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.

Streptococcus pneumoniae is a common respiratory pathogen that is the leading cause of community-acquired pneumonia (). 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 (,). 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) (). Previous studies have shown that most of the S. pneumoniae strains with reduced susceptibility to the fluoroquinolone levofloxacin exhibit a multidrug-resistant (MDR) phenotype (,). Levofloxacin resistance was closely associated with epidemic MDR clones (). 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 (,). 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, university-affiliated hospital in South Korea. We determined MICs by using the broth microdilution method according to Clinical and Laboratory Standards Institute guidelines (). 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 (). We sequenced QRDRs of the gyrA, gyrB, parC, and parE genes in each isolate (). We performed multilocus sequence typing to investigate the genetic backgrounds of fluoroquinolone-resistant pneumococci () and assigned allele numbers and STs by using the PubMLST database (http://pubmlst.org/spneumoniae).

Of the 608 clinical S. pneumoniae isolates, 16 (2.6%) were levofloxacin resistant (MIC >8 μg/mL). We collected 1 resistant isolate in 2009, 3 in 2012, 5 in 2013, and 7 in 2014. Thirteen isolates were from sputum, and 3 isolates were from bronchial lavage. The mean age of patients was 71 years; 14 were male, and 2 were female.

Serotype 11A (n = 9) was most common among the levofloxacin-resistant isolates, followed by serotypes 13 (n = 2), 19F (n = 2), 23F (n = 2), and 6B (n = 1) (Table 1). The most common STs were ST9875 (n = 5), ST8279 (n = 3), and ST9876 (n = 3), which together accounted for 11 of the 16 levofloxacin-resistant isolates. Nine isolates of ST9875, ST9876, and ST10300 were novel STs and had not been identified before this study.

Table 1

Select characteristics of 16 levofloxacin-resistant Streptococcus pneumoniae clinical isolates identified from patients at a hospital in South Korea, 2009–2014*†

Strain	Age, y/sex of patient	Specimen type	Respiratory disorders	Underlying disorders	Serotype	Sequence type
HM-646	36/M	Sputum	Pneumonia	CVA	11A	9875‡
HM-669	70/M	Sputum	Pneumonia	CVA	11A	9875‡
HM-683	77/M	Sputum	Pneumonia	COPD	6B	3173
HM-688	81/M	Sputum	Pneumonia	Cardiac infarction	23F	9876‡
HM-730	77/M	Sputum	Dyspnea with fever	Cervical pain	13	189
HM-762	76/F	Sputum	Pneumonia	Lung cancer	13	8279
HM-781	70/M	Sputum	Pneumonia	CVA	23F	6721
HM-787	35/M	Sputum	Pneumonia	CVA	11A	9875‡
HM-809	58/M	Sputum	Pneumonia	CVA	11A	9875‡
HM-854	77/M	BL	Pneumonia	Lung cancer	11A	99
HM-878	67/M	BL	Pneumonia	ALS	11A	8279
HM-953	82/M	Sputum	Pneumonia	COPD	11A	9875‡
HM-970	68/M	Sputum	Dyspnea with fever	Bronchiectasis	19F	9876‡
HM-1017	85/M	BL	Dyspnea	Lung cancer	11A	8279
HM-1050	62/M	Sputum	Postop atelectasis	CVA	19F	9876‡
HM-1055	89/F	Sputum	Pneumonia	CVA	11A	10300‡

*ALS, amyotrophic lateral sclerosis; BL, bronchial lavage; COPD, chronic obstructive pulmonary disorder; CVA, cerebrovascular accident.
†Among the 16 isolates, 1 (HM-646) was collected in 2009; 3 (HM-669, HM-683, and HM-688) in 2012; 5 (HM-730, HM-762, HM-781, HM-787, and HM-809) in 2013; and 7 (HM-854, HM-878, HM-953, HM-970, HM-1017, HM-1050, and HM-1055) in 2014.
‡Novel sequence type found in our study.

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 (,). Isolate HM-854, which was penicillin susceptible, had Ser81Phe in gyrA and Asp79Asn in parC mutations. All isolates had >1 mutation in parC. The 2 isolates without the Ser79Phe mutation in parC instead carried Asp83Gly or Asp83Asn. The 4 isolates without the Lys137Asn mutation in parC instead carried the Asn91Asp mutation. Isolate HM-1017 (serotype 11A, ST-8279) had 7 QRDR mutations and exhibited the highest resistance against all antimicrobial agents, including levofloxacin (MIC 64 μg/mL). ST-8279 was associated with 2 different serotypes, 11A (n = 2) and 13 (n = 1). The 3 isolates of novel ST-9876 had the same QRDR amino acid changes but had different serotypes, 19F (n = 2) and 23F (n = 1).

The 16 levofloxacin-resistant isolates were also resistant to ofloxacin (MIC >8 μg/mL) and ciprofloxacin (MIC >8 μg/mL) (Table 2). All isolates except 3 had MICs >16 μg/mL against amoxicillin and ceftriaxone. Fourteen isolates were meropenem-resistant (MIC >1 μg/mL); all these isolates were susceptible to vancomycin and linezolid. Only 3 STs (ST-99, ST-189, and ST-3173) exhibited the lowest levofloxacin MIC (8 μg/mL); all these isolates were susceptible to amoxicillin (MIC <2 μg/mL).

Table 2

Antimicrobial susceptibilities of 16 levofloxacin-resistant Streptococcus pneumoniae clinical isolates identified from patients at a hospital in South Korea, 2009–2014*

Strain	MIC, μg/mL (resistance)
	LEV	OFL	CIP†	PEN	AMX	CRO	MER	ERY	CLI	VAN	LZD	TET	TIG†
HM-646	16 (R)	32 (R)	32	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-669	16 (R)	32 (R)	32	>16 (R)	>16 (R)	>16 (R)	8 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-683	8 (R)	16 (R)	16	4 (I)	2 (S)	2 (I)	1 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-688	16 (R)	32 (R)	32	>16 (R)	>16 (R)	>16 (R)	8 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-730	8 (R)	16 (R)	8	4 (I)	2 (S)	2 (I)	0.5 (I)	>16 (R)	>16 (R)	0.5 (S)	0.5 (S)	>16 (R)	0.03
HM-762	32 (R)	64 (R)	32	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	0.5 (S)	>16 (R)	0.015
HM-781	16 (R)	32 (R)	16	16 (R)	16 (R)	>16 (R)	8 (R)	>16 (R)	>16 (R)	0.5 (S)	0.5 (S)	16 (R)	0.03
HM-787	16 (R)	32 (R)	64	16 (R)	16 (R)	>16 (R)	8 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-809	16 (R)	32 (R)	64	16 (R)	>16 (R)	>16 (R)	4 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-854	8 (R)	16 (R)	16	0.06 (S)	0.06 (S)	0.5 (S)	<0.015 (S)	8 (R)	0.06 (S)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-878	16 (R)	32 (R)	32	16 (R)	>16 (R)	>16 (R)	8 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	4 (R)	0.03
HM-953	16 (R)	32 (R)	64	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-970	32 (R)	64 (R)	32	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-1017	64 (R)	128 (R)	64	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03
HM-1050	32 (R)	64 (R)	64	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	0.5 (S)	0.03
HM-1055	16 (R)	32 (R)	128	>16 (R)	>16 (R)	>16 (R)	16 (R)	>16 (R)	>16 (R)	0.5 (S)	1 (S)	>16 (R)	0.03

*AMX, amoxicillin; CIP, ciprofloxacin; CLI, clindamycin; CRO, ceftriaxone; ERY, erythromycin; I, intermediate; LEV, levofloxacin; LZD, linezolid; MER, meropenem; OFL, ofloxacin; PEN, penicillin; R, resistant; S, susceptible; TET, tetracycline; TIG, tigecycline; VAN, vancomycin.
†No susceptibility breakpoints are established for ciprofloxacin and tigecycline.

Most of the 16 isolates in our study were of serotype 11A (n = 9): 5 isolates of ST-9875, 2 of ST-8279, and 1 each of ST-10300 and ST-99. An XDR ST-8279 (serotype 13) clone described in 2014 () was closely related to the 9 serotype 11A isolates in our study. ST-8279 is a double-locus (aroE and xpt) variant of ST-156, which is closely related to global clone Spain9V-3 (). Spain9V-3 is related to 3 ST-3642 isolates (serotype 11A) reported in Taiwan in 2010 () and to 3 MDR ST-166 isolates (serotype 11A) reported in South Korea in 2013 (). In our study, 3 novel STs of MDR S. pneumoniae were identified (ST-9875, ST-9876, and ST-10300). All the ST-8279, ST-9875, and ST-10300 isolates in our study were serotype 11A, with the exception of 1 of the ST-8279 isolates. The ST-9875 and ST-10300 isolates were single-locus variants (in the spi and gki genes, respectively) of ST-8279. ST-9876 is a 1-locus (aroE) variant of an ST-3384 (serotype 9V) clone registered in the PubMLST database.

Serotypes 19F and 23F are included in the 13-valent pneumococcal conjugated vaccine (PCV13), but serotype 11A is not included in PCV13. Serotype 11A is, however, included in the 23-valent pneumococcal polysaccharide vaccine (PPSV23). The US CDC currently recommends the PPSV23 for all adults >65 years of age and all persons 2–64 years of age who are at high risk for pneumococcal disease (). Through national vaccine programs in South Korea, since 2013, PPSV23 has been provided to all adults >65 years of age, and since 2014, 10-valent pneumococcal conjugated vaccine or PCV13 have been provided to young children free of charge ().

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 third-generation cephalosporins.