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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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  Table of Contents  
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 17-21

Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing

Department of Clinical Microbiology, Christian Medical College and Hospital, Vellore - 632 004, Tamil Nadu, India

Date of Submission02-Nov-2014
Date of Acceptance04-Jun-2015
Date of Web Publication15-Jan-2016

Correspondence Address:
V Balaji
Department of Clinical Microbiology, Christian Medical College and Hospital, Vellore - 632 004, Tamil Nadu
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Source of Support: Christian Medical College and Hospital through fluid research grant,, Conflict of Interest: None

DOI: 10.4103/0255-0857.174113

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 ~ Abstract 

Objective: The aim of this study was to utilize the multilocus sequence typing (MLST) technique to characterise Streptococcus pneumoniae among clinical isolates in India. MLST was used to determine clonality, to establish genetic relatedness, to check for correlation between serotypes and sequence types (STs) and its relevance associated with antibiotic resistance. Methods: Forty consecutive invasive S. pneumoniae isolates in children <5 years were characterised. Preliminary identification of serotype and antibiotic susceptible profile was followed with MLST technique to identify the STs of the isolates. STs were then analysed for clonality using an eBURST algorithm and genetic relatedness using Sequence Type Analysis and Recombinational Tests version 2 software. Results: The most common ST was ST63. Among the forty isolates, we identified nine novel STs, six of which had known alleles but in new combinations, three of which had new alleles in their sequence profile. The new STs assigned were 8501–8509. One clonal complex was found among the 40 strains characterised. The most common serotypes in this study were serotype 19F, 14 and 5. Non-susceptibility to penicillin and erythromycin was observed in 2.5% and 30% of the isolates, respectively. Conclusion: This study shows a significant number of novel STs among the 40 isolates characterised (9/40, 22.5%), however, internationally recognised strains were also circulating in India, indicating, there could be greater geographical variation in pneumococcal STs in India. Molecular epidemiology data is essential to understand the population dynamics of S. pneumoniae in India before the introduction of pneumococcal vaccines in NIP in India.

Keywords: Clonal complex, India, multilocus sequence typing, sequence types, Streptococcus pneumoniae

How to cite this article:
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

How to cite this URL:
Gopi T, Ranjith J, Anandan S, Balaji V. Epidemiological characterisation of Streptococcus pneumoniae from India using multilocus sequence typing. Indian J Med Microbiol [serial online] 2016 [cited 2020 Nov 25];34:17-21. Available from:

 ~ Introduction Top

Streptococcus pneumoniae is among the most important aetiological agents of bacterial pneumonia, meningitis and sepsis in children. In a weekly epidemiological report in 2007, World Health Organisation reported that every year almost 1 million children aged below 5 years die of invasive pneumococcal disease (IPD), most of them from developing countries.[1] In 2006, UNICEF reported that of the 44 million pneumonia cases that occurred in India, more than half were due to S. pneumoniae.[2]S. pneumoniae infections are also the leading cause of death due to vaccine-preventable bacterial illness in children aged <5 years.[3]

Currently, more than 90 immunologically distinct capsular serotypes have been described,[4] which possess distinct epidemiological properties. Some serotypes are associated only with nasopharyngeal (NP) carriage and rarely cause invasive disease. Less than 20 serotypes cause >90% of IPD worldwide.[5] Molecular analysis in the past few decades has identified specific genotypes within particular serotypes that are more prone to cause disease and are also associated with drug resistance. Many of these resistant genotypes have spread globally.[6] Another concern is the phenomenon of 'capsular switching' where the S. pneumoniae exchange their capsules. Studies have identified in vaccinated populations, S. pneumoniae isolates with non-vaccine targeted serotypes that are genetically very closely related to isolates of vaccine serotypes.[7] Therefore, it is of utmost importance that genotyping methods be employed, to better understand the population biology of S. pneumoniae.

Pneumococcal conjugate vaccines (PCVs) are available to prevent pneumococcal disease and introduction of PCV into the routine paediatric immunisation schedule in the United States and other countries has substantially reduced the incidence of IPD due to vaccine serotypes in young children and in unvaccinated older children and adults.[8] PCVs have now been incorporated into routine childhood immunisation in 96 countries. Another 51 countries, many in the developing world, including India plan to introduce PCV in the coming years.[9]

Dynamics of serotype/genotype distribution prior to the introduction of PCV in National immunisation programme is, therefore, important for optimal PCV formulation and continued surveillance will also assist in understanding effects of the vaccine over time, once introduced. In the present study, we aimed at determining S. pneumoniae serotypes and genotypes among paediatric invasive strains isolated in a hospital between 2010 and 2011 in order to provide information on circulating strains in Vellore, South India.

 ~ Materials and Methods Top


This laboratory-based study included, 40 consecutive S. pneumoniae, isolated from sterile body fluids (blood-32, Cerebrospinal fluid-5 [CSF-5] and fluid-3) in <5 years age group, over a period of 2 years from January 2010 to December 2011. During the year 2010, 23 isolates were collected and 17 through 2011. The isolates were collected from a single tertiary care hospital in South India. Non-invasive isolates and invasive isolates from patients >5 years of age were excluded from the study. The study protocol was approved by institutional review board. S. pneumoniae was identified using standard methods such as optochin susceptibility test and bile solubility test.[10] Serotypes of the isolates were determined using co-agglutination technique [11] with Neufeld antisera obtained from Statens Serum Institut, Denmark and sequential conventional multiplex polymerase chain reaction (SMPCR) ( Positive controls for SMPCR were kindly provided by Streptococcus laboratories, Centres for Disease Control and Prevention, Atlanta.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by broth microdilution (VITEK 2 system) for the following antibiotics: Penicillin, cefotaxime, erythromycin and cotrimoxazole. Antimicrobial susceptibility tests were interpreted as per on the Clinical Laboratory Standards Institute (2013).[12]

DNA extraction

Genomic DNA was extracted from S. pneumoniae isolates using commercially available (QIAamp DNA Mini kit-Qiagen) according to manufacturer's instructions.

Multilocus sequence typing

Polymerase chain reaction primers and PCR cycle conditions for the seven housekeeping genes were used as described on the multilocus sequence typing (MLST) website ( Both strands of PCR products were sequenced using ABI 3130 Genetic Analyser. Sequences obtained were uploaded onto MLST website and allele and sequence types (STs) for the isolates were assigned. The STs were then analysed using the eBURST algorithm,[13] along with the STs for isolates from India already available at MLST website (as of July 22nd, 2014).

Sequences and computation

DNA editing was performed with the following web tools. Nucleic acid massager ( (Attotron Biosenser Corporation, USA) and Finch Tv software (PerkinElmer, USA) were used for viewing and trimming DNA. ClustalW2 Multiple Sequence Alignment was used for aligning the forward and reverse sequence to get the consensus sequence ( The consensus sequence was submitted to the to assign the allele number for each of the seven housekeeping genes. The allelic profile was submitted and ST assigned. Analytical software-Sequence Type Analysis and Recombinational Tests version 2 (Hosted by University of Oxford, UK) was used for the analysis of MLST data available on website.[14]

 ~ Results Top

Multilocus sequence typing identified 28 different STs. ST63 was the most common ST with six isolates (15%); All six ST63 belonged to serotype 14. Seven STs namely, ST236, ST289, ST2854, ST4209, ST4219, ST4221 and ST7089 had two isolates (5%) in this study. All seven paired STs belonged to same serotypes; with the only close exception of ST4209 having serotypes 15B and 15C. 20 (50%) isolates were represented by singletons in this study (81, 386, 473, 4990, 5034, 5077, 5080, 6025, 6397, 6784, 6787, 8501, 8502, 8503, 8504, 8505, 8506, 8507, 8508, 8509) [Table 1]. Four international clones described by Pneumococcal Molecular Epidemiology Network (PMEN) [] namely ST63 (6 isolates), ST81 (one isolate), ST236 (two isolates) and ST289 (two isolates) were seen in eleven of the isolates (27.5%) in this study [Table 1] and [Figure 1].
Table 1: Clinical characteristics, serotype, sequence type and antimicrobial resistance profile

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Figure 1

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Nine new STs (8501, 8502, 8593, 8504, 8505, 8506, 8507, 8508 and 8509) were determined in this study [Figure 1]. Three isolates had novel alleles; ST 8505 had new allele in ddl gene; ST8506 had new allele in gki gene and ST8509 having two new alleles in aroE and gdh genes. The other six new STs were with known alleles as well as new combinations of alleles. Noval STs accounted for 22.5% of isolates.

One clonal complex was found within the 40 isolates characterised in this study. The 40 isolates in this study along with the 123 Indian isolates already submitted to the MLST website showed 14 clonal complexes, shown in [Figure 1].

Clinically, (11) 27.5% of the patients had meningitis in this study group. Among the isolates causing meningitis STs 4221, 4219 and 7089 were found in two of the isolates each. The STs in the meningitis causing group were due to others and there was no one ST that seemed to be occurring predominantly in this group.

Among the 20 patients (50%) who had septicaemia due to S. pneumoniae, ST 63 was found in three and ST 4209 in two of the isolates each. Interestingly, STs (8502–8507), that is six of the nine newly discovered STs in this study, were found in the other S. pneumoniae that caused septicaemia group. Other clinical syndromes such as pneumonia, peritonitis, empyema and bacteraemia accounted for 22.5% (9) of the cases in this study group, but no single ST accounted for most of these disease presentations.

The 40 isolates belonged to 19 different serogroups/serotypes with 19F, 14 and 5 (n = 19; 47.5%) being the most common serotypes. Among these isolates, only one isolate (2.5%) from CSF was found to be penicillin non-susceptible (MIC 0.25 µg/ml) and 12 isolates (30%) were non-susceptible to erythromycin (MIC ≥0.5 µg/ml). All the isolates were resistant to co-trimoxazole and susceptible to cefotaxime [Table 1].

The only penicillin-resistant isolate encountered in this study belonged to ST63, which is an internationally disseminated clone (Sweden 15A-25), well described for its role in the global spread of penicillin resistance. This penicillin-resistant clone was also resistant to erythromycin. Erythromycin resistance was also encountered in two other global clones, namely ST81 (Spain 23F-1) and ST236 (Taiwan 19F-14).

 ~ Discussion Top

Pneumococcal conjugate vaccine formulations are based on phenotypic (serotype) characters, but with genetic recombination and horizontal transfer of genetic material between S. pneumoniae isolates, phenotypically similar S. pneumoniae can show differences in genotypes. It is, therefore, important to generate genotype data to completely understand the epidemiology and population biology of S. pneumoniae, especially with the introduction of pneumococcal vaccines. Despite India, being the country with largest burden of pneumococcal disease, data available on genotypes and the clonal structure of S. pneumoniae isolates circulating in India is very limited, particularly from children under 5 years of age. The MLST database currently has 163 isolates characterised from various regions of India, including the 40 isolates from this study. An eBURST analysis of these isolates [Figure 1] shows extensive genetic diversity within India, with many STs unique to this region. It is evident from this study that international clones were also circulating in India along with the significant number of new clones

In this study we identified 28 different STs, of which nine were novel genotypes and four were international clones. The serotype 14 capsular variant of Sweden 15A-25 (ST63) was the most common ST identified in six study isolates. Three other well described antibiotic-resistant internationally encountered PMEN clones (Spain 23F-1 ST81; Taiwan 19F-14 ST236 and Colombia 5-19 ST289) were also present among our isolates from Indian children;[15] ( Serotype 5 is highly clonal and the four isolates in this study, which comprised two related STs (ST289 and ST4219), belonged to the major Colombia 5-19 clonal complex. Serotype 19F, which is a common serotype in IPD in children, showed much greater diversity having seven different STs among the 19F serotypes.

Clonal complexes (consisting of ≥2 STs) were observed from the eBURST analysis of the 163 S. pneumoniae isolates from India uploaded in MLST database. Two complexes had four and five isolates each. Three complexes had three isolates, and nine complexes had two isolates [Figure 1]. The 40 isolates in this study produced only one clonal complex (ST 289 and 4219) with a large number of singletons, suggesting a high genetic variation among Indian S. pneumoniae isolates.

In this study, we did not find a single most common ST responsible for any of a particular pneumococcal disease. However, six out of nine new STs were responsible for causing septicaemia. Among the isolates tested, we identified one CSF isolate (serotype 14) with reduced susceptibility to penicillin which belonged to ST63. Penicillin resistance in this study is low compared to rates reported from other parts of the world.[16] However, our study findings are in concordance with other studies published in this region showing low rates of penicillin resistance in India.[17] A much higher prevalence of macrolide resistance has been reported worldwide, particularly in Asia and 30% of isolates in this study were non-susceptible to erythromycin.[16]

Three serotypes (19F, 14 and 5) accounted for 47.5% of isolates and although our numbers were relatively small, we did not see any significant difference in serotype coverage between PCV10 (62.5%) and PCV13 (67.5%) among the study isolates. This coverage is similar when compared to the available serotype prevalence data from our centre for PCV10 and 13 as 64% and 74%, respectively (unpublished data).

One of the limitations of the current study is the relatively small sample size. However, with limited resources for MLST we restricted the study to include only consecutively isolated invasive pneumococcal strains from children <5 years of age, the target group for vaccination. The other limitation is the non-inclusion of the (NP) carriage and the non-invasive isolates. Epidemiological data from studies done elsewhere has explained that almost all (NP) carriage clones were identical to invasive isolates indicating (NP) carriage clones are also associated with invasive isolates.[18],[19] Data from this study will increase the knowledge on circulating genotypes of pneumococcal isolates in India and this study has also served as a pilot study for future genetic studies on S. pneumoniae in India which will include non invasive and NP carriage strains.

 ~ Conclusion Top

This study shows significant genetic diversity within S. pneumoniae isolates identifying novel STs along with internationally well-characterised clones. Knowledge of serotype distribution and information on the clonal structure of S. pneumoniae is important for our understanding of the epidemiology of pneumococcal infections and to provide data to policy makers for universal PCV introduction in India. Continued molecular surveillance of S. pneumoniae is necessary to monitor pneumococcal population dynamics and capsular switching events at the genetic level prior to and after PCV introduction in NIP.

 ~ References Top

Pneumococcal conjugate vaccine for childhood immunization – WHO position paper. Wkly Epidemiol Rec 2007;82:93-104.  Back to cited text no. 1
The United Nations Children's Fund, World Health Organization. Pneumonia the Forgotten Killer of Children. The United Nations Children's Fund (UNICEF)/World Health Organization; 2006. Available at:  Back to cited text no. 2
Whitney CG. More evidence for use of pneumococcal conjugate vaccines. Lancet 2013;381:182-3.  Back to cited text no. 3
Song JY, Nahm MH, Moseley MA. Clinical implications of pneumococcal serotypes: Invasive disease potential, clinical presentations, and antibiotic resistance. J Korean Med Sci 2013;28:4-15.  Back to cited text no. 4
Hausdorff WP, Feikin DR, Klugman KP. Epidemiological differences among pneumococcal serotypes. Lancet Infect Dis 2005;5:83-93.  Back to cited text no. 5
Zemlickova H, Jakubu V, Urbaskova P, Motlova J, Musilek M, Adamkova V. Serotype-specific invasive disease potential of Streptococcus pneumoniae in Czech children. J Med Microbiol 2010;59:1079-83.  Back to cited text no. 6
Temime L, Boelle PY, Opatowski L, Guillemot D. Impact of capsular switch on invasive pneumococcal disease incidence in a vaccinated population. PLoS One 2008;3:e3244.  Back to cited text no. 7
Rosen JB, Thomas AR, Lexau CA, Reingold A, Hadler JL, Harrison LH, et al. Geographic variation in invasive pneumococcal disease following pneumococcal conjugate vaccine introduction in the United States. Clin Infect Dis 2011;53:137-43.  Back to cited text no. 8
VIMS Report: Global Vaccine Introduction – A Report on Current Global Access to New Childhood Vaccines. Johns Hopkins Bloomberg School of Public Health; 2013.  Back to cited text no. 9
Castillo D, Harcourt B, Hatcher C, Jackson M, Katz L, Mair R, et al. Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria Meningitidis, Streptococcus Pneumoniae, and Haemophilus Influenza. WHO Manual. 2nd ed. World Health Organization; 2011. Available at:  Back to cited text no. 10
Lalitha MK, Pai R, John TJ, Thomas K, Jesudason MV, Brahmadathan KN, et al. Serotyping of Streptococcus pneumoniae by agglutination assays: A cost-effective technique for developing countries. Bull World Health Organ 1996;74:387-90.  Back to cited text no. 11
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty third Informational Supplement. CLSI document M100-23. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.  Back to cited text no. 12
Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. eBURST: Inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 2004;186:1518-30.  Back to cited text no. 13
Jolley KA, Feil EJ, Chan MS, Maiden MC. Sequence type analysis and recombinational tests (START). Bioinformatics 2001;17:1230-1.  Back to cited text no. 14
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. 15
Kim SH, Song JH, Chung DR, Thamlikitkul V, Yang Y, Wang H, et al. Changing trends in antimicrobial resistance and serotypes of Streptococcus pneumoniae isolates in Asian countries: An Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Antimicrob Agents Chemother 2012;56:1418-26.  Back to cited text no. 16
Veeraraghavan B, Kurien T. Penicillin resistant Streptococcus pneumoniae in India: Effects of new clinical laboratory standards institute breakpoint and implications. Indian J Med Microbiol 2011;29:317-8.  Back to cited text no. 17
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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. 19


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