Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Isolates Causing Invasive and Noninvasive Pneumococcal Diseases in Korea from 2008 to 2014

Introduction. Streptococcus pneumoniae is an important pathogen with high morbidity and mortality rates. The aim of this study was to evaluate the distribution of common serotypes and antimicrobial susceptibility of S. pneumoniae in Korea. Methods. A total of 378 pneumococcal isolates were collected from 2008 through 2014. We analyzed the serotype and antimicrobial susceptibility for both invasive and noninvasive isolates. Results. Over the 7 years, 3 (13.5%), 35 (10.8%), 19A (9.0%), 19F (6.6%), 6A (6.1%), and 34 (5.6%) were common serotypes/serogroups. The vaccine coverage rates of PCV7, PCV10, PCV13, and PPSV23 were 21.4%, 23.3%, 51.9%, and 62.4% in all periods. The proportions of serotypes 19A and 19F decreased and nonvaccine serotypes increased between 2008 and 2010 and 2011 and 2014. Of 378 S. pneumoniae isolates, 131 (34.7%) were multidrug resistant (MDR) and serotypes 19A and 19F were predominant. The resistance rate to levofloxacin was significantly increased (7.2%). Conclusion. We found changes of pneumococcal serotype and antimicrobial susceptibility during the 7 years after introduction of the first pneumococcal vaccine. It is important to continuously monitor pneumococcal serotypes and their susceptibilities.


Introduction
Streptococcus pneumoniae is one of the most common causes of pneumonia, sepsis, and meningitis and is the leading cause of morbidity and death worldwide in adults and children [1][2][3]. Ninety-two capsular serotypes of S. pneumoniae exist, and the prevalence of serotypes differs according to age, region, and time of the surveillance [4,5]. The 92 serotypes differ in virulence; a minority of serotypes is involved in most of invasive pneumococcal diseases and antimicrobial resistances.
The 23-valent polysaccharide vaccine (PPV23) and a 7valent pneumococcal conjugate vaccine (PCV7) were recommended for the elderly (≥65 years old) and children (≤5 years old), respectively. In Korea, PCV7, which protects against the important invasive serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F), was introduced in 2003 for infants and young children. The introduction of PCV7 in the United States produced a decrease in both invasive and noninvasive pneumococcal diseases caused by these vaccine serotypes [6,7]. However, use of PCV7 has led to changes in prevalent serotypes; it tended to increase the PCV7 nonvaccine serotypes, especially 19A, worldwide [8][9][10]. A second pneumococcal conjugate vaccine (PCV10 and PCV7 with serotypes 1, 5, and 7F added) and a 13-valent vaccine (PCV13 and PCV10 with serotypes 3, 6A, and 19A added) were introduced in Korea in 2010. Since May 2014, pneumococcal vaccination has been provided for 2 BioMed Research International free as a routine national vaccine program, including PCV10, PCV13, and PPSV23 in South Korea.
Since the first detection of S. pneumoniae with high resistance to penicillin and other antibiotics in 1977, high rates of antimicrobial resistance in S. pneumoniae have been a serious concern worldwide [11][12][13]. In Asian countries, betalactam and macrolide resistance are very high, and multidrug resistance (MDR) also is common [4,[14][15][16]. In 2008, the Clinical Laboratory and Standard Institute (CLSI) guideline changed the resistance breakpoint of nonmeningitis S. pneumoniae for penicillin from ≥2 g/mL to ≥8 g/mL [17]. Later, the resistance rate to penicillin decreased significantly; however, the high resistance rates to other antimicrobial agents have continued [18][19][20]. The aim of this study was to evaluate the changes in the prevalence of serotypes and their antimicrobial resistance during the past 7 years in Korea since the introduction of the vaccines.

Clinical Isolates.
All 378 S. pneumoniae isolates collected from patients at a tertiary-care hospital in Korea from January 2008 to June 2014 were included. The isolates were identified by colony morphology, gram staining, optochin susceptibility, and other biochemical reactions using VITEK2 system. All isolates were stored at −70 ∘ C using 10% skim-milk until use.

Antimicrobial Susceptibility.
Antimicrobial susceptibilities were determined using the Microscan system, and the susceptibility interpretive criteria were those published in the relevant guidelines of the Clinical and Laboratory Standards Institute (CLSI). Separate interpretive breakpoints were used to define the resistance to penicillin, cefepime, cefotaxime, and ceftriaxone for meningeal isolates. The following antimicrobial agents were tested: amoxicillin, azithromycin, cefaclor, cefepime, cefotaxime, ceftriaxone, cefuroxime, chloramphenicol, clindamycin, erythromycin, levofloxacin, meropenem, penicillin, tetracycline, sulfamethoxazole/trimethoprim (SXT), and vancomycin. The Food and Drug Administration defines multiresistance as resistance to two or more of the five classes of antibacterial agents represented by erythromycin, cefuroxime, SXT, penicillin, and tetracycline [22]. The resistance to cefotaxime and ceftriaxone was analyzed instead of that to cefuroxime and SXT. Macrolide resistance was defined by the erythromycin susceptibility test results.
We assessed differences in serotypes by age group, clinical specimens, surveillance periods, and resistance types. The data was analyzed with the software IBM SPSS version 22, using chi-square test.
The nonsusceptibility rate to penicillin was 26.8%, including 9.0% resistant and 17.8% intermediate. In children, the rates of penicillin resistance and intermediate susceptibility (20.0% and 46.7%, resp.) were significantly higher than in the elderly (8.5% and 17.1%) and younger adults (8.5% and 15.5%). In invasive S. pneumoniae isolates, the resistant and intermediate rates for penicillin were 6.7% and 16.7%, respectively. The rate of resistance of cefuroxime was high as 66.1%, and the resistance rates to cefotaxime and ceftriaxone were low, 4.1% and 4.7%, respectively. The resistance rate to levofloxacin was 7.2%, with the highest rate being 10.3% in the elderly. Also we found that the resistance rate to levofloxacin increased from 3.6% in period I to 11.7% in period II among all isolates ( value = 0.003).
Among the major serotypes, serotype 3 expressed lowlevel resistance to nine major antimicrobial agents. Serotypes   was decreased to 53 isolates (29.4%) in period II when the vaccines were available. Although the resistance rate to penicillin was significantly decreased, to 3.6% from 13.3%, the resistance rate to levofloxacin was increased to 11.7% from 3.6%.

Discussion
This study describes the serotype and antimicrobial resistance of S. pneumoniae isolates in a tertiary-care hospital in Korea. We evaluated the change in the common serotypes and antimicrobial susceptibility patterns between 2008 and 2014.  [25]. However, we also verified the change in the serotype's distribution after the introduction of PCV10 and PCV13, including the decline in the prevalence of serotype 19A from 11.1% in 2008-2010 to 6.7% in 2011-2014. On the other hand, serotypes 3 and 6A were slightly increased between 2008-2010 and 2011-2014.
We could see differences in the distribution of serotypes between invasive and noninvasive isolates. Ten serotypes (1,8,37, 11B, 15A, 17A, 17F, 18C, 23B, and 23F) were detected only among noninvasive isolates (7.1%), whereas serotype 12F was detected only as an invasive isolate. Also, the prevalence of serotype 19F was higher among noninvasive (7.7%) than invasive (1.5%) isolates, and serotype 22F was more common among invasive (5.9%) than noninvasive (1.9%) isolates. We also found a difference in the serotypes of S. pneumoniae by age. The common serotypes in children were 19A, 11A, and 23A, whereas serotypes 3 and 35 were predominant in adults. The lower occurrence of serotype 3 among the children has been reported in another study also [26]. A limitation of this study was the small numbers (only 4.2%) of isolates from children under 5 years who are the primary target age group for PCV vaccination.
Our findings confirm the previously reported high rates of resistance to macrolides such as erythromycin (73.3%), azithromycin (72.0%), and clindamycin (56.9%). The resistance rate to penicillin was higher (9.4%) in the invasive isolates in period I than in the ANSORP study [4] in Korea from 2009 to 2010 (0.3%). Also, the nonsusceptibility rate to penicillin was high in invasive isolates (23.4%) compared with the report of Park et al. [21] (10.7%) from 2009 to 2014. However, we found that the resistance rate to penicillin declined from 9.4% in period I (2008-2010) to 3.6% in period II (2011-2014) among invasive isolates. The interesting thing was that the intermediate resistance rate to penicillin increased slightly over the course of the study, from 15.6% to 17.9% among invasive isolates. We found that the resistance rate to levofloxacin increased significantly, from 3.6% in period I to 11.7% in period II among all isolates ( value = 0.003). Therefore, we need to continuously investigate the susceptibility rates for penicillin and levofloxacin.
The resistance rates in children were higher than those in other age groups, with 20% of isolates resistant to penicillin. Resistance to levofloxacin was not detected in children, whereas resistance was common in those more than 65 years old (10.3%). We do not know the exact reason why the resistant rates to levofloxacin have increased in the older age group. However, we guess the increasing use of levofloxacin is strongly associated with the higher resistance rates because the fluoroquinolone is commonly used as a choice of drug in adults for the respiratory tract infection while that is hardly used in young children. The resistance rates of noninvasive isolates were higher than those of invasive isolates; the rate of levofloxacin resistance was twice as high.
The proportion of MDR S. pneumoniae isolates, 34.7%, in all isolates and serotypes 19A, 19F, and 6A showed predominant serotypes in MDR S. pneumoniae. In children, the proportion of MDR S. pneumoniae was higher (68.8%) than that in other age groups (36.5% and 29.1% for younger adults and the elderly, resp.). The rate of MDR S. pneumoniae was decreased from 39.4% in 2008-2010 to 29.4% in 2011-2014. The reduction of MDR S. pneumoniae was associated with a decline in the proportion of 19A serotypes.
In this study, we evaluated the change of serotype distribution and antimicrobial susceptibility of all S. pneumoniae isolates in Korea over 7 years. We found a decrease of serotypes 19A and 19F and an increase in nonvaccine serotype 35. There were characteristic findings showing a high nonsusceptibility rate to penicillin in children and high resistance rates to levofloxacin. Therefore, we need continuous monitoring for changes of serotype and appropriate main antimicrobial agents.