Novel Levofloxacin-Resistant Multidrug-Resistant Streptococcus pneumoniae Serotype 11A Isolates, South Korea

Of 608 Streptococcus pneumoniae clinical strains isolated at a hospital in South Korea during 2009–2014, sixteen (2.6%) were identified as levofloxacin resistant. The predominant serotype was 11A (9 isolates). Two novel sequence types of multidrug-resistant S. pneumoniae with serotype 11A were identified, indicating continuous diversification of resistant strains.

S treptococcus pneumoniae is a common respiratory pathogen that is the leading cause of community-acquired pneumonia (1). Although β-lactam antibiotics have long been used for the treatment of respiratory diseases, the increasing prevalence of antibiotic-resistant S. pneumoniae strains has hampered treatment in recent decades (2,3). Resistance to fluoroquinolones has emerged in S. pneumoniae and is caused by mutations within short DNA sequences of gyrA and parC genes that encode the type II topoisomerase subunits known as quinolone-resistance determining regions (QRDRs) (1). Previous studies have shown that most of the S. pneumoniae strains with reduced susceptibility to the fluoroquinolone levofloxacin exhibit a multidrug-resistant (MDR) phenotype (2,4). Levofloxacin resistance was closely associated with epidemic MDR clones (3). Although fluoroquinolone resistance rates remain low in S. pneumoniae in most countries, some extensively drug-resistant (XDR) S. pneumoniae isolates have emerged; this resistance is defined as nonsusceptibility to >1 agent in all but <2 antimicrobial categories (2,4). We examined S. pnemoniae isolates from patients in South Korea to determine antimicrobial resistance. We found novel sequence types (STs) of MDR serotype 11A S. pneumoniae that exhibit resistance to second-line antibiotics such as levofloxacin, ceftriaxone, and meropenem.

The Study
During January 2009-December 2014, we isolated 608 S. pneumoniae clinical strains at a 698-bed, universityaffiliated hospital in South Korea. We determined MICs by using the broth microdilution method according to Clinical and Laboratory Standards Institute guidelines (5). We performed antimicrobial resistance tests for levofloxacin, ofloxacin, ciprofloxacin, penicillin, amoxicillin, ceftriaxone, meropenem, erythromycin, clindamycin, vancomycin, linezolid, tetracycline, and tigecycline. We used S. pneumoniae ATCC 49619 as a control strain. We defined MDR as resistance or intermediate resistance to >3 antimicrobial agents.
We determined serotypes by using the multiplex PCR assay recommended by the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/ pcr.htm). Reactions also included an internal positive control targeting all known pneumococcal cpsA regions (6). We sequenced QRDRs of the gyrA, gyrB, parC, and parE genes in each isolate (7). We performed multilocus sequence typing to investigate the genetic backgrounds of fluoroquinolone-resistant pneumococci (8) and assigned allele numbers and STs by using the PubMLST database (http://pubmlst.org/spneumoniae).
All 16 levofloxacin-resistant isolates contained at least 2 amino acid alterations in the QRDRs of the gyrA, parC, and parE genes. Four QRDR mutations occurred with high frequency: Ser81Phe in gyrA was present in all 16 isolates; Ser79Phe and Lys137Asn in parC were present in 14 and 11 isolates, respectively; and Ile460Val in parE was found in 15 isolates. However, Lys137Asn in parC and Asp435Val and Ile460Val in parE are mutations not involved in resistance, according to previous reports (9,10). Isolate HM-854, which was penicillin susceptible, had Ser81Phe in gyrA and Asp79Asn in parC mutations.

Conclusions
In South Korea, serotype 11A was the most predominant serotype of the 16 levofloxacin-resistant and XDR S. pneumoniae isolates we found. Seven levofloxacin-resistant S. pneumoniae strains were isolated in 2014 alone; the dominant serotype was again 11A (n = 5). All except 1 of these 7 serotype 11A isolates were resistant to the 9 different antimicrobial agents tested. We identified 3 novel STs of MDR serotype 11A S. pneumoniae in our study. S. pneumoniae serotype 11A isolates with novel STs require careful monitoring to combat the increasing prevalence and diversification of MDR pneumococcal strains, especially those with resistance to fluoroquinolones, β-lactams, and thirdgeneration cephalosporins.