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 ~ Introduction
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 ~ Results
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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 358-362

Clonal similarities and sequence-type diversity of invasive and carriage Streptococcus pneumoniae in India among children under 5 Years

1 Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Paediatrics, Christian Medical College, Chennai, Tamil Nadu, India
3 ICMR, National Institute of Epidemiology, Chennai, Tamil Nadu, India
4 Department of Medicine, Christian Medical College, Vellore, Tamil Nadu, India

Date of Submission04-Dec-2019
Date of Decision04-Dec-2019
Date of Acceptance12-Dec-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. Balaji Veeraraghavan
Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_19_348

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 ~ Abstract 

Background: Pneumococcal pneumonia is one of the major causes of mortality in children less than 5 years in Asia, especially in India. Available PCVs have less serotype coverage in India compared to western countries. Moreover, the baseline pneumococcal serotype and sequence type data is limited and available data doesn't represent the entire India. With this background we aimed to characterize invasive and carriage isolates of S. pneumoniae from a tertiary care hospital in South India. Materials and Methods: A total of 221 S. pneumoniae isolates, invasive (n=138) and carriage (n=83) between the time period of 2012-2018 were included. Isolates was identified and confirmed using standard laboratory protocols. Serotyping was performed by Customized sequential multiplex PCR and MLST as described in Results: The major serotypes were 19F, 6B, 14, 6A and 19A and the sequence types (ST) were ST63, 236 and 230. Predominant STs in invasive was ST 63 whereas in carriage were ST4894 and 1701. High level ST diversity in carriage was observed. Majority of the STs were SLVs or DLVs of previously reported STs or PMEN clones. Phylogenetic analyses of the STs revealed gradual expansion of three PMEN CCs CC320, 63 and 230. Conclusion: The vaccine serotypes were the predominant ones found to be associated with IPD, PMEN clones, new STs and antimicrobial resistance. Accordingly, PCV13 is expected to provide invasive serotype coverage of 75% in Indian children less than 5 years. This study provides baseline serotype and sequence type data prior to the introduction of PCV in South India.

Keywords: India, multilocus sequence typing, pneumococcal conjugate vaccines 13, pneumococcal sequence types, Streptococcus pneumoniae

How to cite this article:
Varghese R, Neeravi A, Subramanian N, Pavithra B, Kavipriya A, Kumar JL, Girish Kumar C P, Jeyraman Y, Karthik G, Verghese VP, Veeraraghavan B. Clonal similarities and sequence-type diversity of invasive and carriage Streptococcus pneumoniae in India among children under 5 Years. Indian J Med Microbiol 2019;37:358-62

How to cite this URL:
Varghese R, Neeravi A, Subramanian N, Pavithra B, Kavipriya A, Kumar JL, Girish Kumar C P, Jeyraman Y, Karthik G, Verghese VP, Veeraraghavan B. Clonal similarities and sequence-type diversity of invasive and carriage Streptococcus pneumoniae in India among children under 5 Years. Indian J Med Microbiol [serial online] 2019 [cited 2020 Oct 25];37:358-62. Available from:

 ~ Introduction Top

Streptococcus pneumoniae is a major cause of pneumonia and invasive disease worldwide. The World Health Organisation estimates that around 15 million invasive pneumococcal disease (IPD) cases result in approximately 500,000 deaths worldwide that occur annually in children aged <5 years, particularly in developing countries.[1]S. pneumoniae has been classified currently up to 98 serotypes based on the composition of its capsular polysaccharide, which is the major virulence factor.[2]S. pneumoniae shows serotype diversity with respect to age, geography and disease potential.[3] With the ability of natural transformation, S. pneumoniae has become highly recombinant undergoing intra- and inter-species recombination during its carriage in the nasopharynx.[4],[5] This has led to the exchange of capsule, virulence factors and antimicrobial resistance genes which caused challenges in identification and epidemiological characterisation. Thus, several molecular methods such as pulsed-field gel electrophoresis,[6] BOX typing,[7] ribotyping,[8] multilocus enzyme electrophoresis[9] and multilocus sequence typing (MLST)[10] were used for characterising S. pneumoniae. MLST is widely used due to its unambiguity and reproducibility combined with the advantage of comparable data worldwide.

Despite this serotype diversity, the majority of IPD are caused only by about 6 to 11 serotypes.[11] Pneumococcal conjugate vaccines (PCVs) are commonly designed based on these predominant serotypes. Vaccine introduction has led to the emergence of vaccine escape recombinants in developed countries. Vaccine escape recombinants are strains that have a non-vaccine serotype but the same sequence type (ST). Moreover, the disease potential depends on serotype as well as ST.[12] Worldwide, MLST data to date have recognised highly invasive clones associated with certain serotypes as well as antimicrobial resistance clones. Some of these multidrug-resistant clones have spread globally.[13] ST as well as serotype of the prevalent pneumococci have to be considered in the design of new vaccines. Although a significant reduction in IPD cases was observed in several countries after the introduction of PCV in the early 2000s,[14],[15],[16],[17] the implementation of PCV in the Indian immunisation schedule was implemented in 2017 in a phased manner. This study was initiated with the aim of characterising pneumococcal isolates using MLST: (1) to assess the baseline serotype and ST data for invasive and carriage isolates in India, (2) to determine the relationship between Indian STs and (3) to compare between the Indian and global STs in the pre-vaccine era.

 ~ Materials and Methods Top

Bacterial isolates

Two hundred and twenty-one S. pneumoniae isolates of both invasive (n = 138) and non-invasive (n = 83) obtained from <5-year children were included in the study. The majority of the isolates were from children who were admitted in the Christian Medical College and Hospital, Vellore, between the years January 2012 and June 2018. Few invasive isolates (n = 34) were from other states: Kerala, Delhi, Karnataka and Shimla.

Identification and serotyping

S. pneumoniae was identified using standard methods such as optochin susceptibility test and bile solubility test.[18] Serotyping of the isolates was performed using the co-agglutination technique with Neufeld antisera obtained from Statens Serum Institut, Denmark, and sequential conventional multiplex polymerase chain reaction (PCR) as recommended by the CDC ([19]

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by the VITEK 2 system for the following antibiotics: penicillin, cefotaxime, erythromycin and cotrimoxazole only for the invasive isolates. The results were interpreted based on the Clinical Laboratory Standards Institute guidelines.

Multilocus sequence typing

From the isolates, genomic DNA was extracted manually using the commercial kit (QIAamp DNA Mini kit Qiagen) according to the manufacturer's instructions. PCR primers and thermal profile for the seven housekeeping genes were used as described at the MLST website ( Both strands of PCR products were sequenced using ABI 3130 Genetic Analyser. Sequences were edited using FinchTV, aligned using Clustal Omega ( and were uploaded onto MLST website ( pneumoniae/) and allele and STs for the isolates were assigned. The study STs were then compared along with the global and Indian STs that are available at S. pneumoniae PubMLST website (as of October 5, 2019) using PHYLOViZ software version 2.0 PHYLOViZ team, Lisboa, Portugal.

The phylogenetic trees based on MLST were drawn by the maximum likely hood method, using MEGA 7.0.21 free software and labelled using iTOL software Version 5.4, by Letunic and Bork biobyte solutions GmbH, Germany. ( Concatenated allele sequences except ddl locus of all the Indian STs (n = 395) were obtained from the PubMLST database.

 ~ Results Top

One thirty-eight invasive and 83 carriage isolates of S. pneumoniae were characterised. Among the 138 invasive isolates, 103 were from blood, 31 from cerebrospinal fluid and 4 from pleural fluid. Majority of the invasive strains were isolated from clinical cases of sepsis (n = 52), followed by meningitis (n = 53) and pneumonia (n = 28).

A total of 37 serotypes were identified among the 138 invasive isolates. The most common serotypes were 14, 6B, 19F, 6A and 23F. Among the 83 carriage isolates, a total of 21 different serotypes were found, of which the major serotypes were 19F, 6B, 6A, 23F, 23B and 15C. Among the IPD serotypes, the PCV13 serotype coverage was 75% (n = 103/138).

Penicillin and cefotaxime non-susceptibilities among the meningeal isolates were 45% and 19%, respectively. On the other hand, non-susceptibility to penicillin and cefotaxime was observed in only 2% of the non-meningeal isolates. In addition, resistance to erythromycin and trimethoprim/sulphamethoxazole was 48% and 100%, respectively. Vaccine serotypes 14, 19F, 6B and 6A were found to be resistant to penicillin, cefotaxime and erythromycin. The only invasive non-vaccine serotype that was found to be associated with resistance to the above-mentioned drugs was 10A.

Sequence type analysis

A total of 99 different STs were identified among the invasive isolates (n = 138), of which 43 are new STs. The major STs observed were ST 63 (n = 12), 4219 (n = 4), 236 (n = 4), 11921 (n = 4) and 3135 (n = 4). In the case of carriage isolates (n = 83), a total of 64 different STs were found, of which 47 were new STs. The major STs among carriage isolates were 4894 (n = 4), 1701 (n = 4) and 236 (n = 3). STs observed both in invasive and carriage were 236, 386, 1701, 473, 320, 271, 5612 and 90. A comparison between serotypes and STs showed a high ST diversity within serotype 6B followed by serotype 19F [Figure 1], while multiple serotypes within STs were observed among 63, 386 and 1701. Interestingly, majority of the new STs were associated with vaccine serotypes. The non-vaccine serotypes associated with new STs were 23B, 35B, 20, 34, 39, 10C, 10F, 11A, 15B, 15C and 18A.
Figure 1: Sequence-type distribution among the major serotypes

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goeBURST analysis

goeBURST analysis was carried out to understand clonal relatedness between the Indian and global isolates. The analysis within the study ST shows three clonal complexes (CCs) and 45 singletons. The major CCs and STs associated with invasive and non-invasive STs within the study isolates are shown in [Figure 2]. In comparison with global 800 CC profiles available in the S. pneumoniae PHYLOViZ database (, the study isolates spread across 36 CCs and 22 singletons. Furthermore, 54% of the total study isolates were associated with eight CCs (CC320, CC230, CC63, CC217, CC289, CC473, CC8164 and CC315). Geographically, majority of the STs observed in India were similar to the STs seen in Asia and Europe to a lesser extent in Africa. Within Asia, the Indian STs match with the Southeast Asian countries, mainly Thailand, Nepal and Bangladesh.
Figure 2: geoBURST image of the study isolates

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Phylogenetic analyses of Indian sequence types

Maximum likelihood phylogenetic tree constructed using concatenated MLST allele sequences (except ddl locus to reduce the diversity) revealed mainly seven sequence clusters (SCs). Among these, SC7, 1 and 6 comprise several novel STs that belong to the Pneumococcal Molecular Epidemiology Network (PMEN) CCs such as 230, 320 and 63, respectively [Figure 3]. This is in accordance with the goeBURST observations.
Figure 3: Maximum likelihood tree created using concatenated sequences of multilocus sequence typing genes excluding ddl gene of all Indian isolates

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 ~ Discussion Top

This is the first report to characterise a large number of invasive and carriage isolates of S. pneumoniae from India, both serotype and ST wise. The most common serotypes (19F, 6B, 14 and 6A) observed were similar to the earlier studies reported from India.[20],[21],[22],[23] Overall, 75% of the invasive and 54% of the carriage isolates belong to vaccine serotypes. Most of the vaccine serotypes were highly associated with ST of PMEN clones (68%) and the remaining were new novel clones (32%). The diverse non-vaccine serotypes observed in this study such as 10A, 15B and 23B were similar and serotypes such as 35A, 34 and 15C were different from the earlier reports.[24],[25],[26],[27] Interestingly, the only non-vaccine serotype with multidrug resistance observed in the invasive study collection belongs to serotype 10A, which is alarming.

MLST analysis revealed a higher (90/231; 39%) number of new STs compared to the previously reported.[21] More number of new STs were observed in carriage isolates (46/83) than in invasive (44/138), which reflects increased genetic diversity and continuously evolving carriage population. This is similar to the observation of Brueggmann et al.[12] Only STs that are related to PMEN clones (ST236, 271, 320, 90, 386, 5612, 1701 and 473) were seen both in invasive and carriage. Of these, ST63, ST236 and ST1701 had more number of isolates which corresponds to PMEN-resistant clones. The majority of the new STs were found to be the single-locus variants or double-locus variants of the PMEN clones that have been previously reported mainly in India and other Southeast Asian countries. Interestingly, a new combination of allelic profiles was more than new alleles, which indicates highly evolving pneumococcal clones. This attributes to the clonal expansion of the pre-existing clones or PMEN clones. Most of the study STs were similar to those found in South Asia and Africa. However, we observed that certain STs were specific to India; this includes ST874, ST5672, ST13585 and ST9597.

Further, whole-genome analysis should be performed on these isolates to define global pneumococcal SCs to understand vaccine-driven serotype replacement and antimicrobial resistance.

 ~ Conclusion Top

To conclude, most of the invasive isolates were of vaccine serotypes with penicillin non-susceptibility. STs among vaccine serotypes are highly evolving, which is of concern. The observed ST diversity is due to the expansion of pre-existing PMEN clones that are mainly CC320 (236 Taiwan19F-14), CC63 (Sweden15A- 25) and CC230 (Denmark14-32).

Financial support and sponsorship

MLST for a subset of isolates between the time period of 2012-2014 were supported by IIR grant from Pfizer Reference#W1178713.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

O'Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, McCall N, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: Global estimates. Lancet 2009;374:893-902.  Back to cited text no. 1
Jauneikaite E, Tocheva AS, Jefferies JM, Gladstone RA, Faust SN, Christodoulides M, et al. Current methods for capsular typing of Streptococcus pneumoniae. J Microbiol Methods 2015;113:41-9.  Back to cited text no. 2
Hausdorff WP, Feikin DR, Klugman KP. Epidemiological differences among pneumococcal serotypes. Lancet Infect Dis 2005;5:83-93.  Back to cited text no. 3
Hakenbeck R, Balmelle N, Weber B, Gardès C, Keck W, de Saizieu A. Mosaic genes and mosaic chromosomes: Intra- and interspecies genomic variation of Streptococcus pneumoniae. Infect Immun 2001;69:2477-86.  Back to cited text no. 4
Shak JR, Vidal JE, Klugman KP. Influence of bacterial interactions on pneumococcal colonization of the nasopharynx. Trends Microbiol 2013;21:129-35.  Back to cited text no. 5
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.  Back to cited text no. 6
van Belkum A, Sluijuter M, de Groot R, Verbrugh H, Hermans PW. Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains. J Clin Microbiol 1996;34:1176-9.  Back to cited text no. 7
Kell CM, Jordens JZ, Daniels M, Coffey TJ, Bates J, Paul J, et al. Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya. Infect Immun 1993;61:4382-91.  Back to cited text no. 8
Hall LM, Whiley RA, Duke B, George RC, Efstratiou A. Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: Analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns. J Clin Microbiol 1996;34:853-9.  Back to cited text no. 9
Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae: Identification of clones associated with serious invasive disease. Microbiology 1998;144(Pt 11):3049-60.  Back to cited text no. 10
Johnson HL, Deloria-Knoll M, Levine OS, Stoszek SK, Freimanis Hance L, Reithinger R, et al. Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five: The pneumococcal global serotype project. PLoS Med 2010;7. pii: e1000348.  Back to cited text no. 11
Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG. Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-specific differences in invasive disease potential. J Infect Dis 2003;187:1424-32.  Back to cited text no. 12
McGee L, McDougal L, Zhou J, Spratt BG, Tenover FC, George R, et al. Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network. J Clin Microbiol 2001;39:2565-71.  Back to cited text no. 13
Lepoutre A, Varon E, Georges S, Gutmann L, Lévy-Bruhl D. Impact of infant pneumococcal vaccination on invasive pneumococcal diseases in France, 2001-2006. Euro Surveill 2008;13. pii: 18962.  Back to cited text no. 14
Grijalva CG, Griffin MR. Population-based impact of routine infant immunization with pneumococcal conjugate vaccine in the USA. Expert Rev Vaccines 2008;7:83-95.  Back to cited text no. 15
Ingels H, Rasmussen J, Andersen PH, Harboe ZB, Glismann S, Konradsen H, et al. Impact of pneumococcal vaccination in Denmark during the first 3 years after PCV introduction in the childhood immunization programme. Vaccine 2012;30:3944-50.  Back to cited text no. 16
Rodenburg GD, de Greeff SC, Jansen AG, de Melker HE, Schouls LM, Hak E, et al. Effects of pneumococcal conjugate vaccine 2 years after its introduction, the Netherlands. Emerg Infect Dis 2010;16:816-23.  Back to cited text no. 17
World Health Organization, Centers for Disease Control and Prevention. Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus Influenzae. World Health Organization; 2011.  Back to cited text no. 18
Veeraraghavan B, Jayaraman R, John J, Varghese R, Neeravi A, Verghese VP, et al. Customized sequential multiplex PCR for accurate and early determination of invasive pneumococcal serotypes found in India. J Microbiol Methods 2016;130:133-5.  Back to cited text no. 19
Shariff M, Choudhary J, Zahoor S, Deb M. Characterization of Streptococcus pneumoniae isolates from India with special reference to their sequence types. J Infect Dev Ctries 2013;7:101-9.  Back to cited text no. 20
Gopi T, Ranjith J, Anandan S, Balaji V. Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing. Indian J Med Microbiol 2016;34:17-21.  Back to cited text no. 21
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Molander V, Elisson C, Balaji V, Backhaus E, John J, Vargheese R, et al. Invasive pneumococcal infections in Vellore, India: Clinical characteristics and distribution of serotypes. BMC Infect Dis 2013;13:532.  Back to cited text no. 22
Thomas K. Prospective multicentre hospital surveillance of Streptococcus pneumoniae disease in India. Lancet 1999;353:1216-21.  Back to cited text no. 23
Ladhani SN, Collins S, Djennad A, Sheppard CL, Borrow R, Fry NK, et al. Rapid increase in non-vaccine serotypes causing invasive pneumococcal disease in England and Wales, 2000-17: A prospective national observational cohort study. Lancet Infect Dis 2018;18:441-51.  Back to cited text no. 24
Chochua S, Metcalf BJ, Li Z, Walker H, Tran T, McGee L, et al. Invasive Serotype 35B Pneumococci Including an Expanding Serotype Switch Lineage, United States, 2015-2016. Emerg Infect Dis 2017;23:922-30.  Back to cited text no. 25
Kandasamy R, Voysey M, Collins S, Berbers G, Robinson H, Noel I, et al. Persistent circulation of vaccine serotypes and serotype replacement after five years of UK infant immunisation with PCV13. J Infect Dis 2019;Apr.  Back to cited text no. 26
Galanis I, Lindstrand A, Darenberg J, Browall S, Nannapaneni P, Sjöström K, et al. Effects of PCV7 and PCV13 on invasive pneumococcal disease and carriage in Stockholm, Sweden. Eur Respir J 2016;47:1208-18.  Back to cited text no. 27


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