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Year : 2020  |  Volume : 38  |  Issue : 1  |  Page : 78--86

Molecular capsular typing and multi locus sequence typing of invasive, non-invasive and commensal Streptococcus pneumoniae isolates from North India

Shefali Jain1, Bimal Kumar Das1, Neeraj Mahajan1, Arti Kapil1, Rama Chaudhry1, Seema Sood1, Sushil Kumar Kabra2, Sada Nand Dwivedi3,  
1 Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Paediatrics, All India Institute of Medical Sciences, New Delhi, India
3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Prof. Bimal Kumar Das
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 110 029
India

Abstract

Purpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods: S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer's disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer's disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.

How to cite this article:
Jain S, Das BK, Mahajan N, Kapil A, Chaudhry R, Sood S, Kabra SK, Dwivedi SN. Molecular capsular typing and multi locus sequence typing of invasive, non-invasive and commensal Streptococcus pneumoniae isolates from North India.Indian J Med Microbiol 2020;38:78-86

How to cite this URL:
Jain S, Das BK, Mahajan N, Kapil A, Chaudhry R, Sood S, Kabra SK, Dwivedi SN. Molecular capsular typing and multi locus sequence typing of invasive, non-invasive and commensal Streptococcus pneumoniae isolates from North India. Indian J Med Microbiol [serial online] 2020 [cited 2020 Sep 22 ];38:78-86
Available from: http://www.ijmm.org/text.asp?2020/38/1/78/290683

Full Text



 Introduction



Streptococcus pneumoniae (or Pneumococcus) is a Gram-positive, alpha haemolytic and capsulated bacterium that colonizes in the upper respiratory tract of healthy individuals. Although colonization with pneumococci is mostly asymptomatic, the progression from asymptomatic colonization to disease depends on several factors which are characteristic of specific pneumococcal strains, the status of the host defence as well as depends on the age, geographical area, genetic background and socio-economic conditions. It is able to cause serious infections such as meningitis, sepsis, pneumonia and otitis media. Each year >1.6 million people die due to pneumococcal diseases worldwide and most of the deaths occur in the developing countries.[1] Globally, India has the highest number of deaths because of pneumococcal infections among children <5 years of age.[2] A report from The United Nations International Children's Emergency Fund (UNICEF) in 2006, estimated that 44 million pneumonia cases occurred in India and more than half are due to S. pneumoniae.[3]

As of now, >94 distinct serotypes have been identified.[4] Of these, a few serotypes are associated with invasive disease, a few with carriage while others with both invasive disease and carriage.[5] Globally, 70%–80% invasive pneumococcal diseases are caused by 13 serotypes, which are included in the pneumococcal conjugate vaccine (PCV) 13 vaccine.[6]

Currently, preventative strategies for pneumococcal infection include targeted use of the 23-valent polysaccharide pneumococcal vaccine for individuals older than 2 years of age and routine immunisation of infants and children with the 7, 10 and 13-valent polysaccharide-protein conjugate pneumococcal vaccine. However, increased use of vaccine leads to serotype replacement, rather than reducing its overall burden. Therefore, epidemiological surveillance is important to understand the population dynamics of S. pneumoniae. A large number of molecular methods have been developed and applied for the population structure studies of S. pneumoniae,[7] of these, multilocus sequence typing (MLST) is a reliable, precise, unambiguous and portable tool for characterising and monitoring disease-causing and antibiotic-resistant lineages of S. pneumoniae.

There are very few studies available on the epidemiological characterisation of S. pneumoniae from North-India. Furhermore, there are less data available in the literature on the comparative analysis of invasive with commensal pneumococcal isolates. The present study was undertaken to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of S. pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection in our population.

 Materials and Methods



The study was conducted in the Department of Microbiology, All India Institute of Medical Sciences (AIIMS), Delhi, from February 2012 to December 2016. S. pneumoniae isolates were collected from various samples, including blood, cerebrospinal fluid (CSF), pleural fluid (PF), bronchoalveolar lavage (BAL), sputum and throat swab. The isolates were identified and confirmed by using standard methods. In brief, samples were cultured on commercially available Columbia sheep blood agar plates (Biomerieux, France) and incubated in an atmosphere containing 5% CO2 at 37°C for 18–24 h. Alpha haemolytic colonies with morphology suggestive of S. pneumoniae were subcultured on to a fresh blood agar plate with an optochin disc and were incubated at 37°C with 5% CO2 for another 18–24 h. Isolates that exhibit an inhibition zone ≥14 mm around the optochin disc were identified as S. pneumoniae and were further confirmed by the bile solubility test.

The study was approved by the Ethics Committee, AIIMS, New Delhi, India (Ref. No. IESC/T-59/03.01.2014 and RT-59/03 January 2014).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by the KirbyBauer's disc diffusion method for the following antibiotics: penicillin (1 μg oxacillin), vancomycin (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), tetracycline (30 μg), levofloxacin (5 μg), gatifloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg) and chloramphenicol (30 μg). Antimicrobial susceptibility tests were interpreted as per the Clinical Laboratory Standards Institute (CLSI) guidelines 2012.[8]

Minimum inhibitory concentration determination

Minimum inhibitory concentration (MIC) was determined for penicillin by E-test strips (Biomerieux, France) as recommended for the oxacillin non-susceptible isolates. The MIC values were interpreted according to the CLSI MIC breakpoints 2012.[8]S. pneumoniae ATCC 49619 was used as a control strain.

Serotyping

Serotyping was carried out by quellung reaction using antisera obtained from Statens Serum Institut (SSI, Denmark) as per the manufacturer's instructions.

Isolation of genomic DNA

DNA was extracted using commercially available QIAamp DNA Mini kit as per the manufacturer's instructions and stored at −20°C.

Molecular capsular typing

Seven sequential multiplex polymerase chain reactions (PCR) were performed for the detection of capsular types [Table 1]. A 25 μl reaction was put up with 1X PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs, 2 U Taq polymerase and primers with specified concentrations.[9] The amplified products were visualised by running on 2% agarose gel (w/v) prepared in 0.5 X TBE (Tris-borate ethylene diamine tetra acetic acid) buffer and stained with ethidium bromide (0.5 μg/ml).{Table 1}

Multi locus sequence typing

All the internal fragments of seven housekeeping genes aroE, gdh, gki, recP, spi, xpt and ddl were amplified using respective primers of PCR and PCR cycle conditions as described on the multilocus sequencing typing website (www.mlst.net). The PCR products were purified using Qiagen MinElute gel extraction kit (Qiagen Sciences Inc., USA). Purified products were sequenced by using the BigDye Terminator v3.1 Cycle sequencing kit on 3130xl genetic analyzer (ABI, CA, USA). Sequence analysis was performed by BioEdit v 7.2.5. Phylogenetic analysis was performed using MEGA X.

 Results



A total of 125 isolates were collected in the Department of Microbiology and Department of Paediatrics in AIIMS, New Delhi. Of these 125 isolates, 20 blood, 13 throat swab, 11 sputum, 4 CSF, 1 Pleural fluid and 1 BAL were obtained from patients while 75 isolates from nasopharyngeal swab of healthy children. Sputum, BAL and throat swabs were collected from those children having cold, cough and fever. The patients ranging in age from 2 months to 65 years. Nasopharyngeal swabs were collected from children aged 2 months to 60 months attending paediatric outpatient department at AIIMS. Thirty isolates were from children aged between 2 and 13 years and the rest of the isolates from adults. Thus, 25 isolates were invasive (blood, CSF, PF), 75 were commensal (nasopharyngeal swab) and rest of the 25 isolates (Sputum, BAL and throat swab) considered as non-invasive. All isolates were confirmed by optochin sensitivity and bile solubility test.

Antimicrobial susceptibility testing

Out of 125 S. pneumoniae isolates, 18 isolates (14.4%) were resistant, while 19 isolates (15.2%) were intermediately resistant to penicillin by KirbyBauer's disc diffusion method. Resistance to erythromycin, tetracycline, chloramphenicol, trimethoprim-sulphamethoxazole (Co-trimoxazole), levofloxacin, ciprofloxacin and gatifloxacin were observed in 15 (12%), 11 (8.8%), 7 (5.6%), 102 (81.6%), 6 (4.8%), 5 (4%) and 3 (2.4%) isolates respectively [Table 2]. No resistance was seen to vancomycin. Multidrug resistance (MDR) was observed in 16 (12.8%) isolates. Strains were considered as MDR when they showed resistance to 3 or more antibiotics. The majority of multiple resistance was observed to penicillin, co-trimoxazole, erythromycin and tetracycline.{Table 2}

Among 25 invasive isolates, 3 (12%) were resistant to oxacillin, while 6 (16%) were resistant to erythromycin while in 25 non-invasive isolates, 5 (20%) were resistant to oxacillin and 3 (12%) were resistant to erythromycin. Out of 75 commensal isolates, 10 (13.3%) were resistant to oxacillin, while 6 (8%) were resistant to erythromycin by Kirby-Bauer's disc diffusion method.

Minimum inhibitory concentration

According to the CLSI guidelines 2012, oxacillin resistant and intermediate isolates were further tested for MICs of penicillin by E-test [Table 3]. The MIC values for these resistant and intermediate isolates were compared with the MIC breakpoints by CLSI guidelines 2012. A total of 37 isolates (18 resistant and 19 intermediate resistant) were resistant by Kirby-Bauer's disc diffusion method. Out of these 37 isolates, all 19 intermediate resistant isolates by disc diffusion method were sensitive by E-test. Out of 18 resistant isolates by disc diffusion method; only 4 (3.2%) isolates (two from non-invasive and one each from invasive and commensal) were observed to be resistant (≥2 mg/L) while 14 isolates (11.2%) were intermediate resistant (0.1–1 mg/L) to penicillin by E-test. The invasive resistant isolate was isolated from blood.{Table 3}

Serotyping

A total of 75 S. pneumoniae isolates (25 each Invasive, Non-Invasive and Commensal) were serotyped by multiplex PCR as well as by quellung reaction. By multiplex PCR, 59 isolates were serotyped, while 61 isolates serotyped by quellung reaction. Serogroup that could not be discriminated by multiplex PCR was discriminated by quellung reaction like serogroup 6A/B and 15B/C. Some serotypes which were not included in multiplex PCR were analysed by quellung reaction; these were serogroup/type 5, 24B and 33B. Some serotypes were not identified by quellung reaction but amplified by multiplex PCR, for example, serotype 35B and 31. The isolates were not identified by the quellung reaction, but amplified by PCR; the reason could be the isolate has little or no capsule because after 4–5 subculture capsule becomes thin or lose. Serotypes that could not be identified by multiplex PCR and quellung reaction were defined as non-typeable (NT). Therefore, 16 isolates were not identified by multiplex PCR, while 14 isolates were not identified by quellung, but in total, ten isolates were not identified by both methods. A total of 18 serogroup/types were identified in our isolates; these serogroup/types were: 6A/B (n = 9), 14 (n = 8), 15B/C (n = 6), 19A (n = 5), 3 (n = 5), 4 (n = 5), 19F (n = 4), 9V (3), 11A (n = 3), 1 (n = 3), 7 (n = 3), 35B (n = 2), 10A (n = 2), 33B (n = 2), 31 (n = 2), 5 (n = 1), 16F (n = 1) and 24B (n = 1) [Figure 1]. Among invasive isolates serotype, 14 was found to be the most prevalent followed by serotype 1, 15B, and 3 and serotypes 14, 4, 19A, 19F and 3 were the most common in non-invasive isolates. In commensal isolates, serotypes 6A/B, 14 and 19A were the most common serotypes [Figure 1].{Figure 1}

Multi locus sequence typing

The sequences of seven housekeeping genes of 15 S. pneumoniae isolates (5 each invasive, non-invasive and commensal) were amplified by using the previously described method.[10] The PCR products of each gene were sequenced using both forward and reverse primers. The generated sequences of each gene were aligned using BioEdit v 7.2.5 submitted to the MLST database (https://pubmlst.org/spneumoniae). For each housekeeping gene, the different sequences present within a bacterial species were assigned as distinct alleles and for each isolate, the alleles at each of the seven loci define the allelic profile or ST. The 15 isolates tested were assigned into 15 different STs, of which 6 new STs (ST13826, ST13827, ST13823, ST13824, ST13961 and ST13825) were determined in this study [Table 4]. Two new STs were found among invasive (ST13826, ST13827), 3 in non-invasive (ST13823, ST13824, and ST13961) and one in commensal (ST13825) isolates. The invasive, non-invasive and commensal isolates were found to form different groups or as singletons. Three of the invasive isolates were found in groups 17, 153 and 2, while two were found as singletons. All non-invasive were found as singletons while commensal isolates were found in groups 32, 442, 59 and 184 while one ST was found as singleton. Commensal isolates were found to be more genetically diverse with more number of novel alleles present in our isolates, followed by invasive and non-invasive isolates.{Table 4}

 Discussion



The study demonstrates the antimicrobial susceptibility pattern, serotype distribution and ST of S. pneumoniae isolates from North-India. The work was designed to study the phenotypic and genotypic characteristics of invasive, non-invasive and commensal S. pneumoniae isolates; to detect that S. pneumoniae causing invasive diseases are genotypically distinct from the commensal isolates of pneumococci and to know whether any particular pneumococcal serotypes or individual pneumococcal strain can cause infection or if some have a greater propensity to cause disease, by comparing the isolates from invasive, non-invasive and commensal/nasopharyngeal colonizers by using phenotypic and genotypic methods.

In the present study, 3.2% S. pneumoniae isolates were observed to be resistant (≥2 mg/L), while 11.2% isolates were intermediate resistant (0.1–1 mg/L) to penicillin by E-test. Though 14.4% were resistant and 15.2% were intermediately resistant to penicillin by disc diffusion method, which is low compared to studies from other parts of the world.[11],[12],[13],[14] Similar reports were made by other authors from India.[4],[15],[16],[17],[18],[19] However, a recent study reported that penicillin resistance has been increased from 9.5% in 2008 to 42.8% in 2016.[20]

In this study, 12% of the isolates were found to be resistant and 7.2% were found to be intermediate resistant to erythromycin. Macrolide resistance is low in our study and it is also reported from other studies from India.[15],[16],[18],[21] In contrast, other Indian studies reported erythromycin resistance ranging from 20% to 37%.[20],[22],[23]

We found the highest resistance for co-trimoxazole (81.6%) in our isolates, which is similar to other studies from India.[16],[17],[18],[21] Recently, another study from India reported 97% of resistance for co-trimoxazole.[20] In contrast, a very low resistance rates reported in other India studies.[22],[23],[24]

Among 75 isolates, 25 invasive isolates of S. pneumoniae were distributed into different serotypes, i. e. 1, 3, 4, 5, 6B, 7, 9V, 10A, 14, 15B, 16F, 19A, 19F, 24B, 31, 33B and 35B. Apart from 1 isolate, all the invasive S. pneumoniae isolates were serotyped into 17 types. In invasive isolates serotype 14 was found to be the most common serotype followed by serotype 1 and 3. Non-invasive S. pneumoniae isolates were distributed into 1, 3, 4, 6A, 6B, 7, 9V, 11, 14, 15B, 15C 19A, 19F and 35B serotypes. Of these 25 non-invasive isolates, 4 isolates were NT. We observed that five serotypes were the most common in non-invasive isolates, i.e., 14, 4, 19A, 19F and 3.

A systematic review of South Asian countries, where the author reported the most common serotype 1 in Nepal, serotype 14 in Bangladesh and India, serotype 19F in Sri Lanka and Pakistan.[25] A surveillance study[4] and a systematic review from India[17] also identified serotype 14, 1 and 19F are the most common serotypes in invasive pneumococcal isolates.[20] In contrast, another study from India reported serotype 6 as the most common serotype among invasive pneumococcal isolates.[18]

Commensal S. pneumoniae isolates were distributed into 3, 4, 6A, 6B, 9V, 10, 11, 14, 15, 19A, 19F and 31 serotypes. Four isolates were non-typeable. Among the commensal isolates serogroup, 6A was found to be the most common serotype. Some of the commensal serotypes, i.e., 6 and 19 also reported from the nasopharynx of normal healthy children from Indian studies and most other parts of the world.[26],[27],[28],[29],[30],[31] Overall, the most common serogroup/types in our study were 6, 14, 19, 4, 3 and 1. The result of our study is almost similar to the IBIS study in 1999, with the difference is in order of prevalence.[19] In our study, Serotype 14 was observed more prevalent in invasive and non-invasive isolates. Whereas, serogroup 6A/B was observed in commensal isolates.

In this study, resistant isolates were not restricted to any particular serotype. Serotype 1, 19A, 9V and 14 were associated with penicillin-resistant isolates. Serotype 14, 6A/B, 15B/C, 19A, 3, 4 were found in erythromycin-resistant isolates. Multiple drug resistance isolates were observed to be associated with serogroup/type 14, 19, 6, 1 and 4.

Serotypes included in PCV 10 and PCV 13 vaccines cover approximately 60% serotypes in our study. This is similar to a report where the serotypes in PCV 10 and PCV 13 included 62% and 70% of serotypes in India.[25] The non-vaccine serotypes (15B/C, 11A, 35B, 31, 10A, 33, 24B and 16F) that were found in our isolates are a relatively rare cause of invasive disease. The presence of non-vaccine serotypes is worrying as they are not included in available vaccines and it could play a role in serotype replacement dynamics.

MLST is the most widely used typing method for the characterisation of S. pneumoniae due to its ability to discriminate against bacterial strains and easily accessible database (http://pubmlst.org/spneumoniae/). The database also provides a platform for inter laboratory comparative analysis of epidemiological data. There are over 4,617 different sequences with 14,276 MLST profiles available on the database globally. The data available on S. pneumoniae isolates circulating in India is finite although it harbors the largest population with pneumococcal disease. The MLST database currently has 268 isolates characterised from different parts of India with 199 different MLST profiles. Depending on allelic profile-based analysis (eBURST, http://spneumoniae.mlst.net), the entire S. pneumoniae MLST database has been grouped into 575 groups, of which 1837 STs were singletons. The invasive, non-invasive and commensal isolates in this study were found to form different groups or as singletons. Three of the invasive isolates were found in groups 17, 153 and 2, while two were found as singletons. All non-invasive were found as singletons while commensal isolates were found in groups 32, 442, 59 and 184 while one ST was found as singleton.

MLST in the present study revealed three previously reported STs (ST5191, ST874 and ST1396) and two new STs (ST13826, ST13827) among the invasive S. pneumoniae isolates tested. ST1396 was observed previously in the United States, while ST5191 and ST874 were previously observed in India only. ST1331, ST2839 and ST6983, which were single-locus variants (SLVs) of ST5191, were however reported from Ghana, The Gambia (ST1331); Niger (ST2839); and Israel (ST6983). Similarly, SLVs of ST874 were reported from China (ST5445); Nepal (ST7584, ST7585 and ST6771); Malaysia (ST9599); South Africa (ST7585, ST10775) and Japan (ST6771). Furthermore, SLVs of ST1396 were reported from South Korea (ST271, ST2398); USA (ST271, ST1843, ST1463, ST1396, and ST1430); Vietnam (ST271); China (ST271); Finland (ST271) and South Africa (ST1453. The SLVs for new STs ST13826 were reported from Spain, UK, Denmark, Canada, Poland, Uruguay, France, Netherland, Czech Republic, Sweden, Brazil, Italy, Hungary (ST156); Germany, France, UK, Portugal (ST1569) and Spain (ST44) while ST13827 were reported from Spain (ST6520) (http://spneumoniae.mlst.net).

Among the 5 non-invasive isolates tested through MLST, 3 STs were novel (ST13823, ST13824, and ST13961) while two (ST11667 and ST12480) were available on the database. ST11667 was previously observed in the UK while no records were found for ST12480. SLVs of ST11667 were reported from France, Finland, Saudi Arabia and Malawi (ST2678). In contrast, SLVs of ST 12480 were reported from India (ST4908) and Kenya (ST4908 and ST5758) The SLVs for new ST13823 were reported from Singapore and South Africa (ST6193); while DLVs for ST13824 were observed from Gambia (ST3578), Peru (ST5581, ST6139) and Israel (ST13910) and for ST13961 were reported in Qatar and Canada (ST6354) (http://spneumoniae.mlst.net).

In 5 commensal isolates, 4 STs were available on the database and previously observed from The Gambia, Kenya (ST914); Nepal (ST7645); Niger, South Africa, Thailand, Malawi (ST2213); and India (ST1725) while 1 was novel (ST13825). SLVs of ST914 were observed in The Gambia (ST912, ST913, ST5519); Ethiopia (ST2906, ST6470); UK, Nigeria (ST4340); Egypt (4940) and Kenya (ST5354, ST5386). SLVs of ST2213 were observed in Qatar, South Africa, Thailand (ST2712); UK (ST4437, ST11672); Kenya (ST4437); Malawi (ST9554); Qatar (ST11455) and Israel (ST139541). SLVs of ST1725 were reported from Kenya (ST1210); India, Thailand (ST4209); and Nepal (ST7522, ST8162, ST6020, ST6038). The SLVs for new ST13825 was reported from Nepal (ST11028) (http://spneumoniae.mlst.net).

Genetic variation was found to be highest in commensal isolates in the present study. Genetic variation was found to be less common amongst invasive and non-invasive isolates. New alleles were reported in all invasive, non-invasive and commensals. By sequence comparison of invasive, non-invasive and commensal isolates through the phylogenetic tree, it was observed that invasive isolates were found to be clustered with non-invasive and commensal isolates.

Comparative analysis of all seven genes by constructing dendrogram with combining gene sequences in the order aroE, ddl, gdh, gki, recP, spi and xpt, where invasive, non-invasive and commensal isolates were found to be clustered with each other also suggests the possibility of genetic relatedness and exchange of genetic material between these groups at some point during the course of evolution. A comparison of our data with other Indian isolates [Figure 2] authenticates our results. The results of this study on a limited number of isolates, testifies the genetic relatedness among invasive, non-invasive and commensal isolates. Further studies on a large number of isolates are needed to substantiate the same.{Figure 2}

 Conclusion



Though India has a low incidence of penicillin-resistant pneumococci, but increase in the incidence of penicillin intermediate resistance is worrisome because, at any time, high resistance to penicillin can occur in S. pneumoniae. Increase resistance of co-trimoxazole is possibly due to extensive use of antibiotics in hospitals and inappropriate dosage in the community might be the probable cause of resistance. Thus, there is a need for continuous screening of antimicrobial susceptibility in S. pneumoniae isolates. The presence of non-vaccine serotype is great concern as they are not included in current vaccines, this could play a role in serotype replacement dynamics. Our study implies the importance of continuous screening of serotypes and antimicrobial susceptibility patterns of S. pneumoniae isolates.

The result of the present study indicate that commensal S. pneumoniae isolates carry the same phenotypic and genotypic markers as invasive pneumococcal isolates. Although the number of isolates was few for the evolutionary relationship; invasive, non-invasive and commensal isolates were found to be clustered with each other signify the possibility of genetic relatedness and exchange of genetic material between these groups at some point during evolution. To prevent pneumococcal infection, we should prevent the nasopharyngeal colonisation by S. pneumoniae because colonisation is the first step towards invasive disease.

Acknowledgement

The authors sincerely acknowledge the Indian Council of Medical Research (ICMR) for providing financial assistance and gratefully acknowledge to Mr. Sonu Tyagi and Mr. Sambuddha Kumar, Department of Microbiology, AIIMS, New Delhi, India for their technical support.

Financial support and sponsorship

This work was supported by grant no. 80/834/2013/ECD-I from Indian Council of Medical Research (ICMR), Department of Health Research, Ministry of Health and Family Welfare, Government of India

Conflicts of interest

There are no conflicts of interest.

References

1Levine OS, O'Brien KL, Knoll M, Adegbola RA, Black S, Cherian T, et al. Pneumococcal vaccination in developing countries. Lancet 2006;367:1880-2.
2Sjöström K, Spindler C, Ortqvist A, Kalin M, Sandgren A, Kühlmann-Berenzon S, et al. Clonal and capsular types decide whether pneumococci will act as a primary or opportunistic pathogen. Clin Infect Dis 2006;42:451-9.
3World Health Organization. Pneumococcal conjugate vaccine for childhood immunization – WHO position paper. Wkly Epidemiol Rec 2007;82:93-104.
4Manoharan A, Manchanda V, Balasubramanian S, Lalwani S, Modak M, Bai S, et al. Invasive pneumococcal disease in children aged younger than 5 years in India: A surveillance study. Lancet Infect Dis 2017;17:305-12.
5Donkor ES, Adegbola RA, Wren BW, Antonio M. Population biology of Streptococcus pneumoniae in West Africa: Multilocus sequence typing of serotypes that exhibit different predisposition to invasive disease and carriage. PLoS One 2013;8:e53925.
6McIntosh ED, Reinert RR. Global prevailing and emerging pediatric pneumococcal serotypes. Expert Rev Vaccines 2011;10:109-29.
7Donkor ES. Molecular typing of the pneumococcus and its application in epidemiology in sub-Saharan Africa. Front Cell Infect Microbiol 2013;3:12.
8Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Second Informational Supplement. CLSI 2012:M100-S22. Clinical and Laboratory Standards Institute; 2012.
9Pai R, Gertz RE, Beall B. Sequential multiplex PCR approach for determining capsular serotypes of Streptococcus pneumoniae isolates. J Clin Microbiol 2006;44:124-31.
10Enright 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.
11Cherazard R, Epstein M, Doan TL, Salim T, Bharti S, Smith MA. Antimicrobial resistant Streptococcus pneumoniae: Prevalence, mechanisms, and clinical implications. Am J Ther 2017;24:e361-9.
12Kim 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.
13Jenkins SG, Brown SD, Farrell DJ. Trends in antibacterial resistance among Streptococcus pneumoniae isolated in the USA: Update from PROTEKT US Years 1-4. Ann Clin Microbiol Antimicrob 2008;7:1.
14Reinert RR, Reinert S, van der Linden M, Cil MY, Al-Lahham A, Appelbaum P. Antimicrobial susceptibility of Streptococcus pneumoniae in eight European countries from 2001 to 2003. Antimicrob Agents Chemother 2005;49:2903-13.
15Chawla K, Gurung B, Mukhopadhyay C, Bairy I. Reporting emerging resistance of Streptococcus pneumoniae from India. J Glob Infect Dis 2010;2:10-4.
16Shariff 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.
17Singh J, Sundaresan S, Manoharan A, Shet A. Serotype distribution and antimicrobial susceptibility pattern in children≤5 years with invasive pneumococcal disease in India-A systematic review. Vaccine 2017;35:4501-9.
18Kumar KL, Ganaie F, Ashok V. Circulating serotypes and trends in antibiotic resistance of invasive Streptococcus pneumoniae from children under five in Bangalore. J Clin Diagn Res 2013;7:2716-20.
19Prospective multicentre hospital surveillance of Streptococcus pneumoniae disease in India. Invasive Bacterial Infection Surveillance (IBIS) Group, International Clinical Epidemiology Network (INCLEN). Lancet 1999;353:1216-21.
20Verghese VP, Veeraraghavan B, Jayaraman R, Varghese R, Neeravi A, Jayaraman Y, et al. Increasing incidence of penicillin- and cefotaxime-resistant Streptococcus pneumoniae causing meningitis in India: Time for revision of treatment guidelines? Indian J Med Microbiol 2017;35:228-36.
21Molander 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.
22Jyothilakshmi G, Latha SG. Emerging resistance and antibiotic susceptibility patterns in Streptococcus pneumoniae. Int J Curr Med Appl Sci 2015;7:30-3.
23Nagaraj S, Kalal BS, Manoharan A, Shet A. Streptococcus pneumoniae serotype prevalence and antibiotic resistance among young children with invasive pneumococcal disease: Experience from a tertiary care center in South India. Germs 2017;7:78-85.
24Nandini L, Shantala. GB, Ambika R. Study of penicillin resistance and multidrug resistance of Steptococcus pneumoniae in a tertiary care hospital. Int J Curr Microbiol App Sci 2017;6:913-8.
25Jaiswal N, Singh M, Das RR, Jindal I, Agarwal A, Thumburu KK, et al. Distribution of serotypes, vaccine coverage, and antimicrobial susceptibility pattern of Streptococcus pneumoniae in children living in SAARC countries: A systematic review. PLoS One 2014;9:e108617.
26Raman R, Sankar J, Putlibai S, Raghavan V. Demographic profile of healthy children with nasopharyngeal colonisation of Streptococcus pneumoniae: A research paper. Indian J Med Microbiol 2017;35:607-9.
27Devi U, Ayyagari A, Devi KR, Narain K, Patgiri DK, Sharma A, et al. Serotype distribution & sensitivity pattern of nasopharyngeal colonizing Streptococcus pneumoniae among rural children of Eastern India. Indian J Med Res 2012;136:495-8.
28Verani JR, Massora S, Acácio S, Dos Santos RT, Vubil D, Pimenta F, et al. Nasopharyngeal carriage of Streptococcus pneumoniae among HIV-infected and -uninfected children<5 years of age before introduction of pneumococcal conjugate vaccine in Mozambique. PLoS One 2018;13:e0191113.
29Wang L, Fu J, Liang Z, Chen J. Prevalence and serotype distribution of nasopharyngeal carriage of Streptococcus pneumoniae in China: A meta-analysis. BMC Infect Dis 2017;17:765.
30Menezes AP, Azevedo J, Leite MC, Campos LC, Cunha M, Carvalho Mda G, et al. Nasopharyngeal carriage of Streptococcus pneumoniae among children in an urban setting in Brazil prior to PCV10 introduction. Vaccine 2016;34:791-7.
31Ip M, Chau SS, Lai LS, Ma H, Chan PK, Nelson EA. Increased nasopharyngeal carriage of serotypes 6A, 6C, and 6D Streptococcus pneumoniae after introduction of childhood pneumococcal vaccination in Hong Kong. Diagn Microbiol Infect Dis 2013;76:153-7.