JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1758–1764 0095-1137/11/$12.00 doi:10.1128/JCM.02628-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 5
Comparison of Capsular Genes of Streptococcus pneumoniae Serotype 6A, 6B, 6C, and 6D Isolates䌤 Jae-Hoon Song,1,2† Jin Yang Baek,2† and Kwan Soo Ko2,3* Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea1; Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, South Korea2; and Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, South Korea3 Received 28 December 2010/Returned for modification 5 February 2011/Accepted 2 March 2011
Recently, Streptococcus pneumoniae serotypes 6C and 6D have been identified. It is thought that they emerged by the replacement of wciN in the capsular loci of serotypes 6A and 6B, respectively. However, their evolution has not been unveiled yet. To investigate the evolution of four serotypes of S. pneumoniae serogroup 6, four genes of the capsular polysaccharide synthesis (cps) locus, wchA, wciN, wciO, and wciP, of isolates of S. pneumoniae serotypes 6A, 6B, 6C, and 6D were sequenced. Multilocus sequence typing (MLST) was performed to investigate their genetic backgrounds. The wchA gene of serotype 6C and 6D isolates was distinct from that of serotype 6A and 6B isolates, which may suggest cotransfer of wchA with wciN. Otherwise, serotypes 6C and 6D displayed different genetic backgrounds from serotypes 6A and 6B, which was suggested by MLST analysis. In addition, serotype 6C isolates showed distinct wciP polymorphisms from other serotypes, which also indicated that serotype 6C had not recently originated from serotype 6A. Although serotype 6D shared the same amino acid polymorphisms of wciO with serotype 6B, wciP of serotype 6D differed from that of serotype 6B. The data indicate the implausibility of the scenario of a recent emergence of the cps locus of serotype 6D by genetic recombination between serotypes 6B and 6C. In addition, five serotype 6A and 6B isolates (6X group) displayed cps loci distinct from those of other isolates. The cps locus homogeneity and similar sequence types in MLST analysis suggest that most of the 6X group of isolates originated from the same ancestor and that the entire cps locus might have recently been transferred from an unknown origin. Serotype 6B isolates showed two or more cps locus subtypes, indicating a recombination-mediated mosaic structure of the cps locus of serotype 6B. The collective data favor the emergence of cps loci of serotypes 6A, 6B, 6C, and 6D by complicated recombination.
Streptococcus pneumoniae is an important human pathogen responsible for several invasive and noninvasive bacterial diseases. According to the World Health Organization (WHO), S. pneumoniae is responsible for approximately 1.6 million deaths each year, with nearly 1 million involving children ⬍5 years of age living in developing countries (16). So far, more than 90 different capsular serotypes, including newly identified serotypes 6C, 6D, and 11E, have been recognized (5, 9, 13). S. pneumoniae serogroup 6 isolates are commonly found in invasive diseases. Both serotypes 6A and 6B have a capsule composed of oligosaccharide subunits of rhamnose-ribitolgalactose-glucose (1, 10). Genetic studies have revealed the near identity of the capsular loci of serotypes 6A and 6B, with only a single nucleotide difference in the wciP gene (G instead of A at residue 584), resulting in an amino acid substitution (Ser195Asn) in the encoding sequence and different rhamnose-ribitol linkages (1 3 3 in 6A and 1 3 4 in 6B) (10). In addition, there are two other polymorphisms between the two serotypes: Ala192 and Arg254 in 6A and Ser192 and Gly254 in 6B. Serotype 6C is very similar to serotype 6A. These two
serotypes differ in the replacement of a galactose in 6A by glucose in 6C because the wciN gene in serotype 6C encodes a different glycosyl transferase. The wciN gene of serotype 6C is about 200 bp shorter than the wciN gene of serotype 6A (13). Another new serotype, 6D, is differentiated from 6B by the wciN gene, as in serotype 6C (3, 5). While serotype 6B is included in the 7-valent pneumococcal conjugate vaccine (PCV-7), serotypes 6A, 6C, and 6D are not. Both 6A and 6B are included in the recently developed 13-valent vaccine (PCV-13). It has been postulated that serotypes 6C and 6D emerged by the replacement of wciN in the capsular loci of serotypes 6A and 6B, respectively. However, few studies have addressed the genetic characteristics of the serotypes. In this study, we sequenced the same portion of the capsular polysaccharide synthesis (cps) loci of isolates of serotypes 6A, 6B, 6C, and 6D and compared them. In addition, the genetic background of the serotypes was investigated using multilocus sequence typing (MLST).
* Corresponding author. Mailing address: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea. Phone: 82-31-299-6223. Fax: 82-31-299-6229. E-mail:
[email protected]. † J.-H. Song and J. Y. Baek contributed to this work equally as first authors. 䌤 Published ahead of print on 16 March 2011.
A total of 41 S. pneumoniae isolates were investigated: 12 serotype 6A, 11 serotype 6B, 4 serotype 6C, and 14 serotype 6D isolates (Table 1). All isolates were collected as part of a multinational Asian Network for Surveillance of Resistant Pathogens (ANSORP) surveillance study on invasive pneumococci. S. pneumoniae isolates were collected from clinical specimens representative of normally sterile body sites, such as blood, cerebrospinal fluid, ascitic fluid, pleural fluid, joint fluid, sinus aspirates, and middle ear aspirates. Isolates from lower
MATERIALS AND METHODS
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TABLE 1. S. pneumoniae serogroup 6 isolates used in this study, their geographic origins, and MLST data Serotype
Isolate no.
Locality
Isolation yr
ST (allelic profilea)
6A
HK02-77 M06-6 K13-73 J01-2 J01-16 M18-12 TW03-260 TW03-284 K08-52 K13-75 HK02-14 J01-5
Hong Kong Malaysia South Korea Japan Japan Malaysia Taiwan Taiwan South Korea South Korea Hong Kong Japan
2008 2008 2009 2008 2008 2008 2008 2008 2008 2009 2008 2008
1876 (7-25-4-12-15-20-28) 5863 (5-16-6-1-9-14-1) 83 (4-4-2-4-6-1-1) 2756 (8-8-19-16-77-1-68) 282 (30-4-2-4-4-1-1) 457 (2-8-51-41-15-60-8) 81 (4-4-2-4-4-1-1) 81 (4-4-2-4-4-1-1) 81 (4-4-2-4-4-1-1) 81 (4-4-2-4-4-1-1) 90 (5-6-1-2-6-3-4) 1165 (5-6-1-8-6-3-4)
6B
K13-21 M12-6 V03-9 K13-51 J01-4 TW02-294 TW03-253 M12-8 TW03-254 TW03-308 TW06-5
South Korea Malaysia Vietnam South Korea Japan Taiwan Taiwan Malaysia Taiwan Taiwan Taiwan
2008 2008 2008 2008 2008 2009 2008 2008 2008 2009 2008
1624 (5-6-1-2-6-3-1) 95 (5-6-33-2-6-3-4) 1404 (7-25-4-16-6-20-28) 4760 (7-30-19-6-14-6-157) 2224 (7-12-7-1-116-14-29) 5864 (7-30-2-6-4-39-14) 902 (2-13-2-1-6-121-121) 5864 (7-5-33-5-10-6-16) 76 (2-13-9-15-6-19-42) 76 (2-13-9-15-6-19-42) 76 (2-13-9-15-6-19-42)
6C
K04-4 K13-111 K13-133 V07-123
South Korea South Korea South Korea Vietnam
2008 2009 2009 2007
5866 5867 4597 5871
6D
K15-60 K15-99 K15-115 K15-129 TW02-238 07-056 07-077 07-107 B0704-047 K15-17 K13-22 K13-108 K13-109 K13-110
South Korea South Korea South Korea South Korea Taiwan South Korea South Korea South Korea South Korea South Korea South Korea South Korea South Korea South Korea
2009 2009 2009 2009 2008 2007 2007 2007 2007 2008 2008 2009 2009 2009
4762 (30-4-2-4-30-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 3595 (30-4-2-1-4-4-1) 282 (30-4-2-4-4-1-1) 3171 (8-13-9-6-78-119-14) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1) 282 (30-4-2-4-4-1-1)
a
(7-30-41-6-6-6-31) (7-9-2-6-1-6-384) (7-30-8-4-6-6-14) (2-8-9-164-25-1-9)
aroE-gdh-gki-recP-spi-xpt-ddl.
respiratory tract specimens were also included only if they were cultured from adequate respiratory specimens from patients with clinical and radiographic findings of pneumonia. Serotype was determined using the capsular Quellung reaction with commercial antisera (Statens Serum Institute, Copenhagen, Denmark), as recommended by the manufacturer. In addition, a previously described serotype-specific PCR method (8) was used to identify serotypes 6C and 6D. Four genes of the cps locus, wchA, wciN, wciO, and wciP, were sequenced using the primers listed in Table 2. Evolutionary trees of the four genes were reconstructed on the basis of the nucleotide sequences by the method of neighbor joining. The robustness of the grouping was estimated by bootstrap analysis (1,000 replications). MICs for all S. pneumoniae isolates were determined by the broth microdilution method, according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) (7). In vitro susceptibility was tested for 14 antimicrobial agents: penicillin, amoxicillin, amoxicillin-clavulanate, ceftriaxone, cefuroxime, erythromycin, azithromycin, clarithromycin, levofloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, clindamycin, and trimethoprim-sulfamethoxazole. Penicillin resistance was evaluated by applying the CLSI criteria for oral penicillin therapy (i.e., intermediate, 0.12 to 1 mg/liter; resistance, ⱖ2 mg/liter). S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922 were used as control strains. MLST was performed as described previously (11) to investigate the genetic backgrounds of serogroup 6
isolates. All MLST data, including new sequence types (STs), were submitted to the S. pneumoniae MLST database (www.mlst.net/spneumoniae). Nucleotide sequence accession numbers. The sequences of the wchA, wciN, wciP, and wciO genes have been deposited in the GenBank database under accession numbers HQ662178 to HQ662218.
RESULTS wchA gene. The amino acid sequences of the wchA genes of serotypes 6C and 6D isolates were clearly differentiated from those of serotype 6A and 6B isolates (Fig. 1A). Especially, Lys62, Ile110, Gly149, Ile178, Lys240, and Cys352 were unique in serotype 6C and 6D isolates. However, one serotype 6C isolate, V07-123, showed two different amino acids (Glu240 and Arg352). Two 6A isolates (HK02-14 and J01-5) and three 6B isolates (K13-21, M12-6, and V03-9), hereafter designated the 6X group, also showed distinct amino acid sequence differences from other serotype 6A and 6B isolates. Two amino
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SONG ET AL.
J. CLIN. MICROBIOL. TABLE 2. Primers used in this study
Primer
Gene
Sequence
Reference or source
Forward 5113F 5101F 5106F 5103F2 wciP584gS
wze wciN wchA wciO wciP
5⬘-GGG AAA AAT AAA AAA TAG GTC GGG-3⬘ 5⬘-ATT TGG TGT ACT TCC TCC-3⬘ 5⬘-TAC CAT GCA GGG TGG AAT GT-3⬘ 5⬘-GGA ACT TAC TAG ATG GAG TAG-3⬘ 5⬘-ATT TAT ATA TAG AAA AAC TGG CTC ATG ATA G-3⬘
13 13 13 This study 9
Reverse 3122R2 3101R 6A/B-r 3103R
wchA wciO wciP wzy
5⬘-CGT CCA AGC TAG TCT TCC GTA T-3⬘ 5⬘-CCA TCC TTC GAG TAT TGC-3⬘ 5⬘-TTA GCG GAG ATA ATT TAA AAT GAT GAC TA-3⬘ 5⬘-AAC CCC TAA CAA TAT CAA AC-3⬘
This study 10 6 13
acids (Leu449 and Lys451) were the same as those of serotype 6C and 6D isolates. In addition, D430 was in common in the five 6X group isolates, serotype 6C and 6D isolates, and the other three serotype 6B isolates (J01-4, TW02-294, and TW03253). wciN gene. As expected, S. pneumoniae serotypes 6A/6B and 6C/6D could be clearly differentiated by the wciN gene. Among serotype 6A and 6B isolates, the 6X group of isolates (HK0214, J01-5, K13-21, M12-6, and V03-9) showed wciN amino acid sequences distinct from those of other 6A and 6B isolates, as for the wchA gene (Fig. 1B). At 25 amino acid sites, they showed common amino acid sequences that were different from those of the other 6A and 6B isolates. They also contained inserted sequences of about 300 bp between wciN and wciO. At two amino acid sites (121 and 137), amino acid sequence polymorphisms unrelated to the serotypes were found. Among serotype 6C and 6D isolates, only one amino acid sequence polymorphism was identified in two 6C isolates: Glu368 in K13-111 and V07-123. wciO gene. Serotypes 6C and 6D were not clearly differentiated from serotypes 6A and 6B in the wciO gene (Fig. 1B). Only one amino acid sequence (Thr136) was unique to the four serotype 6C isolates. However, the 6X group of isolates showed distinct amino acid sequences, as in wchA and wciN. Of interest, Lys5 and Leu6, which were different from Arg5 and Ile6 of the main serotype 6A and 6B isolates, were commonly present in the 6X group and in serotype 6C/6D isolates. Four 6B isolates (K13-51, J01-4, TW02-294, and TW03-253) displayed distinct amino acid sequences at two or three sites (Ala77, Thr136, and Met186). Of these, Thr136 was common to the sequences of the serotype 6C isolates. wciP gene. The wciP gene showed more complicated amino acid sequence polymorphisms than the other three genes (Fig. 1B). The sequences for 10 amino acids, Val47, Glu117, Asn132, Asn142, Ile237, Leu238, Glu268, Ser270, Csy295, and Ser328, were unique to five isolates of the 6X group. Lys134 was shared among the 6X group of isolates and four serotype 6B isolates (K13-51, J01-4, TW02-294, and TW03-253). The sequences for two amino acids, Gln158 and Ala318, were found only in four isolates of serotype 6C. Consistent with the findings of previous studies (10, 14), Ser195 was present in serotype 6A and 6C isolates and Asn195 was present in serotype 6B and 6D isolates. These studies demonstrated that Ala192 and Arg254 in serotype 6A (and serotype 6C) and
Ser192 and Gly254 in serotype 6B (and serotype 6D) were distinct polymorphisms. However, Ser192 and Gly254 were identified in two 6A isolates (HK02-14 and J01-5 of the 6X group). In addition, Arg254 was also identified in four 6B isolates (K13-51, J01-4, TW02-294, and TW03-253). Met254 was also present in serotype 6A isolate HK02-77. Although Leu126, which was common in the serotype 6D isolates, was found in two 6B isolates, K13-51 and TW02-294. Gene trees. Trees of the wchA, wciN, wciO, and wciP genes were reconstructed on the basis of their nucleotide sequences (Fig. 2). In all gene trees, five isolates of the 6X group formed a distinct cluster, which was robustly supported by bootstrap analysis (100% in all gene trees). While the isolates of serotypes 6C and 6D clustered into a single group in the wchA and wciN gene trees (Fig. 2A and B), they did not in the wciO and wciP gene trees (Fig. 2C and D). Although serotype 6C isolates displayed close relationships with four isolates of serotype 6B isolates in the wciO gene tree (Fig. 2C), they clustered with serotype 6A isolates in the wciP gene tree (Fig. 2D). MLST. MLST data indicated that serotypes 6A, 6B, 6C, and 6D did not share the same genetic backgrounds (Table 1). The most common clone of serotype 6A was ST81, and ST83 and ST282 were its single-locus variants (Table 1). Although three Taiwanese serotype 6B isolates displayed a common genotype, ST76, serotype 6B isolates showed diverse genotypes. Of interest, four 6X group isolates (HK02-14, J01-5, K13-21, and M12-6) showed similar MLST allelic profiles. That is, they belonged to the same clonal complex, despite their different geographic origins: Hong Kong, Japan, South Korea, and Malaysia. However, the final 6X group isolate (V03-9) showed an allelic profile distinct from the profiles of the other isolates of the 6X group. As evident with serotype 6C, three isolates from South Korea showed similar allelic profiles, indicating a common origin. On the other hand, a 6C isolate from Vietnam showed a distinct allelic profile. All but one 6D isolate showed the same ST (ST282) or its single-locus variant (Table 1). The low variability of 6D isolates may be due to their limited geographic locales of origin: all but one 6D isolate originated from South Korea. However, one 6D isolate from Taiwan also belonged to ST282. Antimicrobial susceptibility. For most of the antimicrobial agents tested, the susceptibilities were not distinct among serotypes (Table 3). Overall, serogroup 6 isolates displayed high penicillin resistance (intermediate, 0.12 to 1 mg/liter;
FIG. 1. Amino acid polymorphisms of the wchA (A) and wciN, wciO, and wciP (B) genes. Dots indicate the same amino acid as that of HK02-77. The amino acid sequences of the wciN gene are represented only for serotype 6A and 6B isolates. The wciN genes of all serotype 6C and 6D isolates showed the same amino acid sequence except Glu368 in K13-11 and V07-123. 1761
FIG. 2. Gene trees of serogroup 6 isolates based on nucleotide sequences of wchA, wciN, wciO, and wciP genes, which were reconstructed by the method of neighbor joining. Due to the absence of a reliable outgroup, the midpoint rooting method was applied to root these trees. Branches supported by values higher than 50% in the bootstrap analysis (1,000 replications) are indicated. The serotypes of the isolates are indicated in parentheses. 1762
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TABLE 3. Antimicrobial susceptibility profiles of S. pneumoniae serogroup 6 isolates used in this study Serotype
Isolate no.
Penicillin susceptibilitya
6A
HK02-77 M06-6 K13-73 J01-2 J01-16 M18-12 TW03-260 TW03-284 K08-52 K13-75 HK02-14 J01-5 K13-21 M12-6 V03-9 K13-51
I S I I I I I I R R I I R I S R
J01-4 TW02-294 TW03-253 M12-8 TW03-254 TW03-308 TW06-5 K04-4 K13-111 K13-133 V07-123 K15-60 K15-99 K15-115 K15-129 TW02-238 07-056 07-077 07-107 B0704-047 K15-17 K13-22 K13-108 K13-109 K13-110
I I S S I I I S S I S R R I I I I I I I I I S I I
6B
6C
6D
Resistant profileb
1763
However, four isolates of the 6X group, with the exception of an isolate from Vietnam, were commonly resistant to cefuroxime, erythromycin, azithromycin, clarithromycin, and trimethoprim-sulfamethoxazole.
FUR-ERY-AZI-CLA FUR-ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-CD FUR-ERY-AZI-CLA ERY-AZI-CLA-CD ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-CD-SXT FUR-ERY-AZI-CLA-SXT FUR-ERY-AZI-CLA-CD-SXT FUR-ERY-AZI-CLA-CD-SXT AMX-A/C-AXO-FUR-ERYAZI-CLA-CD-SXT ERY-AZI-CLA-CD-SXT ERY-AZI-CLA-CD FUR-ERY-AZI-CLA-SXT FUR-ERY-AZI-CLA-SXT FUR-ERY-AZI-CLA-SXT ERY-AZI-CLA-CD-SXT FUR-ERY-AZI-CLA-CD-SXT FUR-ERY-AZI FUR-ERY-AZI-CLA-CIP FUR-ERY-AZI-LEV-GAT-CIP FUR-ERY-AZI FUR-ERY FUR-LEV-GAT-CIP FUR FUR-ERY-AZI-CLA-CD-SXT FUR-ERY-AZI-CLA FUR-ERY-AZI-CLA FUR-ERY-CLA ERY-AZI-CLA-CD FUR-LEV-GAT-CIP LEV-GAT-CIP
a MIC breakpoints of penicillin susceptibility: susceptible (S), ⱕ0.06 mg/liter; intermediate (I), 0.12 to 1 mg/liter; resistant (R), ⱖ2 mg/liter. b AMX, amoxicillin; A/C, amoxicillin-clavulanate; AXO, ceftriaxone; FUR, cefuroxime; ERY, erythromycin; AZI, azithromycin; CLA, clarithromycin; LEV, levofloxacin; MOX, moxifloxacin; GAT, gatifloxacin; CIP, ciprofloxacin; CD, clindamycin; SXT, trimethoprim-sulfamethoxazole.
resistance, ⱖ2 mg/liter) (Table 3). Only eight isolates were susceptible to penicillin (MIC ⬍ 0.12 mg/liter). However, there was a difference in the penicillin resistance among serotypes. While most 6A and 6D isolates were nonsusceptible to penicillin, only one 6C isolate displayed intermediate resistance to penicillin (MIC ⫽ 0.5 mg/liter). Fluoroquinolone-resistant isolates were identified only among serotype 6D isolates. Four or five 6D isolates were resistant to levofloxacin, moxifloxacin, and ciprofloxacin. Clindamycin resistance was also different among serotypes. While 8 of the 12 6A isolates (66.7%) were resistant to clindamycin, only 4 out of 11 6B isolates (36.4%) and 2 out of 14 6D isolates (14.3%) were resistant. Isolates belonging to the 6X group showed no common antimicrobial resistance profiles.
DISCUSSION S. pneumoniae serogroup 6 isolates, including isolates of serotypes 6A, 6B, 6C, and 6D, are important because they are commonly found in infections (13, 12). In South Korea, more than 10% of the pneumococcal isolates recovered in 2007 were serotypes 6A and 6B (15). The discovery of the 6C and 6D serotypes, which are not included in the PCV-7 and PCV-13 vaccine formulations, has increased the urgency to investigate the prevalence, characteristics, and evolution of serogroup 6 isolates. Serotypes 6A and 6B can be differentiated by a single nucleotide substitution in the wciP gene (G584A), which results in an amino acid change (Ser195Asn) (10, 13). Serotype 6C, which was serotyped 6A by classical Quellung reactions, has been thought to arise only by an exchange of wciN for wciN, resulting in the replacement of galactose in 6A with glucose in 6C (2, 4, 13). In addition, serotype 6D is thought to have arisen by the same mechanism: the substitution of wciN of 6B with wciN in 6D (2, 3, 9). However, the present data are consistent with a different explanation concerning the emergence of serotypes 6C and 6D and the existence of a group distinct from classical 6A and 6B isolates. The wchA gene of serotype 6C and 6D isolates was distinct from that of serotype 6A and 6B isolates, in addition to the wciN gene. This finding does not support the scenario that serotypes 6C and 6D emerged only by exchange of wciN to wciN. Two other explanations are plausible. It is conceivable that the wchA gene was independently coexchanged with wciN in serotypes 6A and 6B. That is, serotype 6C differentiated from serotype 6A and serotype 6D differentiated from serotype 6B. A second possibility is that the wchA gene of serotypes 6C and 6D represents a different genetic backbone than the corresponding gene in serotypes 6A and 6B. Put another way, serotypes 6C and 6D are more closely related to each other than serotypes 6A and 6B. MLST analysis indicated that most of the serotype 6C and 6D isolates were not closely related to serotype 6A and 6B isolates. Only one serotype 6A isolate from Japan, J01-16, showed the same ST (ST282) as most of the serotype 6D isolates. This contrasts with the previous notion that serotype 6D emerged from serotype 6B. In addition, serotype 6C isolates showed distinct polymorphisms of the wciP gene compared with other phenotypes, such as Gln158 and Ala318. Although serotype 6A and 6C isolates share Ser195 in the wciP gene, the amino acid sequence of the wciP gene of serotype 6C is different from that of serotype 6A in at least three sites. Thus, it is not plausible that serotypes 6C and 6D recently originated from serotypes 6A and 6B by exchange of some cps genes such as wciN. Amino acid sequences of the wchA and wciN genes were the same among serotype 6C and 6D isolates, except for isolate V07-123 from Vietnam. However, the wciO and wciP genes diverged between the two serotypes. Thus, it is likely that the origin of the wciO and wciP genes of serotypes 6C and 6D is different from that of
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the wchA and wciN genes. That is, the cps loci of serogroup 6 isolates constitute a mosaic structure originating from diverse donors, which may be facilitated due to the natural competence of S. pneumoniae. It has been suggested that the cps locus of serotype 6D may have appeared by a genetic recombination between serotypes 6B and 6C that involved the wciN and wciP genes (4). However, although the wciO gene of serotype 6D isolates is the same as that of some serotype 6B isolates, such as M12-8, TW03-254, TW03-308, and TW06-5, the wciP gene of serotype 6D isolates did not correspond to the wciP gene of any isolates of serotype 6B. Thus, the cps locus of serotype 6D did not emerge by a recent recombination between serotypes 6B and 6C. Presently, some S. pneumoniae serotype 6A and 6B isolates (6X group) displayed cps loci distinct from those of other serotype 6A and 6B isolates. In addition to encoded amino acid polymorphisms, insertion of about 300 bp between the wciN and wciO genes was found only in these five isolates. Distinct cps loci of serotypes 6A and 6B have previously been reported to be class 2 (4, 10). Our 6X group isolates are assumed to be very similar to class 2 isolates described in previous reports (4, 10). The amino acid sequences of four genes of their cps loci were very similar to each other, indicating the same origin. MLST analysis also suggests that most of the isolates originated from the same ancestor, despite different geographic locales of origin, such as Hong Kong, Japan, South Korea, and Malaysia. Only three amino acid sequence polymorphisms of four genes were found among five isolates of the 6X group. The Ser195Asn polymorphism in the wciP gene differentiated these isolates into serotypes 6A and 6B. However, differentiation into serotypes 6A and 6B by Ser195Asn did not correlate with the other wciN polymorphisms (Arg168Lys and Lys183Val). Thus, the entire cps locus might have transferred recently from an unknown origin into the 6X group. The 6X group may represent a serotype different from the other serotypes of serogroup 6. Gene trees support the suggestion that isolates of the 6X group are distinct from other isolates of serogroup 6. Whether they are a new serotype requires further study. There are also some limitations in this study. The number of isolates of each serotype was uneven, and only a few serotype 6C isolates could be investigated. In the surveillance study, 4.2%, 8.6%, 0.9%, and 0.9% of the 1,542 available S. pneumoniae isolates from Asian countries were serotypes 6A, 6B, 6C, and 6D, respectively. The homogeneity of serotype 6C isolates might be due to the limited number of isolates included in this study. In this study, 14 serotype 6D isolates were investigated, but their serotypes were characterized based on those published previously (3). In addition, only four genes of the cps locus were investigated. Further understanding of the evolution of serotype 6C in S. pneumoniae would be possible by sequencing of whole genes of the cps locus.
J. CLIN. MICROBIOL.
In short, this study has ensured that serotypes 6C and 6D emerged by a complicated mechanism, as shown previously (4). The cps loci of serogroup 6 isolates constitute a mosaic structure originating from diverse donors. We also identified the 6X group, consisting of serotype 6A and 6B isolates. They contain homogeneous cps loci and belong to the same clonal complex, except for one isolate. However, they can be clearly differentiated from other serogroup 6 isolates. Their clinical and public health significance warrants investigation. ACKNOWLEDGMENTS S. pneumoniae isolates used in this study were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID; Seoul, South Korea). This study was supported by a Samsung Biomedical Research Institute grant. REFERENCES 1. Aanensen, D. M., A. Mavroidi, S. D. Bentley, P. R. Reeves, and B. G. Spratt. 2007. Predicted functions and linkage specificities of the products of the Streptococcus pneumoniae capsular biosynthetic loci. J. Bacteriol. 189:7856– 7876. 2. Bratcher, P. E., I. H. Park, S. K. Hollingshead, and M. H. Nahm. 2009. Production of a unique pneumococcal capsule serotype belonging to serogroup 6. Microbiology 155:576–583. 3. Bratcher, P. E., K. H. Kim, J. H. King, J. Y. Hong, and M. H. Nahm. 2010. Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. Microbiology 156:555– 560. 4. Bratcher, P. E., et al. 2011. Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6. Microbiology 157:189–198. 5. Calix, J. J., and M. H. Nahm. 2010. A new pneumococcal serotype, 11E, has a variably inactivated wcjE gene. J. Infect. Dis. 202:29–38. 6. Carvalho, M. D. G., et al. 2009. PCR-based quantitation and clonal diversity of the current prevalent invasive serogroup 6 pneumococcal serotype, 6C, in the United States in 1999 and 2006 to 2007. J. Clin. Microbiol. 47:554–559. 7. Clinical and Laboratory Standards Institute. 2009. Performance standards for antimicrobial susceptibility testing, 19th informational supplement, M100-S19. Clinical and Laboratory Standards Institute, Wayne, PA. 8. Jacobs, M. R., et al. 2009. Occurrence, distribution, and origins of Streptococcus pneumoniae serotype 6C, a recently recognized serotype. J. Clin. Microbiol. 47:64–72. 9. Jin, P., et al. 2009. First report of putative Streptococcus pneumoniae serotype 6D among nasopharyngeal isolates from Fijian children. J. Infect. Dis. 200:1375–1380. 10. Mavroidi, A., et al. 2004. Evolutionary genetics of the capsular locus of serotype 6 pneumococci. J. Bacteriol. 186:8181–8192. 11. Moore, M. R., et al. 2008. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J. Infect. Dis. 197: 1016–1027. 12. Nahm, M. H., J. Lin, J. A. Finkelstein, and S. I. Pelton. 2009. Increase in the prevalence of the newly discovered pneumococcal serotype 6C in the nasopharynx after introduction of pneumococcal conjugate vaccine. J. Infect. Dis. 199:320–325. 13. Park, I. H., et al. 2007. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae. J. Clin. Microbiol. 45:1225–1233. 14. Sheppard, C. L., B. Pichon, R. C. George, and L. M. C. Hall. 2010. Streptococcus pneumoniae isolates expressing a capsule with epitopes of both serotypes 6A and 6B. Clin. Vaccine Immunol. 17:1820–1822. 15. Song, J.-H., et al. 2009. Changes of serotype and genotype in Streptococcus pneumoniae isolates from a Korean hospital in 2007. Diagn. Microbiol. Infect. Dis. 63:271–278. 16. World Health Organization. 2007. Pneumococcal conjugate vaccine for childhood immunization—WHO position paper. Wkly. Epidemiol. Rec. 82: 93–104.
