Dominance of Multidrug-Resistant Denmark 14 -32 (ST230) Clone Among Streptococcus pneumoniae Serotype 19A Isolates Causing Pneumococcal Disease in Bulgaria from 1992 to 2013

Abstract

A pneumococcal conjugate vaccine (PCV10) was introduced in Bulgarian national immunization program since April 2010. Clonal composition based on pulsed-field gel electrophoresis and multilocus sequence typing genotyping of 52 serotype 19A Streptococcus pneumoniae isolates was analyzed. These were invasive and respiratory isolates collected between 1992 and 2013 from both children (78.8% <5 years) and adults with pneumococcal infections. Multidrug resistance was found in 82.7% of all 19A isolates. The most prevalent genotype (63.5%) among serotype 19A pneumococcal strains was the multidrug-resistant clonal complex CC230, which is a capsular switched variant of the Denmark¹⁴-32 (ST230) global clone. The most frequent sequence type (ST) was ST230 (48.1%) and together with four other closely related STs (15.4%), belonging to ST1611, ST276, ST7466, and ST2013, which were single- and double-locus variants; they were included in the main CC230. The disappearance of highly drug-resistant ST663 clone and emergence of new clones as CC320 and CC199 was also observed among the rest 19A isolates. A comparison of clonal composition between invasive and noninvasive isolates did not show a great genetic diversity among both kinds of isolates. Continuous surveillance of serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the epidemiology of this serotype.

Introduction

S treptococcus pneumoniae is still a major cause of invasive and respiratory diseases in spite of 7-valent pneumococcal conjugate vaccine (PCV7) introduction. After the implementation of PCV7 vaccination, the incidence of invasive pneumococcal disease (IPD) caused by vaccine serotypes decreased significantly among both children and adults, while nonvaccine serotypes increased.⁶ Particularly, serotype 19A S. pneumoniae has increased in several countries after PCV7 implementation.¹⁶ But, serotype 19A was increasingly found also in countries without PCV vaccination.^8,21 Especially, the increase of multidrug-resistant 19A isolates in many parts of the world is worrisome.^2,3,13,22

At the present two PCVs, the 10-valent (PCV10) and 13-valent (PCV13) vaccines, are available worldwide, since the 7-valent is being progressively removed from the market.²⁸ In April 2010, PCV10 was included in the Bulgarian Immunization Program, being offered as a free-of-charge, universal childhood vaccination. According to the national epidemiological data, more than 90% of newborn children in Bulgaria were vaccinated in 2011–2013 (National Center of Health Information). Of note, PCV7 has been registered in Bulgaria since 2008, but it was not used prior to PCV10 introduction.

Prior to PCV10 implementation, 19A was already common serotype among middle-ear fluids (MEFs), IPD, and noninvasive isolates in Bulgaria. In the prevaccine period, serotype 19A was the third-leading serotype (11%) in children with pneumococcal acute otitis media (AOM) and sixth in rank (5.4%) among children with invasive disease <5 years of age.^17,18 Similar to what was found after PCV7 use in United States and some European countries, the selective pressure of PCV10 in Bulgaria of the resident population can open to isolates that already expressed nonvaccine serotypes like 19A serotype.

The aim of this study was to analyze the clonal composition through pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and antimicrobial susceptibilities of serotype 19A pneumococcal isolates collected in several laboratories in Bulgaria between 1992 and 2013, in order to highlight the epidemiological genetic evolution of that serotype in our country.

Materials and Methods

Isolate collection

Since 1991, our department of microbiology receives pneumococcal isolates on a voluntary basis from several laboratories in Sofia and three university hospital microbiological laboratories throughout Bulgaria (Plovdiv, Pleven, and Varna). We collected as well the data on demographic characteristics, source of isolate, clinical diagnosis, and vaccination status for all received isolates. Our studied pneumococcal population did not represent all sequential strains that had been isolated in collaborating microbiological laboratories, but there were no selection criteria for sending the strains. Among this randomized collection composed of 1,108 S. pneumoniae isolates, 52 strains (4.7%) presented serotype 19A. These isolates were responsible for invasive and noninvasive infections, and they had been isolated from both children and adults for the period from 1992 to 2013. A case of IPD was defined as the recovery of an isolate of S. pneumoniae from a normally sterile site, except for one patient with pneumococcal meningitis. The diagnosis of the last was made by Gram stain and antigen latex agglutination test of the cerebrospinal fluid (CSF) sample but the pneumococcal culture was not obtained from this specimen. At the same time S. pneumoniae was isolated from the throat swab of that patient in pure culture. As a rule, S. pneumoniae as the causative agent is found in the nasopharynx during noninvasive diseases like otitis media or pneumonia,⁴ and we included also pneumococcal isolates obtained from such kind of specimens. Strains were maintained as double skim milk stocks at −80°C and were restored for this analysis. S. pneumoniae strains were confirmed with both methods—optochin susceptibility test and bile solubility.

Serotyping and antimicrobial susceptibility testing

Serogrouping of S. pneumoniae was performed using the latex agglutination method (Pneumotest-Latex kit; SSI). Serotyping was performed with the capsular Neufeld Quellung method using factor antisera (SSI).²³ Additionally, all 19A isolates determined by Quellung reaction were tested for serotype 19A/19F using latex agglutination factor antisera provided from SSI.

The antibiotic susceptibilities were determined by the broth microdilution method according to guidelines of Clinical and Laboratory Standards Institute (CLSI).⁷ Minimal inhibitory concentrations (MICs) were performed on Microtiter plates (Sensititre; Trek Diagnostic Systems Ltd.). STR6F MIC plate was inoculated with cation-adjusted Mueller–Hinton broth and 5% lysed horse blood for S. pneumoniae. Final bacterial density was 5×10⁵ CFU/ml and the plates were incubated at 35°C for 20–24 hr in 5% CO₂. Isolates with penicillin MICs of ≥2 μg/ml and 0.12–1.0 μg/ml were considered to be resistant and intermediately resistant to “oral” penicillin, respectively.⁷ Isolates with cefotaxime MICs ≥2 μg/ml were considered to be resistant to third-generation cephalosporins. For other antibiotics, MICs were interpreted according to CLSI, 2013. Standard quality-control strain was S. pneumoniae ATCC 49619. Resistant and intermediate strains were included in the term “antimicrobial resistance.” Strains resistant to ≥3 classes of antibiotics were defined as multidrug resistant (MDR).

Macrolide resistance genotypes

The presence of ermB and mefA/E was evaluated concurrently in a duplex reaction for all erythromycin-resistant Streptococcus pneumoniae (ERSP) isolates by PCR. The primer sets were as described by Sutcliffe et al.²⁴ Gene amplification was performed using a Techgene-thermal cycler (Techne). MefA and mefE were then distinguished by PCR restriction fragment length polymorphism analysis of the 348-bp mef amplicon with BamHI restriction enzyme (New England Biolabs), which has no restriction site in mefE and one in mefA, generating two fragments of 284 and 64 bp.¹⁵ Mutations in ribosomal protein L4 were determined as described elsewhere.²⁵

Molecular typing by PFGE and MLST

Profiling by PFGE was performed for all serotype 19A isolates included in this study. The preparation of chromosomal DNA, digestion with SmaI, separation, and analysis of the PFGE patterns were performed as described previously.¹⁹ PFGE types and subtypes were defined according to recommendations given by Tenover et al.²⁶

Sequence types (STs) were determined by MLST as previously described.⁹ The amplified fragments of each of the seven genes were directly sequenced in each direction using an ABI3130XL DNA analyzer (Applied Biosystems). Sequencing analysis was done with FinchTV version 1.4.0 software (Geospiza, Inc.). The STs were determined at the MLST database for S. pneumoniae: www.mlst.net. The STs obtained were considered related if they were identical, a single-locus variant (SLV), or a double-locus variant (DLV) of each other. To identify pneumococcal clones, STs were compared with those at Pneumococcal Molecular Epidemiology Network (PMEN), www.sph.emory.edu/PMEN and with those available at the MLST website by using eBURST algorithm to define clonal complexes (CCs).¹⁰

Clustering comparison coefficient (Wallace value) was used to compare the results from PFGE and ST derived from MLST. For the calculation of Wallace value we used Bionumerics script from www.comparingpartitions.info.⁵

Results

Demographic and clinical characteristics of isolates expressing serotype 19A

Of the 52 serotype 19A S. pneumoniae isolates, 41 (78.8%) had been obtained from children <5 years of age, 2 (3.8%) from children aged 6–10 years, and 9 (17.3%) from adults aged from 28 to 84 years. Among these 52 isolates, 9 were from invasive specimens: blood (n=5), CSF (n=2), and pleural and peritoneal fluids (1 each); 16 samples were from MEFs obtained after spontaneous rupture of the tympanum or after paracentesis; and 27 were respiratory tract samples: sputum (n=2), throat (n=5), nasopharynx (n=4), nose (n=13), and eye swab (n=3). These specimens were obtained from patients with invasive and noninvasive community-acquired infections (Table 1). The invasive infections were 10 episodes (19.2%), including bacterial meningitis (n=3), community-acquired pneumonia (n=5), bacteremia, and peritonitis (1 each, respectively). Noninvasive infections were AOM cases (n=16, 30.8%) among children, and upper and lower respiratory tract infections among children and adults (n=26, 50.0%).

Table 1.

Surveillance Population, Antibiotic Resistance, Macrolide Resistance Genotype, and Genetic Structure of 52 Serotype 19a Streptococcus pneumoniae Isolates: 1992–2013

						MIC (μg/ml) ^a
Strain no.	Year isolated	Patient age	Source of isolate	Infection site resistance pattern ^a	Antibiotic	P	CTX	Macrolide R genotype ^a	PFGE pattern	ST
Bul 23	1992	4 years	Nose	Acute sinusitis	P, E, T, SXT, RIF, CTX	8.0	4.0	L4	A	663
Bul 29	1992	1 year 11 months	Nose	CAP	P, E, T, SXT, CTX	8.0	4.0	L4	A	663
Bul 34	1993	1 year	Eye	Conjunctivitis	P, E, T, SXT, CTX	8.0	2.0	L4	A	663
Bul 40	1993	3 years	MEF	AOM	P, E, T, SXT, CTX	8.0	4.0	L4	A	663
Bul 91	1995	4 months	MEF	AOM	P, E, T, SXT, CTX	8.0	4.0	L4	A	663
Bul 105	1995	1 year 9 months	Nose	CAP	P, E, CLI, T, SXT	0.25	≤0.12	ermB	C₁	230
2194 Pl	1997	3 years	Blood	CAP	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
6027 Pir	1998	>18 years	Blood	CAP	LEVO	≤0.03	≤0.12	—	K	423
268 HID	1998	63 years	CSF	Bacterial meningitis	T, SXT	≤0.03	≤0.12	—	B	66
23/1 Sof	2000	10 years	Blood	CAP	E, C, SXT	≤0.03	≤0.12	mefE	B	66
5800 CM	2000	5 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
2402 Pd	2001	1 year	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₄	1611
1458 L	2001	3 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
476 Vr	2001	2 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
08507 R	2002	2 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₃	230
1195 Pd	2003	6 months	Pleural fluid	CAP	P, E, CLI, T, SXT	0.25	≤0.12	ermB	C₅	230
5279 Pd	2004	5 months	MEF	AOM	P, E, CLI, T, SXT	0.25	≤0.12	ermB	E	276
9609 Pir	2005	77 years	Blood	CAP	P	0.12	≤0.12	—	D	3836
1059 Pl	2005	74 years	CSF	Bacterial meningitis	Susceptible	≤0.03	≤0.12	—	G	2632
993 Pd	2005	2 years	Nose	URTI	P, T, SXT	0.25	≤0.12	—	I	1611
2268 Pl	2006	5 months	MEF	AOM	P, E, CLI, T, SXT	0.25	≤0.12	ermB	C₃	230
569 IICH	2006	6 months	Blood	Bacteremia	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
2149 CM	2006	61 years	Sputum	AECB	P, E, CLI, T, LEVO	0.25	≤0.12	ermB	C₁	230
264 AH	2006	84 years	Sputum	COAD	P, E, CLI, T, SXT	0.25	≤0.12	ermB	C₁	230
284 Pd	2007	4 months	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
9611 Pd	2007	3 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₂	230
1049 Pl	2007	25 years	MEF	AOM	P, E, CLI, T, SXT, LEVO	0.25	0.25	ermB	C₁	230
4203 Pl	2007	8 months	Eye	Conjunctivitis	P, E, CLI, T, SXT	0.25	≤0.12	ermB	C₂	230
83 Pl	2007	2 years	Nph	URTI	P, E, CLI, T, SXT	0.25	0.25	ermB	B	7466
374 Pd	2007	1 year	Nph	URTI	P, T, SXT	0.12	≤0.12	—	I	1611
3928 Vn	2007	3 years	Nose	URTI	P, E, CLI, T, SXT	2.0	1.0	ermB	M	352
3663 CM	2007	7 years	Nph	URTI	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
4704 Pl	2007	10 months	Nph	URTI	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₃	230
3618 Pl	2007	10 months	Throat	URTI	P, E, CLI, T, SXT	0.12	≤0.12	ermB	L	315
45 DK-AH	2008	5 years	Throat	CAP	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230
8501 Pd	2008	2 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB+mefE	C₁	230
378 Pl	2008	2 years	Throat	URTI	P, T, SXT	0.25	0.25	—	C₃	2013
395 Pl	2008	3 years	Nose	Acute sinusitis	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₂	230
443 Pl	2008	59 years	Nose	Chronic sinusitis	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₂	7466
529 Pl	2008	2 years	Nose	Chronic sinusitis	P, E, CLI, T, SXT	4.0	1.0	ermB	C₂	230
1366 Pl	2008	5 months	Nose	URTI	P, E, CLI, T, SXT	0.5	0.25	ermB	C₃	230
6295 Kand	2008	2 years	Nose	URTI	P	0.12	≤0.12	—	D	3836
6675 Kand	2008	4 years	Nose	URTI	SXT	≤0.03	≤0.12	—	J	199
1661 CM	2008	1 year 7 months	Nose	URTI	P, T, SXT	1.0	0.5	—	I	1611
318 Kand	2009	3 years	Throat	URTI	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₃	230
345 Pl	2009	2 years	Nose	CAP	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₄	230
2401 Pd	2010	5 months	MEF	AOM	P, E, CLI, T, SXT	1.0	0.5	ermB+mefE	F₁	320
7643 DN	2010	28 years	Peritoneal fluid	Peritonitis	P, E, CLI, T, SXT, CTX	8.0	2.0	ermB	F₂	320
301 HID	2011	8 months	Throat	Bacterial meningitis	P, SXT	2.0	1.0	—	G	2632
581 AH	2011	3 years	Eye	Conjunctivitis	Susceptible	≤0.03	≤0.12	—	H	2365
01 QI	2012	10 months	MEF	AOM	P, SXT	4.0	1.0	—	G	2632
39 QI	2013	5 years	MEF	AOM	P, E, CLI, T, SXT	0.12	≤0.12	ermB	C₁	230

Interpretation according to CLSI, 2013.⁷ Susceptible: to all of antimicrobials tested. CLI, clindamycin; CTX, cefotaxime; E, erythromycin; LEVO, levofloxacin; P, penicillin; RIF, rifampin; SXT, trimethoprim-sulfamethoxazole; T, tetracycline. Resistant and intermediate strains were included. The following breakpoints (μg/ml) for penicillin (oral penicillin V) were used: susceptible ≤0.06, intermediate ≥0.12–1.0, and resistant ≥2.

AECB, acute exacerbation of chronic bronchitis; AOM, acute otitis media; CAP, community acquired pneumonia; CLSI, Clinical and Laboratory Standards Institute; COAD, chronic obstructive airway disease; CSF, cerebrospinal fluid; MEF, middle ear fluid; MIC, minimal inhibitory concentration; Nph, nasopharynx; PFGE, pulsed-field gel electrophoresis; ST, sequence type; URTI, upper respiratory tract infection.

Forty-eight isolates were from the prevaccine period (1992–2010). Two pneumococcal strains isolated in 2010 were included in the prevaccine period because they were obtained from patients who had not received PCV vaccine. The four isolates, obtained in the years from 2011 to 2013, were collected after implementation of PCV10 vaccination.

Antimicrobial resistance and genotype of macrolide resistance

As described already in “Materials and Methods” section, the “oral” penicillin breakpoints were used to calculate the penicillin nonsusceptibility for 19A isolates. Applying these breakpoints, a total of 46 isolates (88.5%) were nonsusceptibile to penicillin (MIC ≥0.12 μg/ml), including 10 strains (21.7%) highly resistant to penicillin (≥2 μg/ml). Global nonsusceptibility rate to cefotaxime was 11.5%. Nonsusceptibility rates also were high for trimethoprim-sulfamethoxazole (88.5%), and tetracycline (82.7%). Resistance rates to erythromycin and clindamycin were 75.5% and 63.5%, respectively. Multidrug resistance was found in 43 isolates (82.7%) of all serotype 19A. Only two isolates were susceptible to all tested antibiotics, four were resistant to at least one, and three were resistant to at least two (Table 1).

Among the 39 ERSP serotype 19A isolates, 33 (84.6%) carried ermB gene, including two isolates with the presence of both ermB and mefE genes. Five isolates had mutations in L4 ribosomal protein (12.8%), and one strain carried the mefE (2.6%) gene solely (Table 1).

Molecular typing

All 52 serotype 19A isolates were analyzed by PFGE and characterized by MLST. Overall, serotype 19A strains were distributed throughout 13 different PFGE patterns; of which the most abundant (28 isolates) was PFGE pattern type C with several subtypes within it (Table 1). A good correspondence between PFGE and MLST results was found (Table 2). The Wallace value comparing PFGE types and related STs was 0.799 (95% CI: 0.609–0.990). This high value indicates that generally, if two strains belong to the same ST, then they will also have the same PFGE pattern. Despite these, two STs were detected in several PFGE types, such as ST1611 and ST7466.

Table 2.

Distribution of Clonal Complexes and Sequence Types According to PFGE Patterns of 52 Serotype 19A Isolates

Clonal complex ^a	PMEN clone ^b	n (%)	STs within each clone (%)	PFGE pattern (n)
CC230	Denmark¹⁴-32/ST230	33 (63.5)	230 (48.1), 1611 (1.9), 7466 (1.9), 2013 (1.9)	C (28)
			1611 (5.8)	I (3)
			276 (1.9)	E (1)
			7466 (1.9)	B (1)
CC663	Colombia^23F-26/ST338	5 (9.6)	663 (9.6)	A (5)
ST2632^c	None	3 (5.8)	2632 (5.8)	G (3)
CC320	Taiwan^19F-14/ST236	3 (5.8)	320 (3.8)	F (2)
			352 (1.9)	M (1)
CC199	Netherlands^15B-37/ST199	3 (5.8)	199 (1.9)	J (1)
			3836 (3.8)	D (2)
CC66	Tennessee¹⁴-18/ST67	2 (3.8)	66 (3.8)	B (2)
CC315	Poland^6B-20/ST315	1 (1.9)	315 (1.9)	L (1)
ST2365^c	None	1 (1.9)	2365 (1.9)	H (1)
CC9 (CC20)^d	England¹⁴-9/ST9 (CSR¹⁴-10/ST20)	1 (1.9)	423 (1.9)	K (1)

CC as defined using information from the PMEN (www.sph.emory.edu/PMEN). For CCs, a cutoff point of five identical loci to the predicted founder of its eBURST group was used.

STs were considered related to a PMEN reference clone if they were identical, SLVs or DLVs.

ST without defined CC but belonging to a known eBURST group.

ST423 is a DLV of two eBURST groups.

CCs, clonal complexes; DLV, double-locus variant; PMEN, Pneumococcal Molecular Epidemiology Network; SLV, single-locus variant.

The 19A isolates belonged to 15 different STs, one of which ST230 was the most frequent (48.1%). All ST230 isolates were represented by identical PFGE cluster C. ST230 isolates showed intermediate resistance to penicillin, except one strain isolated in 2008, which was obtained high level of penicillin resistance (MIC=4 μg/ml). Additionally, ST230 isolates were resistant to erythromycin, clindamycin, tetracycline, and frequently to trimethoprim-sulfamethoxazole, and carried ermB gene. The second in rank ST663 (five isolates) was found only in the period between 1992 and 1995, and the strains showed a unique PFGE cluster A. ST663 isolates showed high level of penicillin resistance (8 μg/ml), resistance to ceftriaxone (2–4 μg/ml), and resistance to erythromycin, tetracycline, and trimethoprim-sulfamethoxazole, and the isolates had mutations in ribosomal protein L4. The rest 19A isolates had highly diverse STs.

Most of STs and PFGE patterns were found among both invasive and noninvasive pneumococcal isolates, except for three invasive strains, ST423 (n=1) and ST66 (n=2), and seven noninvasive isolates—ST663 (n=5), ST2365 (n=1), and ST315 (n=1)—which were found only in the respective group.

Four STs were represented in the vaccine period 2011–2013: ST2632 (n=2), ST230, and ST2365 (1 each). ST2632 was first emerged in 2005 when it was susceptible to all tested antimicrobials, but in the vaccine period the two ST2632 isolates have obtained high-level penicillin resistance and resistance to trimethoprim-sulfamethoxazole.

A population snapshot of the 52 isolates, based on eBURST analysis (Fig. 1), showed a major clonal complex CC230 containing 25 isolates of ST230 (48.1%) and 8 isolates (15.4%), representing four other closely related SLV and DLV forms of ST230. They were ST1611 (DLV, n=4), ST276 (predicted founder, SLV, n=1), ST7466 (DLV, n=2), and ST2013 (DLV, n=1).

FIG. 1.

Population snapshot of 52 serotype 19A isolates comparing sequence types (STs) found in 1992–2013. Each circle represents a single ST, and circle size is proportional to the number of isolates. Primary founder is positioned centrally in the cluster. Solid lines between STs represent single-locus variant (SLV) and gray lines indicate double-locus variants (DLV). STs were considered related to a PMEN reference clone if they were identical, SLVs or DLVs. PMEN, Pneumococcal Molecular Epidemiology Network.

Table 2 shows distribution of CCs as defined at the MLST website. Serotype 19A isolates were grouped into seven CCs using a cutoff point of five identical loci to the predicted founder of its eBURST group, and two STs without defined CCs but belonging to a known eBURST group. The most frequent genotype was the multidrug-resistant CC230, representing 63.5% of all isolates, which is a capsular switched variant of the worldwide-established Denmark¹⁴-32 (ST230). The second-in-rank clone was multidrug-resistant CC663 containing ST663 (five isolates), which is a DLV of the Colombia^23F-26/ST338 clone. This clone emerged in Bulgaria between 1992 and 1995, and was not detected any more in this collection. The isolates CC320 (n=3) are variants of the worldwide-established Taiwan^19F-14 (ST236) clone. These isolates showed high resistance level to penicillin and multiresistance, carried ermB or both ermB plus mefE genes. Next was clonal complex CC199 (three isolates) represented by one isolate ST199 identifier of the Netherlands^15B-37/ST199 clone, and two ST3836 strains that were SLV. The antibiotic pattern of CC199 clone showed susceptibility to penicillin for one isolate (ST199), and low level of penicillin resistance (MIC=0.12 μg/ml) for two ST3836 isolates. Additionally, we observed CC66 containing two ST66 isolates that were SLV of the Tennessee¹⁴-18 (ST67) clone, and one ST315 isolate identifier of the Poland^6B-20/ST315 clone.

Discussion

This is a retrospective study about clonal composition of invasive and noninvasive serotype 19A S. pneumoniae isolates obtained mainly in prevaccine period. PCV10 was introduced in our national vaccination plan since 2010, but the vaccine does not include the serotype 19A. Additionally, the pneumococcal conjugate vaccines have shown a modest cross-protection against antibiotic nonsusceptible isolates of serotype 19A.¹² A considerable increase in pneumococcal infections caused by serotype 19A isolates has been recorded after the introduction of PCV7 worldwide.^1,2,13,16,27 In contrast, multiresistant serotype 19A isolates have been also identified in Israel, Finland, and Bulgaria before the large-scale use of conjugate vaccines.^8,18,21

Similar to what was found in other studies, multidrug resistance was the main feature of our serotype 19A isolates (83% in this study). The high antibiotic consumption in any country represents the major pressure for resistance selection.⁸ Other factors, such as vaccine pressure by the use of PCVs, clonal expansion of existing clones, and the emergence of new clones, have been implicated in the expansion of antibiotic resistance in serotype 19A isolates.^1,22,27

Genotype structure based on PFGE and MLST analyses in this study showed that serotype 19A pneumococcal infections were mainly due to the spread of clones CC230, CC663, CC320, and CC199. There are five major international CCs associated with serotype 19A (CC81, CC193, CC199, CC230, and CC320), three of which were found in our study.¹⁶ A comparison of clonal composition between invasive and noninvasive strains did not show a great genetic diversity among both kinds of isolates.

The dominant ST was ST230 (48.1%), representing international Denmark¹⁴-ST230 clone. Capsular switching of the Denmark¹⁴-32/ST230 clone with serotypes 19F, 19A, and 24F has been reported in many countries.^2,8,13 ST230 was detected as far back as 1995 and it is successfully disseminated throughout the study period including after introduction of PCV10, suggesting clonal spreading in Bulgaria. When categorized according to CC, CC230 was the most prevalent (63.5%) and successfully disseminated all over the period. Four additional STs were included in the CC230, which were SLV and DLV of the ST230 and were first detected in 2001 and later on. This suggests that as the main ST230 remains stable over the entire period, it gradually diversifies into new MLST-locus variants.

Several studies from southwest European countries and one from Israel have identified also representatives of clone Denmark¹⁴-32/ST230 as a major cause of serotype 19A diseases in children and adults, but they found, in particular, ST276 as a major ST of CC230 clone. An increased isolation of multiresistant ST276 has been documented in Spain, Portugal, and France.^1,8,13,14 In contrast, we found an increased isolation of MDR ST230 among our 19A isolates. Until now this multiresistant clone has rarely been found in the United States.^3,11

CC320, related to well-known multidrug-resistant Taiwan^19F-ST236 clone, was found between 2007 and 2010 before pneumococcal vaccination in our country. The emergence of ST320 among MDR serotype 19A isolates increased in United States and some European countries in the post-PCV7 era.^2,3,11 However, multidrug-resistant ST320 has spread rapidly among serotype 19A isolates in some Asian countries before PCV7 introduction.²⁰ Another international CC199 clone has been observed to increase slightly in United States^3,11 and in Spain,¹⁴ due to the spread of ST199 isolates, which were susceptible to antibiotics. Similar to what was found in these studies our few CC199 isolates did not show multidrug resistance.

In conclusion, although serotype 19A pneumococci were genetically heterogeneous in this study, the most predominant was CC230 (ST230), which is due, as was shown, to the clonal spreading of this successfully disseminated Bulgaria clone during the last 20 years. Continuous surveillance of the pneumococcal serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the evolution of this serotype.

Footnotes

Acknowledgments

The study was partially supported by funds from Medical University of Sofia (Grant No. D31/2013). The authors thank all microbiological laboratories for their cooperation and for providing the isolates included in this study.

Disclosure Statement

No competing financial interests exist.

References

Aguiar

I.S.

, Pinto

F.R.

, Nunes

, Serrano

, Melo-Cristino

, Sá-Leão

, Ramirez

, and de Lencastre

. 2010. Denmark¹⁴-230 clone as an increasing cause of pneumococcal infection in Portugal within a background of diverse serotype 19A lineages. J. Clin. Microbiol., 48:101–108.

Ardanuy

, Rolo

, Fenoll

, Tarrago

, Calatayud

, and Liñares

2009. Emergence of a multidrug-resistant clone (ST320) among invasive serotype 19A pneumococci in Spain. J. Antimicrob. Chemother., 64:507–510.

Beall

B.W.

, Gertz

R.E.

, Hulkower

R.L.

, Whiney

C.G.

, Moore

M.R.

, and Brueggemann

A.B.

. 2011. Shifting genetic structure of invasive serotype 19A pneumococci in the United States. J. Infect. Dis., 203:1360–1368.

Bogaert

, De Groot

, and Hermans

P.W.

2004. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect. Dis., 4:144–154.

Carriço

J.A.

, Silva-Costa

, Melo-Cristino

, Pinto

F.R.

, de Lencastre

, Almeida

J.S.

, and Ramirez

. J. Clin. Microbiol., 44:2524–2532.

[CDC] Centers for Disease Control and Prevention. 2005. Direct and indirect effects of routine vaccination of children with 7-valent pneumococcal conjugate vaccine on incidence of invasive pneumococcal disease—United States, 1998–2003. MMWR Morb. Mortal. Wkly. Rep., 54:893–897.

[CLSI] Clinical and Laboratory Standards Institute. 2013. Performance standards for antimicrobial susceptibility testing, 23th ed. Approved standard M100–S23. CLSI, Wayne, PA.

Dagan

, Givon-Lavi

, Leibovitz

, Greenberg

, and Porat

2009. Introduction and proliferation of multidrug-resistant Streptococcus pneumoniae serotype 19A clones that cause acute otitis media in an unvaccinated population. J. Infect. Dis., 199:776–785.

Enright

M.C.

, and Spratt

B.G.

. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology, 144:3049–3060.

10.

Feil

E.J.

, Li

B.C.

, Aanensen

D.M.

, Hanage

W.P.

, and Spratt

B.G.

. 2004. eBURST: Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol., 186:1518–1530.

11.

Hanage

W.P.

, Bishop

C.J.

, Lee

G.M.

, Lipsitch

, Stevenson

, Rifas-Shiman

S.L.

, Pelton

S.I.

, Huang

S.S.

, and Finkelstein

J.A.

. 2011. Clonal replacement among 19A Streptococcus pneumoniae in Massachusetts, prior to 13 valent conjugate vaccination. Vaccine, 29:8877–8881.

12.

Hausdorff

W.P.

, Hoet

, and Schuerman

. 2010. Do pneumococcal conjugate vaccines provide any cross-protection against serotype 19A?. BMC Pediatr., 10:4.

13.

Mahioub-Messai

, Doit

, Koeck

J.L.

, Billard

, Evrard

, Bidet

, Hubans

, Raymond

, Levy

, Robert Cohen

, and Bingen

. 2009. Population snapshot of Streptococcus pneumoniae serotype 19A isolates before and after the introduction of 7-valent pneumococcal vaccination in French children. J. Clin. Microbiol., 47:837–840.

14.

Marimon

J.M.

, Alonso

, Rolo

, Ardanuy

, Liñares

, and Pérez-Trallero

. 2012. Molecular characterization of Streptococcus pneumoniae invasive serotype 19A isolates from adults in two Spanish regions (1994–2009). Eur. J. Clin. Microbiol. Infect. Dis., 31:1009–1031.

15.

Montanari

M.P.

, Mingoia

, Giovanetti

, and Varaldo

P.E.

. 2003. Phenotypes and genotypes of erythromycin-resistant pneumococci in Italy. J. Clin. Microbiol., 41:428–431.

16.

Reinert

, Jacobs

M.R.

, and Kaplan

S.L.

2010. Pneumococcal disease caused by serotype 19A: review of the literature and implications for future vaccine development. Vaccine, 28:4249–4259.

17.

Setchanova

, Alexandrova

, Mitov

, Nashev

, Kantardjiev

, and The Group for Microbiological surveillance of pneumococci. 2012. Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates in bulgaria before the introduction of pneumococcal conjugate vaccine. J. Chemother., 24:12–17.

18.

Setchanova

, Kostyanev

, Alexandrova

, Mitov

, Nashev

, and Kantardjiev

2013. Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines. Ann. Clin. Microbiol. Antimicrob., 12:6.

19.

Setchanova

, and Tomasz

1999. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria. J. Clin. Microbiol., 37:638–648.

20.

Shin

, Baek

J.Y.

, Kim

S.H.

, Song

J.H.

, and Ko

K.S.

. 2011. Predominance of ST320 among Streptococcus pneumoniae serotype 19A isolates from 10 Asian countries. J. Antimicrob. Chemoter., 66:1001–1004.

21.

Siira

, Jalava

, Tissari

, Vaara

, Kaijalainen

, and Virolainen

2012. Clonality behaind the increase of multidrug-resistance among non-invasive pneumococci in Southern Finland. Eur. J. Clin. Microbiol. Infect. Dis., 31:867–871.

22.

Song

J.-H.

, Dagan

, Klugman

K.P.

, and Fritzell

. 2012. The relationship between pneumococcal serotypes and antibiotic resistance. Vaccine, 30:2728–2737.

23.

Sorensen

U.B.S.

1993. Typing of pneumococci by using 12 pooled antisera. J. Clin. Microbiol., 31:2097–2100.

24.

Sutcliffe

, Grebe

, Tait-Kamradt

, and Wondrack

1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob. Agents Chemother., 40:2562–2566.

25.

Tait-Kamradt

, Davies

, Appelbaum

P.C.

, Depardieu

, Courvalin

, Petitpas

, Wondrack

, Walker

, Jacobs

M.R.

, and Sutcliffe

. 2000. Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumoniae from Eastern Europe and North America. Antimicrob. Agents Chemother., 44:3395–3401.

26.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, and Swaminathan

. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

27.

Van der Linden

, Reinert

R.R.

, Kern

W.V.

, and Imöhl

. 2012. Epidemiology of serotype 19A isolates from invasive pneumococcal disease in German children. BMC Infect. Dis., 13:70.

28.

World Health Organization. 2012. Pneumococcal vaccines—WHO position paper. Wkly. Epidemiol. Rec., 87:129–144.