Vaccinations in Children, Japan, 2010–2013

Pneumococcal conjugate vaccination of children is associated with penicillin-resistant Streptococcus pneumoniae in adults.

S treptococcus pneumoniae can cause invasive pneumococcal diseases (IPDs), which can be treated with generally effective antimicrobial drugs. In adults, well-known risk factors for IPDs include underlying conditions such as splenectomy, functional asplenia, immunodeficiency, and HIV infection. Risk is also increased with age >65 years, diabetes, renal dialysis, and chronic hepatic dysfunction (1,2).
In 2010, the US Advisory Committee on Immunization Practices (ACIP) issued recommendations that all persons >65 years of age should be vaccinated with 23-valent pneumococcal polysaccharide vaccine (PPSV23) to prevent pneumococcal diseases (3). In 2012, the ACIP also recommended that 13-valent pneumococcal conjugate vaccine (PCV13), which the US Food and Drug Administration approved in 2011, be given to adults >19 years of age if they have immunocompromising conditions (4). Recently, the ACIP recommended routine administration of both PCV13 and PPSV23 to all adults >65 years of age (5).
Presently, pneumococcal conjugate vaccines (PCVs) are incorporated into pediatric vaccination schedules in >120 countries, and PCV13 has been approved for adults in >100 countries. Pneumococcal infections in adults have decreased as an indirect effect of widespread use of PCVs for children (2,6,8,9,12). However, in countries where PCV7 or PCV13 was introduced, overall coverage of serotypes by the vaccine gradually decreased because of pneumococcal serotype replacement from vaccine type to nonvaccine type. In particular, increases of nonvaccine serotypes, such as 6C, 15A, 23A, and 35B, have been reported in the United States (13,14) and other countries (15)(16)(17)(18).
In Japan, PCV7 vaccination was introduced for children <5 years of age in November 2010 by the Provisional Fund for the Urgent Promotion of Vaccination. PCV7 was incorporated into routine vaccination schedules in April 2013 and replaced by PCV13 in November 2013. PCV7 rapidly decreased IPD infections in children. However, pneumococcal infections caused by non-PCV7 serotypes (19A, 15A, 15B, 15C, and 24) increased in children during 2012 (19).
Meanwhile, Japan became the first "super-aging" society in the world in 2013, when 25.1% of the nation's population was >65 years of age (Statistics Bureau, Ministry of Internal Affairs and Communications, http:// www.stat.go.jp/english/data/handbook/c0117.htm). Lifestyle-related and other diseases that affect response to infection have increased in older adults, and pneumonia has again become the third leading cause of death in Japan. Nevertheless, the voluntary PPSV23 immunization rate among older persons long remained <18%. In November 2014, the Japanese Ministry of Health, Labour and Welfare began promoting routine vaccination with PPSV23 for adults >65 years of age.
Because of these developments, we sought to clarify changes in serotypes and in genetic antimicrobial resistance in isolates from adults with IPDs after PCV7 for children was introduced in Japan. We describe capsular serotypes, drug-resistance genotypes, and multilocus sequence typing in isolates from adults with IPDs, and we examine the relationships of these factors to specific IPDs.

Patients and Pneumococcal Strains
Our study included adult patients (>19 years of age) with IPD. Pneumococcal isolates from sterile clinical samples such as blood, cerebrospinal fluid, pleural effusion, and joint fluid were collected from clinical laboratories at 341 hospitals participating in this IPD surveillance study. Each hospital had a microbiology laboratory, and the hospitals were distributed nearly uniformly throughout Japan. The hospitals participated in the surveillance project after written permission was obtained from laboratory or hospital directors.
From April 2010 through March 2013, a total of 715 pneumococcal isolates were collected for the study: 275 during April 2010-March 2011, the first surveillance period; 213 during April 2011-March 2012, the second period; and 227 during April 2012-March 2013, the third period. This large-scale IPD surveillance for adults was performed in conjunction with IPD surveillance for children (19). During the first surveillance period, the rate of voluntary PCV7 vaccination for children was ≈10%. The second period occurred simultaneously with the Urgent Promotion of Vaccination incentive for children, and the immunization rate was ≈50%-60%. The third period occurred just before the transition from PCV7 to PCV13 for children, and the PCV7 immunization rate was ≈80%-90%. However, the rate of voluntary vaccination with PPSV23 in adults remained <18% throughout the surveillance periods.
The collected pneumococcal isolates were sent promptly from each clinical laboratory to our laboratory (Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan), accompanied by survey forms completed by attending physicians but not identifying patients in compliance with ethical guidelines for epidemiology in Japan. The surveys collected information on patient's age at illness onset, sex, clinical manifestations, blood test results at hospitalization, and outcome. Clinical manifestations and diagnoses were verified in all patients, according to diagnostic criteria and guidelines for sepsis and related conditions (20).

Serotypes and Antimicrobial Drug-Resistant Genotypes
Serotypes were determined by the capsular quellung reaction by using antiserum purchased from the Statens Serum Institute (Copenhagen, Denmark). By using real-time PCR methods described previously (21), alterations were identified in 3 penicillin-binding protein (PBP) genes mediating β-lactam resistance in S. pneumoniae: pbp1a (encoding enzyme PBP1A), pbp2x (encoding PBP2X), and pbp2b (encoding PBP2B). Amino acid substitutions within or near each PBP's conserved amino acid motifs, such as serinethreonine (Thr)-methionine-lysine (STMK), were detected by using real-time PCR. The mef (A) and erm (B) genes mediating macrolide resistance were also identified by real-time PCR (21). Quinolone resistance was analyzed by sequencing the quinolone-resistance-determining region in the genes gyrA, gyrB, parC, and parE in strains with MICs of levofloxacin of >4 μg/mL (22).
Identification of capsular type by the quellung reaction and resistance genotyping by real-time PCR was performed within 1 day of sample collection for each strain. Results were reported immediately to the medical staff at the referring hospital.

Antimicrobial Susceptibility Testing
MICs of 7 intravenous antimicrobial agents (penicillin, ampicillin, cefotaxime, ceftriaxone, meropenem, panipenem, and vancomycin) were determined for all pneumococcal isolates by agar-dilution methods (23). We obtained each agent from the respective manufacturer.

Multilocus Sequence Typing
Multilocus sequence typing (MLST; http://www.MLST. net) was performed for nonvaccine serotype strains, except for PPSV23 serotypes, according to previously described methods with minor modifications (24). Primers used were based on sequences listed by the US Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/ biotech/strep/alt-MLST-primers.htm). Analyses by using MLST and eBURST (Department of Infectious Disease Epidemiology, Imperial College of London, UK) were performed, as described by the MLST website (http://spneumoniae.mlst.net).

Statistical Analysis
We assessed differences in serotypes by age groups, clinical symptoms and signs, resistance types, and surveillance periods. To determine whether differences were statistically significant, we performed χ 2 tests or the Fisher exact test by using Ekuseru-Toukei 2012 software for statistics (Social Survey Research Information, Tokyo, Japan).

Yearly Changes in Pneumococcal Serotypes
During each annual surveillance period (April-March) during 2010-2013, pneumococcal serotypes from IPD isolates varied (Table 1). All strains were isolated from sterile samples, such as blood, cerebrospinal fluid, pleural effusion, or joint fluid.
During all surveillance periods, the estimated voluntary inoculation rate for PPSV23 in adults was <18% in Japan. In February 2010, the vaccination incentive for PCV7 in children began on a voluntary basis. In November 2010, the Special Fund for the Urgent Promotion of Vaccination was initiated, and PCV7 vaccination of children <5 years of age became an official priority throughout Japan. Estimates of the PCV7 immunization rate in children was <10% in 2010, 50%-60% in 2011, and 80%-90% in 2012 (19). PPSV23 coverage of serotypes found in IPD isolates decreased significantly each year, from 82.2% in 2010 to 77.5% in 2011 to 72.2% in 2012 (p = 0.036). Respective coverages with PCV7 and PCV13 also decreased significantly, from 43.3% and 73.8% in 2010 to 39.4% and 60.6% in 2011 to 23.8% and 54.2% in 2012, respectively (p<0.001). Although serotype 22F increased significantly in IPD isolates, yearly decreases of serotype 6B contributed markedly to decreases in PPSV23 coverage. Nonvaccine serotypes (i.e., non-PPSV23) increased significantly during the surveillance periods from 13.5% in 2010 to 20.7% in 2011 to 24.2% in 2012 (p = 0.007).

Yearly Changes in Resistance Genotypes and Serotypes
Yearly changes in genotypes and serotypes affecting β-lactam resistance also occurred during the 3 periods (Figure). Decreases closely related to a reduction of serotypes 6B, 19F, and 23F, but not of serotype 14, were found for a number of gPRSP. Emergence of gPRSP was evident among 6 non-PPSV23 serotypes (6D, 15A, 15C, 16F, 23A, and 35B), although such resistance strains were few. Almost all non-PPSV23 serotype strains showed macrolide resistance (online Technical Appendix Figure 2), which did not change significantly from year to year (Table 2).

Serotype Distribution by Disease
A comparison of proportions of serotypes included in PCV7, PCV13, PPSV23, and non-PPSV23 by disease category (i.e., pneumonia, bacteremia and sepsis, meningitis, and others) showed that frequency of serotypes differed considerably by type of IPD (Table 4). Prevalence of serotypes 3, 14, and 19A was higher in isolates of patients with pneumonia than in those from patients with other illnesses (p value range <0.001-0.076). In contrast, serotypes 10A, 15A, 23A, and 23F were significantly more prevalent in isolates of patients with meningitis than in those from patients with other illnesses (p value range <0.001-0.004).

Antimicrobial Drug Susceptibility by Genotype
The online Technical Appendix Table shows susceptibilities (50% MIC, 90% MIC, and MIC range) of 6 parenteral β-lactam agents for S. pneumoniae strains (n = 710). PBP gene alterations affect MICs of penicillin, ampicillin, cefotaxime, and meropenem (online Technical Appendix Figure 3). Most strains in this study were gPISP with pbp2x gene alterations that reduced susceptibility to cephalosporin agents rather than to penicillin. MICs of cefotaxime and ceftriaxone were affected more by pbp2x alterations than by pbp2b alterations. In contrast, MICs of penicillin, ampicillin, and meropenem were affected more by pbp2b alterations than by pbp2x alterations. Strains showing MICs >4μg/mL to cefotaxime had pbp2a gene alteration and also had pbp1a, pbp2x, and pbp2b gene alterations (data not shown).

MLST in Non-PCV13 Serotype Strains
MLST results for 92 strains (non-PCV13 serotypes) collected during 2012 and results for 2 gPRSPs (serotypes 15B collected during 2010 and 23A collected during 2011) indicate that sequence types (STs), 8 of which were new, and clonal complexes (CCs) varied ( and Thr445Ala adjacent to the serine-serine-asparagine amino acid motif in PBP2B, respectively.

Discussion
Wide use of PCV7 or PCV13 in young children has contributed to a decline in IPD (6-9) and other pneumococcal diseases, including pneumonia (25,26) and otitis media (27,28). The decline resulted from both direct vaccine effect and herd immunity.
In the United States, introduction of PCV7 led to decreased rates of IPD, resulting from vaccine-related strains in children and in unvaccinated adults because of indirect effects of pediatric use (6,29,30). After increased incidence of serotype 19A among causative pathogens in children, multivalent PCV13 replaced PCV7 in 2010 (11). PCV13 now has been introduced in >120 countries, and various pneumococcal diseases have decreased as a result. Unfortunately, serotype replacement by non-PCV13 serotypes such as 6C, 15B, 22F, 23A, and 35B has also ensued (13,14,(16)(17)(18)31).
In Japan, incorporation of PCV7 and Haemophilus influenzae type b conjugate vaccine into routine vaccination schedules for children occurred later than in other countries and was spurred by the Provisional Special Fund for the Urgent Promotion of Vaccination, initiated in November 2010. The vaccination rate for children improved rapidly from <10% in 2010 to 50%-60% in 2011 and 80%-90% in 2012. Routine PCV7 for children was implemented officially in April 2013 and was replaced with PCV13 in November 2013.
Although PPSV23 was approved in 1988, few (<18% in 2012) adults voluntarily received PPSV23 vaccination. Since October 2014, vaccination with PPSV23 in adults >65 years of age has been supported by public government funding. However, at this time, vaccination rates are unclear because these older persons can receive 1 PPSV23 vaccination over a 5-year period.
In our large-scale surveillance, we aimed to clarify molecular epidemiology for pneumococci and β-hemolytic streptococci and to collect background data on patients with IPD throughout Japan during April 2010-March 2013. Pediatric IPD caused by PCV7 serotypes and other PCV7related 6A serotypes decreased significantly soon after *Includes empyema (n = 17) and pleuritis (n = 16). †Bacteremia (n = 39), sepsis (n = 51), severe sepsis (n = 58), and septic shock (n = 24). ‡Cellulitis (n = 7), arthritis (n = 6), spondylitis (n = 4), peritonitis (n = 3), and other conditions. vaccine implementation (19), but the non-PCV7 serotypes 19A, 15A, 15B, 15C, and 24 increased each year. These results reflect simultaneous replacement of PCV7 serotypes with non-PCV7 serotypes among pneumococci that colonize the nasopharynx of children (K. Ubukata et al., unpub. data). Consequently, frequency of transmission of PCV7 serotypes from children to adults would be expected to decrease. In this study, we found epidemiologic changes among pneumococcal strains isolated from adult patients with IPD; vaccine-type strains decreased significantly (6)(7)(8)(9). However, the prominent decrease in serotype 6B detected by our surveillance differed from findings in the United States, where serotype 14 decreased most prominently after PCV7 introduction (14). IPD cases in Japan resulting from serotype 14 decreased significantly for adults during our ongoing surveillance in 2014. Given Japan's high population density, effects of herd immunity were likely present in children and adults. However, non-PCV7 types, especially serotype 22F, increased.
A decrease in penicillin-nonsusceptible S. pneumoniae strains has been associated with serotype changes occurring after vaccine introduction (29,30,32). In our previous genetic studies, the gPRSP with 3 PBP alterations encoded by the pbp1a, pbp2x, and pbp2b genes also decreased significantly (19). However, our current study found increases in the gPISP with pbp2x gene alterations that reduce susceptibilities to cephalosporin agents (e.g., cefotaxime and ceftriaxone), rather than reducing susceptibility to penicillin. This trend likely reflects preference among medical practices in Japan for cephalosporin and macrolide agents.
We also identified some gPRSPs not previously reported as new STs. Of 7 STs identified in gPRSP and non-PCV7 serotypes, 4 STs had been posted in the pneumococcal database of the MLST website: ST558 (CC558) of serotype 35B, identified in the United States (33); ST63 (CC63) of serotype 15A derived from Sweden 15A -25; ST282 (CC81) of serotype 6D in South Korea (34,35); and ST83 (CC81)   (37,38) indicated occurrence of capsular switching by genetic recombination among strains of different serotypes. Recombination of regions consisting of the capsular cps gene locus and 2 adjacent pbp1a and pbp2x genes results in capsular switching and penicillin resistance. A pneumococcal strain arising from such an event will show high penicillin resistance from mutations in pbp genes because of selection pressure from antimicrobial drugs. MLST and genotypic results of gPRSPs identified in non-PCV13 strains in this study support occurrence of such genetic events.
In relation to serotypes and IPDs in adults, PPSV23 coverage has been reported to differ by disease (39). Our results show respective coverages of PCV13 and PPSV23 to be 73.9% and 84.3% for patients with pneumonia and 45.7% and 69.3% for patients with meningitis. This finding supports recommendations by the ACIP (5) for routine vaccinations with both PCV13 and PPSV23 in all adults >65 years of age, especially those with serious underlying diseases. For pneumococci with a large number of capsular types, development of a novel vaccine against components other than the capsule is expected (40). Overuse of antimicrobial agents should be avoided worldwide to avoid selection of novel resistant strains capable of undermining vaccine efficacy.
This study was funded in part by a grant (Research on Emerging and Re-emerging Infectious Diseases H22-013) from the Japanese Ministry of Health, Labour and Welfare to K. U.
Dr. Ubukata is a microbiologist at Keio University School of Medicine. Her research interests include molecular epidemiology, particularly of respiratory infections, and comprehensive rapid identification of pathogens. *Results of gPISP (pbp2b) (n = 9) and gPISP(pbp1a+2b) (n = 3) were omitted because of a low number of strains.