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The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine
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Diagnostic Microbiology and Infectious Disease 68 (2010) 439 – 444 www.elsevier.com/locate/diagmicrobio
The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine Pak-Leung Hoa,⁎, Irene Anga , Kin-Hung Chowa , Eileen L. Laia , Susan S. Chiub a
b
Department of Microbiology, The University of Hong Kong, Pokfulam Road, Pokfulam, Hong Kong SAR, China Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China Received 20 May 2010; accepted 26 July 2010
Abstract A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA–wciNβ– wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways. © 2010 Elsevier Inc. All rights reserved. Keywords: Streptococcus pneumoniae; Drug resistance; Serotype; Prevalence
1. Introduction Streptococcus pneumoniae is an important cause of bacterial infections involving the respiratory tract, bloodstream, and central nervous system. While over 90 different capsular serotypes are recognized, most studies have found a small number of them dominating the pneumococcal population (Harboe et al., 2010). Accordingly, the dominant serotypes such as those in serogroup 6 have been more extensively studied than others. Recently, a new serotype, 6C, which is closely related to 6A, was discovered because an increase in disease caused by this type was recognized in the United States, following the widespread use of the 7-valent pneumococcal conjugate vaccine (PCV7). Due to cross-reactivity with polyclonal antibodies, these isolates were initially thought to be serotype 6A. Serotypes 6A and ⁎ Corresponding author. Tel.: +852-2855-4897; fax: +852-2855-1241. E-mail address: [email protected] (P.-L. Ho). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.07.020
6C are highly similar. Biochemically, the only difference between the 2 serotypes involved the replacement of galactose in 6A with glucose in 6C (Park et al., 2007b). Sequencing showed that this occurs because the wciN gene in serotype 6C encodes a different glycosyl transferase (Park et al., 2007a). In serotype 6C, the wciNβ (or wciN6c) gene is about 200 base pairs shorter than the 1 in serotype 6A (Park et al., 2007a). More recently, another new serotype, 6D, which differed from 6B by the same 6C associated wciNβ gene, was reported in naturally occurring pneumococci (Bratcher et al., 2010; Jin et al., 2009a). The 2 new subtypes, serotypes 6C and 6D, among serogroup 6 could be distinguished by polymerase chain reaction (PCR) assays (Jin et al., 2009a, 2009b). A factor 6d antiserum has also been introduced for serotyping of 6C (Jacobs et al., 2010). In Hong Kong, studies that employed polyvalent antisera (factors 6b and 6c, provided by the Statens Serum Institut, Copenhagen, Denmark) have shown that 10.6% to 27.7% of all the pneumococcal serotypes
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belonged to serogroup 6 (Chiu et al., 2001; Ho et al., 1999, 2004). In Hong Kong, the PCV7 was registered in year September 2005, and a program for universal use in infant was introduced in September 2009. To obtain an understanding of the subtypes among these serogroup 6 isolates prior to the use of PCV7, we retrieved 3 stored pneumococcal collections for testing. The genotypic characteristics of the 6C and 6D isolates were determined by molecular studies. 2. Materials and methods 2.1. Bacterial strains Three bacterial collections were retrieved for the present investigation. The first collection included 181 isolates obtained from clinical specimens of inpatients treated in 7 public hospitals in 1998 (Ho et al., 1999). The second collection consisted of sterile site isolates causing invasive pneumococcal disease in patients treated in 5 hospitals during 1995 to 2001 (Ho et al., 2004, 2006). The third collection comprised 383 nasopharyngeal isolates collected from children (aged 2–6 years) who attended kindergartens and day care centers in year 2000 (Chiu et al., 2001). The isolates were stored in commercial bacterial preservation system at −80 °C (Microbank; Pro-Lab Diagnostics, Cheshire, United Kingdom). All isolates that remained viable and were previously categorized as serogroup 6 isolate by the Quellung reaction were included in the present study. 2.2. Antimicrobial susceptibility testing MIC of penicillin and cefotaxime for the isolates were determined by Etest (AB Biodisk, Sweden) following the manufacturer's recommendation. The disc diffusion method was used to determine susceptibility for erythromycin, clindamycin, cotrimoxazole, chloramphenicol, and levofloxacin in accordance with the Clinical and Laboratory Standard Institute (2009). ATCC 49619 (S. pneumoniae) and 29213 (Staphylococcus aureus) strains were included as quality controls. 2.3. Serotype-specific PCRs The capsular type of the isolates were previously determined to be either serotype 6A or 6B using polyclonal sera from the Statens Serum Institut and the Quellung reaction. The multiplex PCR algorithm described previously were used to repeat the identification of the serotypes (Jin et al., 2009a, 2009b). The following primer combinations were used: wciP584gS/wciP-r (specificity for 6A/6C), wciP584aS/wciP-r (specificity for 6B/6D), and wciNβS1/ wciNβA2 (specificity for 6C and 6D). 2.4. Sequencing and analysis DNA fragments of the wciP and partial wchA–wciNβ– wciO (wcANO) genes were amplified by PCR and sequenced using primers described previously. Primers wciPS2 and
wcisPA1 were used for amplification of the wciP region (810 bp), and the inner primer pair wciPS1 and wciPA2 were used for sequencing. For the partial wcANO genes, primers 5106 and 3101 were used for amplification (1.8 kb), and primers 3101, 5106, wciNβA2, and wciNβS1 were used for sequencing. The wcANO sequences of the Hong Kong 6C and 6D isolates were compared with 11 non-Hong Kong 6C and 6D isolates (accession no. HM367702 to HM367704) of the corresponding region available in GenBank. These include 3 Brazil 6C isolates, 1 Korea 6C isolate, 1 USA 6C isolate (accession no. EF538714 to EF538718) (Park et al., 2007a), 4 Fijian 6C and 6D isolates (accession no. FJ899597 to FJ899600) (Jin et al., 2009a), and 2 Korea 6D isolates (accession nos. GQ848645 and GQ848646) (Bratcher et al., 2010). All the sequences were aligned according to the USA 6C isolate (accession no. EF538714.). For comparison purpose, each different sequence for the wchA (partial, nucleotide position 6251–6521, 271 bp), wciNβ gene (complete, nucleotide position 6522–7646, 1125 bp), and wciO gene (partial, nucleotide position 7633–7694, 62 bp) was assigned as a distinct allele and given a different allele number. The allele number for the 3 cps genes (wchA– wciNβ–wciO) defined the wcANO profile of the isolate. The sequences for the 3 wcANO fragments were concatenated, and a tree was constructed from the concatenated sequence (1444 bp) by the neighbor-joining method using the Molecular Evolutionary Genetic Analysis software version 4.0 (Tamura et al., 2007). The wciP sequences (nearly complete, nucleotide position 8526–9270, 645 bp) of the Hong Kong (accession nos. HM367705 and HM367706) and non-Hong Kong 6C and 6D isolates (accession nos. FJ899601, FJ899602, GQ848645, GQ848646, and EF538714) were compared against wciP alleles 1 to 12 of 6A and 6B isolates published previously (Mavroidi et al., 2004). A neighbor-joining tree was constructed from the wciP sequences to demonstrate genetic relatedness between the 6C/6D isolates and the 6A/6B isolates. 2.5. Pulsed field gel electrophoresis and multilocus sequence typing The genetic relatedness for the subset of 6C and 6D isolates was studied by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Protocols for PFGE analysis and MLST have been described previously (Ho et al., 2001). SmaI was used for digestion of DNA in the PFGE analysis, and the results were interpreted according to a suggested scheme (Tenover et al., 1995). Strain SP264 representative of the Spain23F-1 clone was used as a control in the PFGE analysis. 3. Results 3.1. Prevalence of serotype 6C and 6D Information for the 3 groups of isolates and the PCR subtyping results is summarized in Table 1. The proportions
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Table 1 Distribution of serotypes within serogroup 6 isolates for 3 pneumococcal collections, Hong Kong Group
1 2 3
Study period
Isolate source
Second half of 1998 1995–2001 December 1999–June 2000
Total no. of isolates obtained
Various clinical specimens, 7 regional hospitals Blood or CSF, 5 hospitals Nasopharyngeal isolates from children aged 2–6 years attending 79 day care centers and kindergartens
Total a
No. of pneumococcal isolates with capsular Serogroupa
Serotype
6
6A
181 265 383
24/24 24/28 106/106
5 2 13
829
154/158
20
6B
6C
6D
17 22 88
0 0 5
2 0 0
127
5
2
No. of viable isolates/no. obtained.
of serogroup 6 isolates among all pneumococci were 13.2% (24/181) for group 1, 10.6% (28/265) for group 2, and 27.7% (106/383) for group 3. Four of the isolates from group 2 were missing. Thus, a total of 154 serogroup 6 isolates were tested. The specimen sources for the isolates were nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2). PCRs with primers specific for serotypes 6A/6C (wciP584gS/wciP-r) and 6B/ 6D (wciP584aS/wciP-r) yielded results that were concordant with conventional serotyping except for 1 isolate. The isolate was initially serotyped as 6B, but PCR indicated that it belonged to 6A/6C. The serotyping was repeated by the quelling reaction and demonstrated to be 6A. Of the 25 isolates that were serotyped as 6A by the quelling reaction, 5 isolates contained the wciNβ gene and were reclassified as serotype 6C. Of the 129 isolates that were serotyped as 6B by the quelling reaction, 2 isolates contained the wciNβ gene and were reclassified as serotype 6D. Overall, serotype 6B was the predominant serogroup 6 subtype, and the proportions did not differ significantly by isolate source group (70.8% [17/24] in group 1, 91.6% [22/24] in group 2, and 83% [88/106] in group 3; P = 0.22), age group (81.6% [115/ 141] for children versus 70.6% [12/17] for adult, P = 0.28),
and specimen type (79.3% [23/29] for invasive isolates versus 80.6% [104/129] for noninvasive isolates, P = 0.87). 3.2. Molecular analysis Antimicrobial susceptibility and genotypic features for the 5 serotype 6C and 2 serotype 6D isolates were summarized in Table 2. All but 2 of the 6C isolates were fully susceptibility to penicillin and cefotaxime. Susceptibility profiles of the isolates for the non–β-lactam antibiotics were variable. MLST suggested 4 genetic lineages and PFGE suggested 5. Two isolates were clonally related (ST76, PFGE pattern A); another 3 isolates that included both 6C and 6D isolates belonged to the same clonal group (ST982 or its single-locus variants) but exhibited variations in the PFGE patterns (B1, B2, and C). The 2 ST76/PFGE A isolates were recovered from children in the same kindergartens. The remaining 2 isolates had distinct typing results, ST4542/PFGE D and ST3087/PFGE E. Polymorphism in the wcANO region among the isolates was investigated (Fig. 1A and B). The analysis showed that relatedness of the sequences exhibited some relationship according to the geographic sources, those from Hong Kong,
Table 2 Phenotypic and genotypic characteristics of serotype 6C and 6D isolates Strain
G81 G821 J119 L35 Q116 S7D1 S9C4
Year
2000 2000 2000 2000 2000 1998 1998
Serotype
6C 6C 6C 6C 6C 6D 6D
Sex/age (years)
Source
F/5 F/6 M/5 F/5 M/6 F/1 M/73
NP NP NP NP NP Sputum Sputum
MIC (μg/mL)
Resistograma
wcANO profileb
wciP allelec
PEN/CTX 1/0.75 1/1 0.023/0.016 0.047/0.047 0.023/0.023 0.023/0.023 0.032/0.032
RSRSS RSRSS RRRRS RRRSS RSRSS RSRSS RRRSS
1-1-1 1-1-1 1-1-1 1-2-1 1-1-1 1-2-1 1-2-1
9 9 9 9 9 5 5
MLST ST
Allelic profile
76 76 4542 982 3087 5085 5086
2-13-9-15-6-19-42 2-13-9-15-6-19-42 7-13-1-6-6-6-14 8-13-9-60-78-119-6 2-12-4-16-6-1-5 8-13-241-60-78-119-6 8-13-9-60-78-1-6
PFGE
A A D B1 E B2 C
ST = sequence type; F = female; M = male; PEN = penicllin G; CTX = cefotaxime. a Resistance pattern for erythromycin, clindamycin, cotrimoxazole, chloramphenicol, and levofloxacin. Test for inducible resistance to clindamycin was negative in all the isolates. b The complete wciNβ gene (1125 bp), partial wciP gene (810 bp), and regions flanking wciNβ (wchA, 271 bp and wciO, 62 bp) were sequenced. The same number was assigned for each unique wchA, wciNβ, wciO, and wciP sequence. c The wciP allele was assigned according to polymorphisms reported previously (Mavroidi et al., 2004). A total of 12 distinct wciP alleles have been reported.
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Fig. 1. Genetic relatedness of the cps genes of 6C and 6D isolates. (A) Neighbor-joining tree showing the relatedness of the concatenated wchA, wciNβ, and wciO (wcANO) genes (1444 bp) among the 6C and 6D isolates collected in different countries and in different periods is shown. The bracket showed the allelic profile of the 3 genes in the order wchA–wciNβ–wciO and the number of isolates. The scales represent a genetic distance of 0.01%. (B) The polymorphic nucleotide sites of the wcANO genes were shown. The nucleotides were numbered in vertical format. The nucleotide at each variable site is shown for the first allele, and only the nucleotides that differed from those in this sequence were shown. (C) Neighbor-joining tree showed the relatedness of the wciP gene (645 bp) among Hong Kong and non-Hong Kong 6C/6D isolates, and to those of the wciP alleles 1 to 12 of the 6A/6B isolates published previously (Mavroidi et al., 2004). The wciP-1, -2, -7, -9, and -11 alleles (boxed) were found exclusively among serotype 6A isolates, whereas wciP-3, -4, -5, and -6 and the more divergent wciP-8 and -12 were restricted to serotype 6B isolates. The serotype association of wciP-10 has not been reported. The scale represented a genetic distance of 0.2%.
Korea, and Fijian form 1 group, whereas those from Brazil and the United States form another group. It is notable that the first group included both serotype 6C and 6D isolates, and that identical sequences were shared by 11 serotype 6D isolates (2 from Hong Kong, 2 from Korea, and 7 from Fijian) and 1 serotype 6C isolate from Hong Kong. Among serotypes 6A and 6B isolates, a total of 12 distinct wciP alleles have been described (Mavroidi et al., 2004). These alleles were assigned according to polymorphisms for a 645-bp internal region of the gene. A phylogenetic tree was constructed to investigate the relationship of the wciP gene among 6C and 6D isolates to those alleles (Fig. 1C). The result showed that all the 6C isolates clustered with the wciP-9 allele, whereas the 6D isolates form a group with the wciP-5 allele.
4. Discussion Recent studies and the results presented here highlight the need to subtype serogroup 6 pneumococcal isolates. Serotype 6B, 1 of the 7 types in PCV7, elicits crossprotection to the related serotype 6A. However, there is no
cross immunity for serotype 6C (Park et al., 2008). Among nasopharyngeal isolates collected from Massachusetts children between 1994 and 2007, there was an increase in the prevalence of serotype 6C and a reduction in 6A after the introduction of PCV7 (Nahm et al., 2009). Similarly, serotype 6C among nasopharynx (NP) isolates increased from 3.1% in year 1 to 13.6% in year 2 in the United Kingdom following implementation of PCV7 in the infant immunization schedule (Tocheva et al., 2010). However, such dramatic increase in serotype 6C is not always observed. In Portugal, there has been no dramatic increase in serotype C between 1996 and 2007 (Nunes et al., 2009). Among Australian children living in remote communities, the proportion of serotype 6C among all nasopharyngeal pneumococci was reported to remain at 4% four years after the universal use of PCV7 in infant immunization program (Leach et al., 2009). The pneumococcal isolates in this study were collected before PCV7 was introduced in Hong Kong. The low prevalence (1.3%) of serotype 6C among nasopharyngeal isolates in our cohort of children was similar to those reported for other countries before the widespread use of PCV7: 0.6% (before
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2001) among Massachusetts children in the United States and 3.2% (2000–2001) among a cohort of household members in Brazil (Campos et al., 2009; Nahm et al., 2009). This study also revealed that serotype 6D has been in existence since 1998 at a low frequency. Regarding this serotype, it was initially constructed experimentally by the group who discovered 6C by replacing the wciN6B gene in the capsular locus of 6B with wciN6c from serotype 6C (Bratcher et al., 2009). Subsequently, the serotype was found in 2 of 14 nasopharyngeal isolates obtained from healthy children in Korea in 2008 (Bratcher et al., 2010) and in 14 of 34 nasopharyngeal isolates from Fijian children collected between 2004 and 2007 (Jin et al., 2009a). In the Fijian children cohort, it was further shown that PCV7 immunization may offer selective advantage for serotype 6D. Eight-six percent of children from whom serotype 6D was isolated had received at least 1 dose of PCV7, compared with 45% of those colonized with 6B (Jin et al., 2009a). To further investigate the genetic relationship between serotypes 6C and 6D, we sequenced the capsular gene loci that distinguish the subtypes among serogroup 6. Analysis of the polymorphic sites revealed 2 different wcANO profiles among our strains. Since the MLST and PFGE results indicated that both groups of serotype 6C and 6D isolates were genetically diverse, such genetic recombination involving the capsular loci has likely occurred on multiple occasions and involving different pneumococcal lineages. Previous studies have shown that the regions flanking wciNβ were highly conserved among serotype 6C collected from a different period from different continents (Park et al., 2007a). Here, we showed that those sequences for isolates form Hong Kong, Korea, and Fijian are more closely related to each other than were those from Brazil and the United States. The wcANO sequences for 4 of the Hong Kong 6C strains were identical and differed from the remaining one by only 2 single nucleotide polymorphisms (SNPs). Identical sequences were shared by the 2 Korea 6C isolates and some of the Fijian 6C isolates. On the other hand, the sequences for the 6C isolates from Fijian children exhibited a number of consistent differences in wchA (6 SNPs), wciN (1 SNP), and wciP (8 SNPs), suggesting different ancestral sources (Jin et al., 2009a). These variations could reflect different ancestral sources from which the wcANO genes were derived or divergence following acquisition from a single ancestral source. We favored the different ancestral source explanation because identical sequence was shared by the 6C isolates from Hong Kong and Korea collected over a 10-year interval. By comparison, different wcANO sequences were found for 6C isolates collected within a few years of each other. Since the number of isolates tested is small, a firm conclusion cannot be drawn at this stage. Population analysis of 6A and 6B strains showed that point mutations in the wciP gene (A/G polymorphism at position 8938 in EF538714) leading to serotype changes
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from 6A to 6B and vice versa occurred (Mavroidi et al., 2004). Theoretically, type 6C and 6D strains can also switch serotypes by the same mechanism. Our finding of one 6C (L35) and two 6D (S7D1 and S9C4) isolates sharing similar MLST/PFGE results and identical wcANO DNA sequences suggest that this may have occurred naturally. The 6B component included in the currently available pneumococcal conjugate vaccines does not confer any cross-immunity against 6C (Nahm et al., 2009). It is not known if it will provide cross immunity to 6D. Interestingly, despite the existence of different wciP alleles among the 6A and 6B strains, the polymorphisms within the wciP genes for the 6C and 6D isolates were quite homogenous. The reason for this is unclear. One possible explanation is that certain wciP sequence may favor recombination event involving the wcANO genes. Furthermore, all of our 6C and 6D isolates were uniformly resistant to erythromycin and cotrimoxazole; 2 were additionally nonsusceptible to β-lactams. The finding highlights the ability of 6C and 6D isolates to develop multidrug resistance. In the United States where serotype 6C is emerging, many of the isolates were found to be nonsusceptible to penicillin and erythromycin (Carvalho et al., 2009). In conclusion, the prevalence of the 2 newly described serotypes 6C and 6D in Hong Kong was low before the introduction of PCV7. Continued surveillance is necessary to guide future vaccine strategies. Acknowledgments This study is supported by research grants from the Research Fund for the Control of Infectious Diseases of the Health and Food Bureau of the Hong Kong SAR Government. Authors had no conflict of interest to declare. References Bratcher PE, Park IH, Hollingshead SK, Nahm MH (2009) Production of a unique pneumococcal capsule serotype belonging to serogroup 6. Microbiology 155:576–583. Bratcher PE, Kim KH, Kang JH, Hong JY, Nahm MH (2010) Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. Microbiology 156: 555–560. Campos LC, Carvalho MG, Beall BW, Cordeiro SM, Takahashi D, Reis MG, Ko AI, Reis JN (2009) Prevalence of Streptococcus pneumoniae serotype 6C among invasive and carriage isolates in metropolitan Salvador, Brazil, from 1996 to 2007. Diagn Microbiol Infect Dis 65: 112–115. Carvalho MG, Pimenta FC, Gertz Jr RE, Joshi HH, Trujillo AA, Keys LE, Findley J, Moura IS, Park IH, Hollingshead SK, Pilishvili T, Whitney CG, Nahm MH, Beall BW (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. Chiu SS, Ho PL, Chow FK, Yuen KY, Lau YL (2001) Nasopharyngeal carriage of antimicrobial-resistant Streptococcus pneumoniae among young children attending 79 kindergartens and day care centers in Hong Kong. Antimicrob Agents Chemother 45:2765–2770.
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Diagnostic Microbiology and Infectious Disease 68 (2010) 439 – 444 www.elsevier.com/locate/diagmicrobio
The prevalence and characteristics of Streptococcus pneumoniae isolates expressing serotypes 6C and 6D in Hong Kong prior to the introduction of the 7-valent pneumococcal conjugate vaccine Pak-Leung Hoa,⁎, Irene Anga , Kin-Hung Chowa , Eileen L. Laia , Susan S. Chiub a
b
Department of Microbiology, The University of Hong Kong, Pokfulam Road, Pokfulam, Hong Kong SAR, China Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China Received 20 May 2010; accepted 26 July 2010
Abstract A study was conducted to determine the prevalence of the 2 newly described types, 6C and 6D, among pneumococcal isolates collected in Hong Kong before availability of the 7-valent pneumococcal conjugate vaccine. A total of 154 serogroup 6 isolates obtained from nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2) during 1995 to 2001 were analyzed by polymerase chain reaction typing. Five nasopharyngeal and 2 sputum isolates were found to belong to 6C and 6D, respectively. The isolates were genetically diverse, but one 6C and two 6D isolates exhibited some clonal relationship. Phylogenetic analysis of the wchA–wciNβ– wciO nucleotide sequences showed that the Hong Kong 6C/6D isolates had 2 allelic profiles, which were more closely related to 6C/6D isolates from Fijian and Korea than were those from Brazil and the United States. However, all of the wciP gene sequences for both Hong Kong and non-Hong Kong isolates clustered together: 6C isolates with the wciP-9 allele and 6D isolates with the wciP-5 allele. In conclusion, the prevalence of the 2 newly described serotypes was low before the era of the pneumococcal conjugate vaccine. Nonetheless, results from the molecular studies indicated that the evolution of the capsular genes have involved complex pathways. © 2010 Elsevier Inc. All rights reserved. Keywords: Streptococcus pneumoniae; Drug resistance; Serotype; Prevalence
1. Introduction Streptococcus pneumoniae is an important cause of bacterial infections involving the respiratory tract, bloodstream, and central nervous system. While over 90 different capsular serotypes are recognized, most studies have found a small number of them dominating the pneumococcal population (Harboe et al., 2010). Accordingly, the dominant serotypes such as those in serogroup 6 have been more extensively studied than others. Recently, a new serotype, 6C, which is closely related to 6A, was discovered because an increase in disease caused by this type was recognized in the United States, following the widespread use of the 7-valent pneumococcal conjugate vaccine (PCV7). Due to cross-reactivity with polyclonal antibodies, these isolates were initially thought to be serotype 6A. Serotypes 6A and ⁎ Corresponding author. Tel.: +852-2855-4897; fax: +852-2855-1241. E-mail address: [email protected] (P.-L. Ho). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.07.020
6C are highly similar. Biochemically, the only difference between the 2 serotypes involved the replacement of galactose in 6A with glucose in 6C (Park et al., 2007b). Sequencing showed that this occurs because the wciN gene in serotype 6C encodes a different glycosyl transferase (Park et al., 2007a). In serotype 6C, the wciNβ (or wciN6c) gene is about 200 base pairs shorter than the 1 in serotype 6A (Park et al., 2007a). More recently, another new serotype, 6D, which differed from 6B by the same 6C associated wciNβ gene, was reported in naturally occurring pneumococci (Bratcher et al., 2010; Jin et al., 2009a). The 2 new subtypes, serotypes 6C and 6D, among serogroup 6 could be distinguished by polymerase chain reaction (PCR) assays (Jin et al., 2009a, 2009b). A factor 6d antiserum has also been introduced for serotyping of 6C (Jacobs et al., 2010). In Hong Kong, studies that employed polyvalent antisera (factors 6b and 6c, provided by the Statens Serum Institut, Copenhagen, Denmark) have shown that 10.6% to 27.7% of all the pneumococcal serotypes
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belonged to serogroup 6 (Chiu et al., 2001; Ho et al., 1999, 2004). In Hong Kong, the PCV7 was registered in year September 2005, and a program for universal use in infant was introduced in September 2009. To obtain an understanding of the subtypes among these serogroup 6 isolates prior to the use of PCV7, we retrieved 3 stored pneumococcal collections for testing. The genotypic characteristics of the 6C and 6D isolates were determined by molecular studies. 2. Materials and methods 2.1. Bacterial strains Three bacterial collections were retrieved for the present investigation. The first collection included 181 isolates obtained from clinical specimens of inpatients treated in 7 public hospitals in 1998 (Ho et al., 1999). The second collection consisted of sterile site isolates causing invasive pneumococcal disease in patients treated in 5 hospitals during 1995 to 2001 (Ho et al., 2004, 2006). The third collection comprised 383 nasopharyngeal isolates collected from children (aged 2–6 years) who attended kindergartens and day care centers in year 2000 (Chiu et al., 2001). The isolates were stored in commercial bacterial preservation system at −80 °C (Microbank; Pro-Lab Diagnostics, Cheshire, United Kingdom). All isolates that remained viable and were previously categorized as serogroup 6 isolate by the Quellung reaction were included in the present study. 2.2. Antimicrobial susceptibility testing MIC of penicillin and cefotaxime for the isolates were determined by Etest (AB Biodisk, Sweden) following the manufacturer's recommendation. The disc diffusion method was used to determine susceptibility for erythromycin, clindamycin, cotrimoxazole, chloramphenicol, and levofloxacin in accordance with the Clinical and Laboratory Standard Institute (2009). ATCC 49619 (S. pneumoniae) and 29213 (Staphylococcus aureus) strains were included as quality controls. 2.3. Serotype-specific PCRs The capsular type of the isolates were previously determined to be either serotype 6A or 6B using polyclonal sera from the Statens Serum Institut and the Quellung reaction. The multiplex PCR algorithm described previously were used to repeat the identification of the serotypes (Jin et al., 2009a, 2009b). The following primer combinations were used: wciP584gS/wciP-r (specificity for 6A/6C), wciP584aS/wciP-r (specificity for 6B/6D), and wciNβS1/ wciNβA2 (specificity for 6C and 6D). 2.4. Sequencing and analysis DNA fragments of the wciP and partial wchA–wciNβ– wciO (wcANO) genes were amplified by PCR and sequenced using primers described previously. Primers wciPS2 and
wcisPA1 were used for amplification of the wciP region (810 bp), and the inner primer pair wciPS1 and wciPA2 were used for sequencing. For the partial wcANO genes, primers 5106 and 3101 were used for amplification (1.8 kb), and primers 3101, 5106, wciNβA2, and wciNβS1 were used for sequencing. The wcANO sequences of the Hong Kong 6C and 6D isolates were compared with 11 non-Hong Kong 6C and 6D isolates (accession no. HM367702 to HM367704) of the corresponding region available in GenBank. These include 3 Brazil 6C isolates, 1 Korea 6C isolate, 1 USA 6C isolate (accession no. EF538714 to EF538718) (Park et al., 2007a), 4 Fijian 6C and 6D isolates (accession no. FJ899597 to FJ899600) (Jin et al., 2009a), and 2 Korea 6D isolates (accession nos. GQ848645 and GQ848646) (Bratcher et al., 2010). All the sequences were aligned according to the USA 6C isolate (accession no. EF538714.). For comparison purpose, each different sequence for the wchA (partial, nucleotide position 6251–6521, 271 bp), wciNβ gene (complete, nucleotide position 6522–7646, 1125 bp), and wciO gene (partial, nucleotide position 7633–7694, 62 bp) was assigned as a distinct allele and given a different allele number. The allele number for the 3 cps genes (wchA– wciNβ–wciO) defined the wcANO profile of the isolate. The sequences for the 3 wcANO fragments were concatenated, and a tree was constructed from the concatenated sequence (1444 bp) by the neighbor-joining method using the Molecular Evolutionary Genetic Analysis software version 4.0 (Tamura et al., 2007). The wciP sequences (nearly complete, nucleotide position 8526–9270, 645 bp) of the Hong Kong (accession nos. HM367705 and HM367706) and non-Hong Kong 6C and 6D isolates (accession nos. FJ899601, FJ899602, GQ848645, GQ848646, and EF538714) were compared against wciP alleles 1 to 12 of 6A and 6B isolates published previously (Mavroidi et al., 2004). A neighbor-joining tree was constructed from the wciP sequences to demonstrate genetic relatedness between the 6C/6D isolates and the 6A/6B isolates. 2.5. Pulsed field gel electrophoresis and multilocus sequence typing The genetic relatedness for the subset of 6C and 6D isolates was studied by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Protocols for PFGE analysis and MLST have been described previously (Ho et al., 2001). SmaI was used for digestion of DNA in the PFGE analysis, and the results were interpreted according to a suggested scheme (Tenover et al., 1995). Strain SP264 representative of the Spain23F-1 clone was used as a control in the PFGE analysis. 3. Results 3.1. Prevalence of serotype 6C and 6D Information for the 3 groups of isolates and the PCR subtyping results is summarized in Table 1. The proportions
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Table 1 Distribution of serotypes within serogroup 6 isolates for 3 pneumococcal collections, Hong Kong Group
1 2 3
Study period
Isolate source
Second half of 1998 1995–2001 December 1999–June 2000
Total no. of isolates obtained
Various clinical specimens, 7 regional hospitals Blood or CSF, 5 hospitals Nasopharyngeal isolates from children aged 2–6 years attending 79 day care centers and kindergartens
Total a
No. of pneumococcal isolates with capsular Serogroupa
Serotype
6
6A
181 265 383
24/24 24/28 106/106
5 2 13
829
154/158
20
6B
6C
6D
17 22 88
0 0 5
2 0 0
127
5
2
No. of viable isolates/no. obtained.
of serogroup 6 isolates among all pneumococci were 13.2% (24/181) for group 1, 10.6% (28/265) for group 2, and 27.7% (106/383) for group 3. Four of the isolates from group 2 were missing. Thus, a total of 154 serogroup 6 isolates were tested. The specimen sources for the isolates were nasopharynx (n = 106), blood (n = 22), respiratory (n = 24), and cerebrospinal fluid (CSF) (n = 2). PCRs with primers specific for serotypes 6A/6C (wciP584gS/wciP-r) and 6B/ 6D (wciP584aS/wciP-r) yielded results that were concordant with conventional serotyping except for 1 isolate. The isolate was initially serotyped as 6B, but PCR indicated that it belonged to 6A/6C. The serotyping was repeated by the quelling reaction and demonstrated to be 6A. Of the 25 isolates that were serotyped as 6A by the quelling reaction, 5 isolates contained the wciNβ gene and were reclassified as serotype 6C. Of the 129 isolates that were serotyped as 6B by the quelling reaction, 2 isolates contained the wciNβ gene and were reclassified as serotype 6D. Overall, serotype 6B was the predominant serogroup 6 subtype, and the proportions did not differ significantly by isolate source group (70.8% [17/24] in group 1, 91.6% [22/24] in group 2, and 83% [88/106] in group 3; P = 0.22), age group (81.6% [115/ 141] for children versus 70.6% [12/17] for adult, P = 0.28),
and specimen type (79.3% [23/29] for invasive isolates versus 80.6% [104/129] for noninvasive isolates, P = 0.87). 3.2. Molecular analysis Antimicrobial susceptibility and genotypic features for the 5 serotype 6C and 2 serotype 6D isolates were summarized in Table 2. All but 2 of the 6C isolates were fully susceptibility to penicillin and cefotaxime. Susceptibility profiles of the isolates for the non–β-lactam antibiotics were variable. MLST suggested 4 genetic lineages and PFGE suggested 5. Two isolates were clonally related (ST76, PFGE pattern A); another 3 isolates that included both 6C and 6D isolates belonged to the same clonal group (ST982 or its single-locus variants) but exhibited variations in the PFGE patterns (B1, B2, and C). The 2 ST76/PFGE A isolates were recovered from children in the same kindergartens. The remaining 2 isolates had distinct typing results, ST4542/PFGE D and ST3087/PFGE E. Polymorphism in the wcANO region among the isolates was investigated (Fig. 1A and B). The analysis showed that relatedness of the sequences exhibited some relationship according to the geographic sources, those from Hong Kong,
Table 2 Phenotypic and genotypic characteristics of serotype 6C and 6D isolates Strain
G81 G821 J119 L35 Q116 S7D1 S9C4
Year
2000 2000 2000 2000 2000 1998 1998
Serotype
6C 6C 6C 6C 6C 6D 6D
Sex/age (years)
Source
F/5 F/6 M/5 F/5 M/6 F/1 M/73
NP NP NP NP NP Sputum Sputum
MIC (μg/mL)
Resistograma
wcANO profileb
wciP allelec
PEN/CTX 1/0.75 1/1 0.023/0.016 0.047/0.047 0.023/0.023 0.023/0.023 0.032/0.032
RSRSS RSRSS RRRRS RRRSS RSRSS RSRSS RRRSS
1-1-1 1-1-1 1-1-1 1-2-1 1-1-1 1-2-1 1-2-1
9 9 9 9 9 5 5
MLST ST
Allelic profile
76 76 4542 982 3087 5085 5086
2-13-9-15-6-19-42 2-13-9-15-6-19-42 7-13-1-6-6-6-14 8-13-9-60-78-119-6 2-12-4-16-6-1-5 8-13-241-60-78-119-6 8-13-9-60-78-1-6
PFGE
A A D B1 E B2 C
ST = sequence type; F = female; M = male; PEN = penicllin G; CTX = cefotaxime. a Resistance pattern for erythromycin, clindamycin, cotrimoxazole, chloramphenicol, and levofloxacin. Test for inducible resistance to clindamycin was negative in all the isolates. b The complete wciNβ gene (1125 bp), partial wciP gene (810 bp), and regions flanking wciNβ (wchA, 271 bp and wciO, 62 bp) were sequenced. The same number was assigned for each unique wchA, wciNβ, wciO, and wciP sequence. c The wciP allele was assigned according to polymorphisms reported previously (Mavroidi et al., 2004). A total of 12 distinct wciP alleles have been reported.
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Fig. 1. Genetic relatedness of the cps genes of 6C and 6D isolates. (A) Neighbor-joining tree showing the relatedness of the concatenated wchA, wciNβ, and wciO (wcANO) genes (1444 bp) among the 6C and 6D isolates collected in different countries and in different periods is shown. The bracket showed the allelic profile of the 3 genes in the order wchA–wciNβ–wciO and the number of isolates. The scales represent a genetic distance of 0.01%. (B) The polymorphic nucleotide sites of the wcANO genes were shown. The nucleotides were numbered in vertical format. The nucleotide at each variable site is shown for the first allele, and only the nucleotides that differed from those in this sequence were shown. (C) Neighbor-joining tree showed the relatedness of the wciP gene (645 bp) among Hong Kong and non-Hong Kong 6C/6D isolates, and to those of the wciP alleles 1 to 12 of the 6A/6B isolates published previously (Mavroidi et al., 2004). The wciP-1, -2, -7, -9, and -11 alleles (boxed) were found exclusively among serotype 6A isolates, whereas wciP-3, -4, -5, and -6 and the more divergent wciP-8 and -12 were restricted to serotype 6B isolates. The serotype association of wciP-10 has not been reported. The scale represented a genetic distance of 0.2%.
Korea, and Fijian form 1 group, whereas those from Brazil and the United States form another group. It is notable that the first group included both serotype 6C and 6D isolates, and that identical sequences were shared by 11 serotype 6D isolates (2 from Hong Kong, 2 from Korea, and 7 from Fijian) and 1 serotype 6C isolate from Hong Kong. Among serotypes 6A and 6B isolates, a total of 12 distinct wciP alleles have been described (Mavroidi et al., 2004). These alleles were assigned according to polymorphisms for a 645-bp internal region of the gene. A phylogenetic tree was constructed to investigate the relationship of the wciP gene among 6C and 6D isolates to those alleles (Fig. 1C). The result showed that all the 6C isolates clustered with the wciP-9 allele, whereas the 6D isolates form a group with the wciP-5 allele.
4. Discussion Recent studies and the results presented here highlight the need to subtype serogroup 6 pneumococcal isolates. Serotype 6B, 1 of the 7 types in PCV7, elicits crossprotection to the related serotype 6A. However, there is no
cross immunity for serotype 6C (Park et al., 2008). Among nasopharyngeal isolates collected from Massachusetts children between 1994 and 2007, there was an increase in the prevalence of serotype 6C and a reduction in 6A after the introduction of PCV7 (Nahm et al., 2009). Similarly, serotype 6C among nasopharynx (NP) isolates increased from 3.1% in year 1 to 13.6% in year 2 in the United Kingdom following implementation of PCV7 in the infant immunization schedule (Tocheva et al., 2010). However, such dramatic increase in serotype 6C is not always observed. In Portugal, there has been no dramatic increase in serotype C between 1996 and 2007 (Nunes et al., 2009). Among Australian children living in remote communities, the proportion of serotype 6C among all nasopharyngeal pneumococci was reported to remain at 4% four years after the universal use of PCV7 in infant immunization program (Leach et al., 2009). The pneumococcal isolates in this study were collected before PCV7 was introduced in Hong Kong. The low prevalence (1.3%) of serotype 6C among nasopharyngeal isolates in our cohort of children was similar to those reported for other countries before the widespread use of PCV7: 0.6% (before
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2001) among Massachusetts children in the United States and 3.2% (2000–2001) among a cohort of household members in Brazil (Campos et al., 2009; Nahm et al., 2009). This study also revealed that serotype 6D has been in existence since 1998 at a low frequency. Regarding this serotype, it was initially constructed experimentally by the group who discovered 6C by replacing the wciN6B gene in the capsular locus of 6B with wciN6c from serotype 6C (Bratcher et al., 2009). Subsequently, the serotype was found in 2 of 14 nasopharyngeal isolates obtained from healthy children in Korea in 2008 (Bratcher et al., 2010) and in 14 of 34 nasopharyngeal isolates from Fijian children collected between 2004 and 2007 (Jin et al., 2009a). In the Fijian children cohort, it was further shown that PCV7 immunization may offer selective advantage for serotype 6D. Eight-six percent of children from whom serotype 6D was isolated had received at least 1 dose of PCV7, compared with 45% of those colonized with 6B (Jin et al., 2009a). To further investigate the genetic relationship between serotypes 6C and 6D, we sequenced the capsular gene loci that distinguish the subtypes among serogroup 6. Analysis of the polymorphic sites revealed 2 different wcANO profiles among our strains. Since the MLST and PFGE results indicated that both groups of serotype 6C and 6D isolates were genetically diverse, such genetic recombination involving the capsular loci has likely occurred on multiple occasions and involving different pneumococcal lineages. Previous studies have shown that the regions flanking wciNβ were highly conserved among serotype 6C collected from a different period from different continents (Park et al., 2007a). Here, we showed that those sequences for isolates form Hong Kong, Korea, and Fijian are more closely related to each other than were those from Brazil and the United States. The wcANO sequences for 4 of the Hong Kong 6C strains were identical and differed from the remaining one by only 2 single nucleotide polymorphisms (SNPs). Identical sequences were shared by the 2 Korea 6C isolates and some of the Fijian 6C isolates. On the other hand, the sequences for the 6C isolates from Fijian children exhibited a number of consistent differences in wchA (6 SNPs), wciN (1 SNP), and wciP (8 SNPs), suggesting different ancestral sources (Jin et al., 2009a). These variations could reflect different ancestral sources from which the wcANO genes were derived or divergence following acquisition from a single ancestral source. We favored the different ancestral source explanation because identical sequence was shared by the 6C isolates from Hong Kong and Korea collected over a 10-year interval. By comparison, different wcANO sequences were found for 6C isolates collected within a few years of each other. Since the number of isolates tested is small, a firm conclusion cannot be drawn at this stage. Population analysis of 6A and 6B strains showed that point mutations in the wciP gene (A/G polymorphism at position 8938 in EF538714) leading to serotype changes
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from 6A to 6B and vice versa occurred (Mavroidi et al., 2004). Theoretically, type 6C and 6D strains can also switch serotypes by the same mechanism. Our finding of one 6C (L35) and two 6D (S7D1 and S9C4) isolates sharing similar MLST/PFGE results and identical wcANO DNA sequences suggest that this may have occurred naturally. The 6B component included in the currently available pneumococcal conjugate vaccines does not confer any cross-immunity against 6C (Nahm et al., 2009). It is not known if it will provide cross immunity to 6D. Interestingly, despite the existence of different wciP alleles among the 6A and 6B strains, the polymorphisms within the wciP genes for the 6C and 6D isolates were quite homogenous. The reason for this is unclear. One possible explanation is that certain wciP sequence may favor recombination event involving the wcANO genes. Furthermore, all of our 6C and 6D isolates were uniformly resistant to erythromycin and cotrimoxazole; 2 were additionally nonsusceptible to β-lactams. The finding highlights the ability of 6C and 6D isolates to develop multidrug resistance. In the United States where serotype 6C is emerging, many of the isolates were found to be nonsusceptible to penicillin and erythromycin (Carvalho et al., 2009). In conclusion, the prevalence of the 2 newly described serotypes 6C and 6D in Hong Kong was low before the introduction of PCV7. Continued surveillance is necessary to guide future vaccine strategies. Acknowledgments This study is supported by research grants from the Research Fund for the Control of Infectious Diseases of the Health and Food Bureau of the Hong Kong SAR Government. Authors had no conflict of interest to declare. References Bratcher PE, Park IH, Hollingshead SK, Nahm MH (2009) Production of a unique pneumococcal capsule serotype belonging to serogroup 6. Microbiology 155:576–583. Bratcher PE, Kim KH, Kang JH, Hong JY, Nahm MH (2010) Identification of natural pneumococcal isolates expressing serotype 6D by genetic, biochemical and serological characterization. Microbiology 156: 555–560. Campos LC, Carvalho MG, Beall BW, Cordeiro SM, Takahashi D, Reis MG, Ko AI, Reis JN (2009) Prevalence of Streptococcus pneumoniae serotype 6C among invasive and carriage isolates in metropolitan Salvador, Brazil, from 1996 to 2007. Diagn Microbiol Infect Dis 65: 112–115. Carvalho MG, Pimenta FC, Gertz Jr RE, Joshi HH, Trujillo AA, Keys LE, Findley J, Moura IS, Park IH, Hollingshead SK, Pilishvili T, Whitney CG, Nahm MH, Beall BW (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. Chiu SS, Ho PL, Chow FK, Yuen KY, Lau YL (2001) Nasopharyngeal carriage of antimicrobial-resistant Streptococcus pneumoniae among young children attending 79 kindergartens and day care centers in Hong Kong. Antimicrob Agents Chemother 45:2765–2770.
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