Characterization of Streptococcus pneumoniae isolates from India with special reference to their sequence types

Introduction: Streptococcus pneumoniae is a major cause of mortality and morbidity in young children and the elderly. In the present study we evaluated antimicrobial susceptibilities, serotypes, and sequence types of pneumococcal isolates recovered in New Delhi, India. Methodology: A total of 126 clinical isolates of Streptococcus pneumoniae were investigated. They were subjected to disk diffusion susceptibility testing, broth microdilution testing, serotyping and multilocus sequence typing. Results: Broth microdilution assay showed that 5%, 20% and 23% of the isolates exhibited resistance to penicillin, erythromycin and ciprofloxacin, respectively. Serotypes19, 1 and 6 were more frequently isolated. Thirty per cent of the strains were comprised of serotypes 1, 3, 5, 19A and 7F, which are not included in the seven-valent vaccine. Fifty-nine isolates were typed using multilocus sequence typing. Thirty new sequence types were encountered in this study. Only one clonal complex with 4 isolates was seen; 11 clonal complexes and 96 sequence types (STs) were observed among 115 Indian isolates. Only 18 of the 96 STs were found globally, of which only 4 STs were found in many countries with larger numbers. Conclusions: This study identifies the non-vaccine serotypes of Streptococcus pneumoniae circulating in India. It is important that an appropriate vaccine which covers all serotypes is used in the region.


Introduction
Streptococcus pneumoniae is a commensal flora of the nasopharynx of humans.However, when it invades the bloodstream, it has the ability to cause serious infections ranging from otitis media, pneumonia, septicemia and meningitis, especially in children and elderly individuals.There are more than 90 serotypes identified.Some of the serotypes are known to cause more virulent infection than others.While various vaccines are available to prevent these infections, the 23-valent polysaccharide vaccine is commonly used, but due to poor immunogenicity in children, other multivalent conjugate vaccines are being developed and in use in the United States of America (USA), Canada, and European countries.A 7-valent conjugate vaccine (PCV-7) was introduced in the USA in 2000 and is widely used.Except for Korea and Taiwan, the PCV7 vaccine is not implemented in many Asian countries including India.Even if licensed in some of these countries, the coverage of the vaccine is very low [1].PCV10 and PCV13 are also in use in some countries.To implement a vaccine effectively, one must be aware of the serotypes prevalent in that region so that an appropriate vaccine can be chosen.There are limited studies to indicate the serotypes of Indian strains.Molecular typing, especially of the sequence types, is available for only a very few strains [2,3].The present study was undertaken to delineate the serotype, antimicrobial susceptibility pattern, and genotype of S. pneumoniae isolates from India and compare them with the global strains.

Isolation and identification
This study was conducted between 2007 and 2010.Various clinical samples (cerebrospinal fluid, pleural fluid, blood, tracheal aspirate, nasopharyngeal samples, and sputum) obtained from patients suffering from meningitis, community acquired pneumonia, septicemia, or acute exacerbation of chronic obstructive pulmonary disease were processed and the Streptococcus pneumoniae isolates identified by standard microbiological methods [4].Briefly, these were cultured on 5% sheep blood agar plates and incubated overnight at 37°C with 5% CO 2 .Draughtsman colonies with alpha hemolysis suspected to be S. pneumoniae were further confirmed by standard procedures for optochin susceptibility and bile solubility [4].For sputum, only those isolates recovered in significant quantities (ascertained by quantitation of sputum) were considered as pathogenic [5].Others were taken to be commensal.

Antibiotic susceptibility of the isolates
Isolates were tested for antimicrobial susceptibility by Kirby-Bauer's disk diffusion test.The test organisms were grown by inoculating two to three colonies in nutrient broth and incubating them at 37°C overnight.The turbidity of the growth obtained was standardized to match the turbidity of 0.5 McFarland's turbidity standards.A blood agar plate was inoculated using a swab impregnated with the bacterial suspension.Antibiotic impregnated disks (Difco, Franklin Lakes, NJ, USA) were placed on the surface of freshly inoculated plates.The plates were incubated at 35°C at 5% to 10% CO 2 for 18 hours.The zone of inhibition was recorded and interpreted per Clinical Laboratory Standards Institute (CLSI) guidelines [6].

Minimum inhibitory concentrations of the antimicrobials
Minimum inhibitory concentrations (MICs) of penicillin, erythromycin and ciprofloxacin were determined using the micro broth dilution assay per CLSI guidelines [7].Briefly, bacteria were grown overnight on Muller-Hinton agar with 5% sheep blood, at 37°C with 5% CO 2 .The bacterial colonies were suspended in 0.9% physiological saline to turbidity equal to the 0.5 McFarland's standard (1.5 x 10 8 CFU/ml).This was diluted 1:30 before use.The assay was performed in cation-adjusted Mueller-Hilton broth containing 0.1 ml of lysed sheep blood and various concentrations of antibiotics.0.01 ml of the bacterial suspension was used to inoculate the MIC panels.These were incubated at 35°C with 5% CO 2 overnight.The microtitre plates were read and the antimicrobial endpoint recorded as the first well showing no readily visible growth or haze as detected by the unaided eye.The isolates were labelled as sensitive, resistant, or intermediate according to CLSI guidelines [7].

Serotyping of S. pneumoniae isolates
Serotyping of S. pneumoniae isolates was performed by latex agglutination test by coating the latex beads using the conventional antisera (Staten serum institute).It was performed by means of the "Chess Board Method" using the manufacturer's instructions.Briefly, the isolates were tested using the antisera present in pools A, B, C, D, E, F, G, P, Q, R, S, T and non-vaccine serotypes.Individual serotypes were determined by reactivity in two pools.
The amplified fragments were sequenced by Ocimum Biosolutions Ltd, Hyderabad, India.The assignment of alleles and sequence types was performed by the software available at the Multi Locus Sequence Typing website's pneumococcal page (http://www.mlst.net).Allelic combinations not already in the database were submitted and assigned new sequence types.The sequences were compared with the sequences of respective alleles downloaded from the website www.mlst.net.Clonal complexes (CC) were identified by grouping all isolates present in the S. pneumoniae database using the eBURST algorithm [10] with the software provided in the Multi Locus Sequence Typing website http://eburst.mlst.net).Clonal complexes consisted of sequence types that shared 6 of 7 alleles with at least one other sequence type in the complex and named after the putative founder (i.e., the sequence type that had the greatest number of single-locus variants) of the group, or after the most frequent sequence type of the group.Sequence types that did not group with others in the database were defined as singletons.

Results
A total of 126 S. pneumoniae isolates, (18 CSF, 3 pleural fluid, 11 blood, 72 sputum, 3 tracheal aspirate, 2 throat swab, and 17 nasopharyngeal samples) were obtained from patients during the study period.The patients ranged in age from 2 to 77 years.Forty isolates were from children aged 2 to 12 years and the rest of the isolates were from adults.The majority of the pediatric isolates were from CSF and blood.Out of the 72 sputum isolates, 49 were recovered as the predominant pathogen while 23 isolates were commensal.In five cases other pathogens were isolated (three Acinetobater, one Pseudomonas, and one Haemophilus) in association with pneumococcus, which were also significant.

Antibiotic susceptibility of the isolates
Antibiotic resistance patterns are shown in Table 1.Among 126 isolates tested, a total of 41 isolates (33%) showed resistance to penicillin by oxacillin disk diffusion test.Ninety-eight (78%) isolates were sensitive, 8 (6%) isolates were intermediately sensitive, and 20 (16%) isolates were found resistant to Erythromycin.Furthermore, 103 (82%) and 38 (30%) isolates were resistant to co-trimoxazole and tetracycline respectively, while 17 (13%) isolates were found resistant to chloramphenicol.Nineteen (15%) isolates showed resistance while 25 (20%) isolates were found intermediately sensitive to ciprofloxacin, and 10 (8%) isolates were found resistant and 9 (7%) intermediately sensitive to cefotaxime.Forty-five (36%) isolates showed resistance to more than three drugs and were considered as multidrug resistant isolates.There was no difference in the preponderance of penicillin resistance seen in either the commensal or the pathogenic sputum isolates.Though Acinetobacter and Pseudomonas showed resistance to most groups of antibiotics, no resistance was observed to those groups in the pneumococcal isolates, which were isolated along with these.

Determination of minimum inhibitory concentration (MIC) of isolates found resistant to antimicrobials
MIC to penicillin and erythromycin was determined in 107 clinical isolates by micro broth dilution test.Five (5%) isolates, two each from CSF and nasopharyngeal samples and one from sputum, showed resistance to penicillin.Though 33% of the strains were resistant by disk diffusion test, only 5% showed resistance in this assay.Using the newer CLSI breakpoints most of the non-susceptible pneumococal isolates were found to be sensitive; hence a marked reduction in the percentage of resistant isolates was found.Twenty-one (20%) isolates were resistant to erythromycin, which were resistant by disk diffusion test also.The MIC of ciprofloxacin was tested in 57 isolates, that were either resistant or intermediatesensitive, and a few sensitive by disk diffusion test.Thirteen isolates (23%) showed resistance to ciprofloxacin and 20 (35%) were found intermediately sensitive; however, 5 isolates that were intermediately sensitive to ciprofloxacin by disk diffusion test showed low MIC levels and considered as susceptible (Table 2).

Multi Locus Sequence Typing
The sequence types and their comparisons with other STs in the MLST database are given in Table 3. Fifty-nine isolates were typed.Out of these 29 were of known sequence types while the remaining 30 showed new sequence types.Twenty-five out of these 30 were submitted to the MLST site and were assigned new ST numbers.The new types were variants of the existing sequence types differing in one or more loci.This observation shows that the serotypes are continuously evolving.ST types 303 (4 isolates), 4217 (2) 1811 (2) and 1669 (4), 6899 (2) and 6896 (2) each had more than one isolate.Some of the new STs in this study are single locus variants (SLVs) of an earlier new type found in India.For example, ST 1702, which was earlier found in the study, was a sensitive clone which has evolved into SLV ST6896 which is resistant to cotrimoxazole.All other types had a single isolate.There was only one clonal complex seen that was comprised of four isolates; the rest were singletons.

Discussion
This study characterizes the Indian isolates with respect to antimicrobial sensitivity, serotypes, and sequence types.Using the new breakpoints for nonmeningeal isolates, many of the non-susceptible isolates were found to be sensitive to penicillin and hence only 5% of the isolates were resistant to penicillin, which is low in comparison to the resistance reported by several investigators from other countries [3,12].Two out of the five resistant isolates were from CSF; however, it is interesting to note that two isolates from nasopharyngeal samples of healthy children were resistant to penicillin with a high MIC of > 4.These were also resistant to erythromycin.Dissemination of these resistant strains in the community can lead to treatment failure to penicillin.Multidrug resistance was seen in 36% of the isolates, which is comparable to resistance rates seen in studies from North America (9% to24%) and Europe (0 to 43%) [3,13,14].However, this rate is much lower than the overall resistance rate of 59.3% reported more recently from Asian countries, with the highest seen in China (83.3%) followed by Vietnam (75.5%).In the present study, resistant isolates were not restricted to any particular serotype; these results differ from those seen in other studies which have reported more than 60% of 19A and 35B to be non-susceptible to penicillin [12,[15][16][17].In the present study, 20% of the isolates were resistant to erythromycin, and this rate is very low compared to the high resistance rate of 70% seen in other Asian countries [18].
It is evident from the serotyping data that only 34% of our strains would be covered by the 7-valent vaccine.Similar results were seen in investigations from other Asian countries, which reported 52.5% of the strains being covered [18].An additional 20% each would be covered by the 10-valent and 13-valent conjugate vaccines.In spite of non-introduction of the vaccine in the regular immunization programme in India, non-PCV7 vaccine serotype 19A (data not shown), 7F and 1 were seen in 6%, 10% and 9% of the  [18].Hence the 13valent pneumococcal vaccine would be a better choice.
The MLST database has a total of 115 Indian isolates (including ours) with 96 STs (as on 25 October 2011, 17:16:13 IST).Among these, only 10clonal complexes (CC) comprised of 38 isolates were seen; the rest all were singletons (Figure 1).These data show the diversity even among the Indian isolates.CC5067 (Group 1) was the largest with 5 STs having 8 isolates.Four of the 5 STs were isolated in this study.This CC is included in the Group 52 of the global strains (Figure 2).The predictor founder of this group (5067) is from India and is a double locus variant (DLV) of 27, which was an older ST recognized in 1995 from Denmark and an SLV of 28, which was recognized in 1994 from the Netherlands and later in 2005 from Germany.Four SLVs of this sequence type have originated in India since, suggesting that though initially acquired from Europe, the ST is evolving and giving rise to newer STs which are restricted to India.
Comparing the Indian STs with the global scenario, it was observed that only 18 of the 96 STs from India were found globally.Among these only 4 STs (303, 989, 236 and 63) were predominantly found in larger numbers in many countries.All other STs were restricted to a few countries including India.Most of these were from Thailand, African countries such as Nigeria, Kenya and Ghana, and neighbouring countries of India including Nepal, Bangladesh and China (www.mlst.net).Most of the STs seen in African countries are also seen in India as such or as their SLVs, and this correlation may be explained by the travel between India and these countries and hence the dissemination of the African strains.Isolates showing the same serotype but different sequence types and vice versa, seen across the globe, were observed in this study, also showing that they are genetically different.For example, isolates with serotype 7F showed 6 different sequence types in this study.Similarly, ST 303 and ST 4217 were present in multiple serotypes.
Out of the three STs, 63, 289 and 236 belonging to the PMEN clone, resistance pattern and serotype of new Delhi, were concordant with corresponding features of PMEN clones ST289 (Colombia 5 -19) and ST 236 ((Taiwan 19F -14).However, ST 63 isolate in this study was antibiotic sensitive and serotype 14, while the corresponding PMEN clone (Sweden15A-25) was resistant and of serotype 15A.ST 63 is seen to be present in various serotypes including 15A, 14, 19A 23F and 19F.The presence of this ST in non-vaccine serotypes, especially 19A, can eventually replace the vaccine serotypes and have the potential to cause disease, as seen in the present study also (unpublished data).After the introduction of pneumococcal vaccine, certain non-vaccine serotypes (11,15,33,35, and 38 etc.), which were normal commensals of the nasopharynx, have been shown to cause serious invasive infections [19,20].Hence continuous monitoring of the isolates worldwide is required to implement appropriate vaccines in a particular geographical area.

Conclusion
The majority of the isolates obtained in this study were sensitive to penicillin and belonged to non-7valent vaccine serotypes.Most of them were of unique sequence types, showing that they were very diverse.Therefore, these facts have to be kept in mind before treating patients and also before introducing the pneumococcal vaccine in India.

Figure 1 .
Figure 1.Population snapshot of115 Indian isolates of Streptococcus pneumoniae based on eBURST analysis

Figure 2 .
Figure 2. Population snapshot of Group 52 (Predictor founder 5067) of Global isolates (inclusive of Group 1 of Indian isolates) of Streptococcus pneumoniae on eBURST analysis

Table 1 .
Antibiotic susceptibility patterns of S. pneumoniae isolates by disk diffusion test

Table 2 .
MIC (minimum inhibitory concentration) of Streptococcus pneumoniae isolates

Table 3 .
Clinical and molecular characteristics of Streptococcus pneumoniae isolates from India and their comparison with isolates recovered from other countries

Table 3
Continued.Clinical and molecular characteristics of Streptococcus pneumoniae isolates from India and their comparison with isolates recovered from other countries