Association Between Pneumococcal Surface Protein A Family and Genetic/Antimicrobial Resistance Traits of Non-Invasive Pneumococcal Isolates from Adults in Northern Japan

Abstract

Pneumococcal isolates from adult patients in northern Japan in 2016 were subjected to molecular investigation related to pneumococcal surface protein A (PspA) and drug resistance determinants. Of the 51 isolates, serotype 3/ST180 was the most prevalent (17.6%), followed by 35B (ST2755/ST558) (11.8%) and 15A (ST63/ST7874/ST13068/ST13785) (9.8%). Coverage of serotypes by 13-valent conjugate vaccine and 23-valent polysaccharide vaccine was 27.5% and 49%, respectively. All the isolates expressed PspA family 1 or 2 (51% and 49%, respectively). Each serotype was associated with either of the PspA families (e.g., serotype 3, PspA family 1; serotypes 35B and 15A, PspA family 2). Multidrug resistance (MDR) was found in 84.3% of the isolates. Minimum of one altered penicillin-binding protein gene was detected in 82.4% of isolates, indicating 25.5% non-susceptibility to penicillin. Serotypes 15A and 35B were predominant and demonstrated MDR. An isolate of serotype 15A/ST13785 (single-locus variant of ST242) was resistant to fluoroquinolones associated with double mutation in the quinolone resistance-determining regions of gyrA and parC. The present study indicates the spread of MDR pneumococci represented by isolates of serotypes 3, 15A, and 35B, and prevalence of both PspA family 1 and 2 in isolates obtained from adult patients.

Introduction

Streptococcus pneumoniae (or pneumococcus) is an important bacterial pathogen that causes both invasive and non-invasive diseases such as septicemia, meningitis, and pneumonia.¹ In Japan, a 7-valent pneumococcal conjugate vaccine (PCV7), licensed for voluntary immunization of children aged <5 years in 2010, reached an immunization rate of 90% in 2 years.² PCV7 was introduced into the national immunization program for 7 months starting from April 2013 to October 2013 until it was replaced by 13-valent pneumococcal conjugate vaccine (PCV13) in November 2013. Since 2014, the 23-valent pneumococcal polysaccharide vaccine (PPSV23) was recommended for routine vaccination at specific ages at every 5 years in the elderly aged ≥65 years. According to the view (2015–2018) proposed by the Japanese Respiratory Society and the Japanese Association for Infectious Diseases, PCV13 was available for voluntary vaccination of unvaccinated elderly or elderly who received PPSV23 vaccine a year ago or earlier. In 2017, the immunization rate of PPSV23 was 54%, despite its inclusion in the routine vaccination program.³

In Japan, although PCV7 serotype-associated pneumococcal diseases decreased shortly after the first introduction of PCV7, there was an increase in non-pneumococcal conjugate vaccine (non-PCV) serotypes-associated pneumococcal diseases.^4,5 Similar findings were reported from the United States, Europe, and Asia.^6–10 As an indirect effect of the widespread use of PCVs in children, there was a persistent increase in the number of non-PCV serotypes within pneumococcal isolates across all age groups.^11–13 In addition, the predominant non-vaccine serotype-related strains were significantly associated with antibiotic resistance.^14,15 Thus, the emergence and spread of pneumococcal clones of non-vaccine serotypes present further concern to public health worldwide.

The current pneumococcal vaccines, PCV13 and PPSV23, provide protection against the only capsular serotypes (13 and 23 serotypes, respectively) included in the vaccines among the nearly 100 different serotypes.¹⁶ Pneumococcal surface protein (PspA), a highly immunogenic surface protein present on virtually all pneumococcal strains,^17–20 is a potential vaccine candidate for the future. PspA immunization can protect against infections caused by various pneumococcal serotypes and has gained particular research interest in recent years.²¹ PspA proteins are classified into three families, which include six clades based on the C-terminal 100 amino acid of the α-helix region (clade-defining region), possessing diverse antigenicity.²² Although PspA protein could induce cross-reactive antibodies to heterologous PspA molecules at varying degrees, in vivo, immunization with a single PspA protein could not confer adequate protective immunity against all the families/clades.²³ Thus, for designing a successful vaccine, information related to the prevalence and antigenic/genetic diversity of pspA among different serotypes is essential. In our previous study, we detected the presence of PspA in nearly all pneumococcal isolates (99.6%) of pediatric patients.²⁴ However, limited information is available on the prevalence of PspA in isolates of non-PCV13/PPSV23 serotypes, non-vaccine types (NVTs) from adult patients.

The present study aimed to analyze the prevalence of serotypes and its relevance to the PspA family. In addition, the molecular characteristics and associated antimicrobial resistance (AMR) of pneumococcal isolates obtained from adult patients 6 years after sequential immunization with 7- and 13-valent vaccines for children were analyzed. Further, the prevalence of serotypes with altered penicillin-binding protein (PBP) genes was compared with that of our molecular epidemiological study related to the PCV7 baseline year 2011.²⁵

Materials and Methods

Pneumococcal isolates

From hospitals and clinics in Hokkaido prefecture, Japan, 51 non-duplicate pneumococcal isolates were recovered from adult patients with non-invasive pneumococcal disease (IPD) (samples collected between June 2016 and November 2016) and analyzed. Isolates were collected from clinical specimens obtained from non-sterile sites such as pus, sputum, nasal, and eye discharge. These were subsets of all pneumococcal isolates obtained from Sapporo Clinical Laboratory, Inc. During the study period, of the 800 isolates recovered from all age groups, the first nearly 100 initial isolates were taken each month for the study. Of the total isolates, 678 were recovered from children, and 51 obtained from adults were analyzed in the present study. Of the 51 isolates, 37 (72.5%) were obtained from patients within the age group of 16–64 years, and 14 (27.5%) were aged ≥65 years. The male-to-female ratio was 1.2. All isolates were stored in Microbank (Pro-lab Diagnostics, Richmond Hill, Canada) at −80°C until further use. Before the analyses, the isolates were grown on Trypticase^® soy agar substituted with 5% sheep blood agar (Nippon Becton Dickinson) for 24 h at 37°C with 5% CO₂.

Molecular typing of S. pneumoniae

Pneumococcal serotypes were assigned using conventional multiplex-PCR (cm-PCR)²⁶ and PCR-based sequencing methods as described previously.^5,24,25,27 Three serotypes, 24F/24A/24B, assigned by cm-PCR were genetically differentiated based on rbsF gene (unique to serotype 24F/B) and mutations in abp1 and rmlC (unique to serotype 24B),²⁸ via PCR with primers designed using the sequences from GenBank (accession nos. CR931688 and CR931687). Primer sequences of rbsF, abp1, and rmlC used in the present study were [24.rbs-F (5′-GTGATTTTATTTGAATGGTC-3′) and 24.rbs-R (5′-ATCTTCAAGATTATAGTCGG-3′)], [24F/B.abp1-F (5′-CCAGAAATTGATGAAATCTGT-3′) and 24F/B.abp1-R (5′-TGTTCGTCTCAACAAGTCACG-3′)], and [24F/B.rmlC-F (5′- GCTTCCACTTGGATTTCCAG-3′) and 24F/B.rmlC-R (5′-GCATACTTGGGTTTGAGTTC-3′)], respectively. PspA family typing was achieved with PCR as described previously.²⁰ PBP genes pbp1a, pbp2x, and pbp2b, and macrolide resistance genes [erm(B) and mef(A/E)] were detected and characterized as described previously.^29,30 PBP genotypes are represented as gPRSP (alteration in three pbp genes), gPISP (alteration in one or two pbp genes), and gPSSP (absence of alteration in the three pbp genes). Altered PBP gene(s) in gPISP are represented in parentheses, for example, gPISP (pbp2b).³¹ Sequence type (ST) of all isolates was determined based on the multilocus sequence typing (MLST) scheme for pneumococcus (http://pubmlst.org/). ST of each isolate compared with the Pneumococcal Molecular Epidemiology Network (PMEN) International Clones (www.sph.emory.edu/PMEN).

Antimicrobial susceptibility testing and genetic analysis of quinolone resistance

Broth microdilution test was used to measure the minimum inhibitory concentration (MIC) within a limited concentration range of ten antimicrobial agents (penicillin [PEN], erythromycin [ERY], tetracycline [TET], clindamycin [CLI], trimethoprim-sulfamethoxazole [SXT], ceftriaxone [CRO], cefaclor [CEC], imipenem [IPM], levofloxacin [LVX], and vancomycin [VAN]) against all isolates with Dry Plate Eiken HW14 (Eiken, Tokyo, Japan). The Clinical and Laboratory Standards Institute (CLSI) criteria³² were applied to MICs to classify the isolates as susceptible (S), intermediate (I), or resistant (R). PEN resistance is defined as the breakpoint for oral PEN V, MIC ≥2 μg/mL. Multidrug resistance (MDR) is defined as resistance to three or more different antimicrobial classes.^33,34

For an isolate (SRP2368) that demonstrated resistance to LVX (MIC ≥8 μg/mL), susceptibility to fluoroquinolones (FQs) (LVX and ciprofloxacin [CIP]) was further analyzed by the standard broth microdilution method, and mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) were analyzed as described previously.³⁵ Full-length nucleotide sequences of gyrA and parC genes were determined with PCR and direct sequencing.

Nucleotide sequence accession numbers

The nucleotide sequences of gyrA and parC of the isolate (ID: SRP2368) were deposited in the GenBank database with accession nos. MH279935 and MH 279936, respectively.

Results

All isolates were classified into 16 serotypes and 2 PspA family types (Table 1). The most prevalent serotype was 3 (17.6%), followed by 35B (11.8%), 15A (9.8%), 19A (7.8%), and 15B (7.8%). PCV13 and PPSV23 serotypes in the isolates accounted for 27.5% and 49.0%, respectively. All isolates expressed either PspA family 1 (51.0%) or family 2 (49.0%). Isolates of serotypes 19F, 3, 10A, 22F/22A, 33F, 6E, 23A, 24F, and 34 had PspA family 1, while those of serotypes 19A, 11A/11D, 35B and serogroup 15 were associated with family 2 PspA. Only isolates of serotype 6C expressed either PspA family 1 (one isolate belonging to ST2924) or family 2 (two isolates belonging to ST2923 and its single locus variant [SLV]).

Table 1.

Distribution of Serotypes with PspA Family Types for All Isolates

Vaccine type	Serotype	No. of isolates
		Total (%)	PspA
		Total (%)	Family 1	Family2
		PCV7 serotype^a	19F	1 (2.0)	1	0
PCV13^b/PPSV23 serotype	3	9 (17.6)	9	0
	19A	4 (7.8)	0	4
PPSV23^c serotype	10A	2 (3.9)	2	0
	11A/11D	3 (5.9)	0	3
	15B	4 (7.8)	0	4
	22F/22A	1 (2.0)	1	0
	33F	1 (2.0)	1	0
Non-vaccine serotype (non-PCV13/PPSV23)	6C	3 (5.9)	1	2
	6E	3 (5.9)	3	0
	15A	5 (9.8)	0	5
	15C	1 (2.0)	0	1
	23A	2 (3.9)	2	0
	24F	3 (5.9)	3	0
	34	3 (5.9)	3	0
	35B	6 (11.8)	0	6
	Total	51	26	25
	(%)	(100)	(51.0)	(49.0)

PCV7: 4, 6B, 9V, 14, 18C, 19F, and 23F.

PCV13: PCV7 + 1, 3, 5, 6A, 7F, and 19A.

PPSV23: PCV7 + 1, 2, 3, 5, 7F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 19A, 20, 22F, and 33F.

PCV7, 7-valent pneumococcal conjugate vaccine; PCV13, 13-valent pneumococcal conjugate vaccine; PPSV23, 23-valent pneumococcal polysaccharide vaccine.

Molecular characteristics with AMR profiles for all isolates are shown in Table 2. None of the isolates was resistant to CRO, IPM, and VAN. High rates of antibiotic non-susceptibly were exhibited toward ERY (98.0%), TET (96.1%), CLI (76.5%), and SXT (74.5%), while 84.3% of all isolates exhibited MDR. The proportion of PEN-non-susceptible S. pneumoniae (PNSP) was higher among the isolates of non-PCV13/PPSV23 serotypes (NVTs) (46.2%) than among PCV13/PPSV23 serotypes, vaccine serotypes (VTs) (12.0%), and all the isolates of NVTs 6E, 15A, and 23A were PNSP. Significantly, an isolate belonging to serotype 15A was resistant to LVX. Macrolide resistance genes, erm(B), mef(A/E), and both the genes together were detected in 82.4%, 19.6%, and 3.9% of the isolates, respectively, while only one isolate did not have any of the macrolide resistance genes. A single altered PBP gene was detected in 82.4% of the isolates. The most prevalent pbp genotype was gPISP (pbp2x) (50.1%), followed by gPRSP (pbp1a+2x+2b) (19.6%) and gPSSP (17.6%). gPRSP was confirmed in serotypes 19F, 19A, 6C, 6E, 15A, and 35B, which were non-susceptible to PEN. All the gPSSP and gPISP (pbp2x) isolates were susceptible to PEN.

Table 2.

Molecular Epidemiological Characterization and Antimicrobial Resistance of 51 Pneumococcal Isolates

Serotype (total no. of isolates)	No. of isolates	PspA family type	ST ^a	MLST allelic profile ^b	ST, registered as PMEN clone	pbp genotype	Macrolide resistance gene		Antimicrobial resistance profiles
							erm(B)	mef(AE)	PEN	ERY	TET	CLI	SXT	CRO	CEC	IPM	LVX	VAN
							PCV7 serotype
19F (1)	1	1	926	15-16-19-15-6-20-14	SLV of ST236/Taiwan^19F-14	gPRSP	−	+	I	R	R	S	I	S	R	S	S	S
PCV13/PPSV23 serotype
3 (9)	9	1	180	7-15-2-10-6-1-22	Netherlands³-31	gPISP(pbp2x)	+	−	S	R	R	R	S	S	S	S	S	S
19A (4)	1	2	2331	10-16-150-1-17-1-29		gPISP(pbp2x)	−	+	S	R	R	S	S	S	S	S	S	S
	1	2	2331	10-16-150-1-17-1-29		gPSSP	+	−	S	R	R	R	I	S	S	S	S	S
	1	2	13784	10-13-150-1-17-121-29		gPISP(pbp2x)	−	+	S	R	R	S	I	S	S	S	S	S
	1	2	3111	61-60-67-16-10-104-14		gPRSP	+	+	I	R	R	R	S	S	R	S	S	S
PPSV23 serotype
10A (2)	1	1	5236	7-12-1-1-10-1-11	DLV of ST113/Netherlands^18C-36	gPISP(pbp2x)	+	−	S	I	R	S	S	S	S	S	S	S
10A (2)	1	1	5236	7-12-1-1-10-1-11	DLV of ST113/Netherlands^18C-36	gPSSP	+	−	S	R	R	R	S	S	S	S	S	S
11A/11D (3)	1	2	99	5-8-4-16-6-1-31		gPISP(pbp1a+2x)	+	+	I	R	R	S	I	S	R	S	S	S
	1	2	99	5-8-4-16-6-1-31		gPISP(pbp1a+2x)	−	+	S	R	R	S	I	S	R	S	S	S
	1	2	99	5-8-4-16-6-1-31		gPSSP	−	+	S	R	R	S	S	S	S	S	S	S
15B (4)	4	2	199	8-13-14-4-17-4-14	Netherlands^15B-37	gPISP(pbp2x)	+	−	S	R	R	R	S	S	S	S	S	S
22F/22A (1)	1	1	433	1-1-4-1-18-58-17		gPISP(pbp2x)	+	−	S	R	R	R	S	S	S	S	S	S
33F (1)	1	1	717	5-35-29-1-45-39-18		gPSSP	+	−	S	R	I	R	S	S	S	S	S	S
Nonvaccine serotype
6C (3)	1	2	2923	2-13-2-5-6-121-29		gPISP(pbp2x)	−	+	S	R	R	S	I	S	S	S	S	S
	1	2	6413	2-13-2-5-6-124-14		gPRSP	−	+	I	R	R	S	I	S	R	S	S	S
	1	1	2924	1-5-2-6-6-1-14		gPISP(pbp2x)	+	−	S	R	R	R	I	S	S	S	S	S
6E (3)	3	1	90	5-6-1-2-6-3-4	Spain^6B-2	gPRSP	+	−	I	R	R	R	R	S	R	S	S	S
15A (5)	1	2	63	2-5-36-12-17-21-14	Sweden^15A-25	gPRSP	+	−	I	R	R	R	S	S	S	S	S	S
	1	2	63	2-5-36-12-17-21-14	Sweden^15A-25	gPISP(pbp1a+2x)	+	−	I	R	R	R	I	S	S	S	S	S
	1	2	7874	2-5-359-12-17-21-14	SLV of ST63/Sweden^15A-25	gPISP(pbp1a+2x)	+	−	I	R	R	R	S	S	I	S	S	S
	1	2	13068	2-5-36-12-17-777-14	SLV of ST63/Sweden^15A-25	gPRSP	+	−	I	R	R	R	I	S	I	S	S	S
	1^c	2	13785	15-29-4-21-14-1-14	SLV of ST242/Taiwan^23F-15	gPRSP	+	−	I	R	R	R	I	S	R	S	R	S
15C (1)	1	2	199	8-13-14-4-17-4-14	Netherlands^15B-37	gPISP(pbp2x)	+	−	S	R	R	R	S	S	S	S	S	S
23A (2)	2	1	5242	7-13-8-6-1-337-8	SLV of ST338/Colombia^23F-26	gPISP(pbp2x+2b)	+	−	I	R	R	R	S	S	R	S	S	S
24F (3)	2	1	2572	7-75-9-6-25-6-14		gPSSP	+	−	S	R	R	R	I	S	S	S	S	S
24F (3)	1	1	2572	7-75-9-6-25-6-14		gPSSP	+	−	S	R	R	R	R	S	S	S	S	S
34 (3)	1	1	3116	10-8-6-1-9-1-279		gPISP(pbp2x)	−	−	S	S	S	S	I	S	S	S	S	S
	1	1	7388	5-5-7-16-9-1-14		gPSSP	+	−	S	R	R	S	I	S	S	S	S	S
	1	1	7388	5-5-7-16-9-1-14		gPSSP	+	−	S	R	R	R	R	S	S	S	S	S
35B (6)	4	2	2755	10-12-2-1-152-28-14		gPISP(pbp2x)	+	−	S	R	R	R	R	S	S	S	S	S
	1	2	2755	10-12-2-1-152-28-14		gPISP(pbp2x)	+	−	S	R	S	R	R	S	S	S	S	S
	1	2	558	18-12-4-44-14-77-97	SLV of ST377/Utah^35B-24	gPRSP	−	+	I	R	R	S	I	S	R	S	S	S
Total	51						Total (%)	R	0	96.1	94.1	76.5	19.6	0	23.5	0	2.0	0
								R + I	25.5	98.0	96.1	76.5	74.5	0	27.5	0	2.0	0

Identified as a new ST in this study, shown in bold.

Gene locus number in SLV and DLV of PMEN clones is underlined.

Isolate with LVX resistance had double mutations within the QRDRs of gyrA (Ser81Phe) and parC (Asp83Try).

PEN, penicillin; ERY, erythromycin; TET, tetracycline; CLI, clindamycin; SXT, trimethoprim-sulfamethoxazole; CRO, ceftriaxone; CEC, cefaclor; IPM, imipenem; LVX, levofloxacin; VAN; vancomycin; S, susceptible; I, intermediate resistance; R, resistance; ST, sequence type; MLST, multilocus sequence typing; PMEN, Pneumococcal Molecular Epidemiology Network; SLV, single locus variant; DLV, double locus variant; QRDR, quinolone resistance-determining region.

The present study identified two novel STs, ST13784 (double locus variant [DLV] of ST2331) belonging to serotype 19A, and ST13785 (SLV of ST242/Taiwan^23F-15) belonging to serotype 15A, from 51 isolates. STs and their SLV/DLV registered as PMEN clones were detected in 28 isolates (54.9%). All the isolates of serotype 3 belonging to ST180 (PMEN clone Netherlands³-31) were non-susceptible to ERY, TET, and CLI. Isolates of serotypes 15B and 15C assigned to ST199 (PMEN clone Netherlands^15B-37) were resistant to ERY, TET, and CLI. Concerning the PNSP isolates of NVTs, the isolates of serotypes 6E and 23A belonging to ST90 (PMEN clone Spain^6B-2) and ST5242 (SLV of ST338/Colombia^23F-26), respectively, were resistant to four or five classes of antibiotics. One of the six isolates of serotype 35B belonging to ST558 (SLV of ST377/Utah^35B-24) was a PNSP.

Four of the five isolates of serotype 15A belonged to either ST63 (PMEN clone Sweden^15A-25) or its SLV that was resistant to ERY, TET and CLI, while the last isolate assigned to ST13785 (SLV of ST242/Taiwan^23F) was resistant to five classes of antibiotics. The only isolate of serotype 15A/ST13785 was resistant to LVX/CIP showing MICs of 16 μg/mL each, associated with double mutations within the QRDRs of gyrA (Ser81Phe) and parC (Asp83Try).

Figure 1 shows the comparison of PBP genotypes in each serotype between our previous study (2011; n = 63)²⁵ and the present study (2016; n = 51). Serotypes 10A, 33F, 15A, 24F, and 34 were found only in 2016. Among the NVT serotypes of 2016, three of the five isolates of serotype 15A (60%) were assigned to gPRSP, while serotype 35B with gPISP (pbp2x) accounted for 83.3% in 2016, which were higher rates than that in 2011. Serotype 3 was the most predominant and represented gPISP (pbp2x).

FIG. 1.

Comparison of PBP genotypes in each serotype between our previous study (2011; n = 63)²⁵ and present study (2016; n = 51). A part of isolates assigned to serotypes 6A (n = 5/6) and 6B (n = 1/2) in the previous study (2011) were confirmed as the novel serotype 6E.²⁷ Pneumococcal surface protein A (PspA) family types 1 and 2 detected in the 2016 isolates are shown in gray and dark gray, respectively. PBP, penicillin-binding protein.

Discussion

In the present study, all the predominant isolates of serotype 3 expressing PspA family 1 and identical antibiotic resistance patterns were assigned to ST180 (Netherlands³-31). The serotype 3/ST180 lineage was found to be predominant globally,^36,37 and was documented as a common lineage in adults.^38–40 This serotype was mainly isolated from IPD adult patients from pre-PCV to post-PCV period.⁴¹ In Greece, serotype 3 accounted for 33% in isolates obtained from PCV13-vaccinated children,⁴² while in Portugal its prevalence in children did not show a significant change.⁴³ A study conducted in Japan indicated a significantly lower antibody response to serotype 3 in the PCV13- than in PPSV23-vaccinated group.⁴⁴ These findings suggest PCV to be potentially less effective against serotype 3 compared with other serotypes. In addition, the lower immunization rate of PPSV23 among Japanese adults aged ≥65 years (∼40% based on the national average in 2016⁴⁵) might be associated with the dominance of serotype 3.

The proportion of PCV13 serotypes was lower (27.5%) in the present study compared with that in our previous surveillance conducted during the PCV7 voluntary immunization period in 2011²⁵ (52.4%) and the PCV7/PCV13 routine immunization period (April 2013–October 2013 and November 2013–November 2014) (50.8% and 43.7%, respectively).⁵ In contrast, 51% of isolates in 2016 were assigned to NVTs, including serotypes 15A and 35B. Recently, a rapid increase of the MDR serotype 15A belonging to ST63 (Sweden^15A-25) was documented in Germany,¹³ the United Kingdom,¹⁵ and Canada.⁴⁶ In our study, all the isolates of serotype 15A exhibited MDR (PNSP with resistance to ERY, TET, and CLI) and mostly belonged to either ST63 or its SLV. Recent studies from Japan have reported the spread of 15A/ST63 MDR clone among children.^47,48 In the present study, an isolate of serotype 35B was assigned to ST558 (SLV of ST377/Utah^35B-24), while the remaining isolates were assigned to ST2755. PCV13 vaccination resulted in the emergence of dominant PNSP 35B/ST558 clone in the United States.⁴⁹ According to the MLST database (http://pubmlst.org.spneumoniae/), serotype 35B/ST2755 clone is found exclusively in Asian countries (China and Japan).^47,48 Thus, our results indicate the spread of 15A/ST63 MDR clone, 35B/ST558, and 35B/ST2755 clones among all age groups during the PCV13 era in Japan.

AMR of pneumococci is a worldwide concern.^45,50 In the present study, most of the isolates (84.3%) were resistant to at least three classes of antibiotics. Recently, comparable high rates of antibiotic resistance were reported in Korea (81.5%)³⁴ and China (88–99.4%).^51,52 In contrast, resistance to FQs, which is due to mutations in the QRDRs, has been rarely reported in Japan, and FQ resistance rate in pneumococci isolated from adult patients was 1.6% based on nationwide surveillance study.⁵³ In the present study, an isolate belonging to serotype 15A/ST13785 (SLV of ST242/Taiwan^23F-15) showed MDR and resistance to FQs, having double mutations within the QRDRs. Such double mutation was described previously in only Taiwan⁵⁴ and Japan.⁵⁵ A recent study from Taiwan has shown a high prevalence of serotype 15A/ST242 pneumococci with FQ-non-susceptibility.⁵⁶ The FQ-resistant 15A/ST13785 (SLV of ST242) isolate identified in the present study was resistant to ERY, TET, CLI, CEC, SXT, and was non-susceptible to PEN representing gPRSP, suggesting the development of significant MDR in pneumococci infecting adults.

In our previous study for pediatric pneumococcal isolates (during the same period as the present study), 99.6% of the isolates expressed PspA (family 1, 42.3%; family 2, 56.6%; family 3, 0.6%).²⁴ These results are comparable with those in the present study for adult pneumococcal isolates (PspA, 100% positive; family 1, 51.0%; family 2, 49.0%). These findings indicate the prevalence of both PspA family 1 and 2 in children and adults during the same period. Accordingly, in the preparation of a successful recombinant PspA vaccine, both family 1 and 2 need to be incorporated.⁵⁷

The limitation of the present study is the small sample size and a subset of isolates collected during the study period that might lead to significant bias in the prevalence of serotypes, drug resistance, and genetic traits. Thus, to recognize the changing trend, these results are insufficient to draw a comparative analysis with our previous study. Continuous surveillance is required to elucidate the trend of genetic traits (serotypes, ST, and PspA) and AMR in the prevailing clones of S. pneumoniae.

Footnotes

Acknowledgment

This study was partially supported by the Japan Society for the Promotion of Science, KAKENHI grant no. 16K09101.

Disclosure Statement

No competing financial interests exist.

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