Acinetobacter baumannii is a significant pathogen responsible for resistant recalcitrant infections and outbreaks in hospitals worldwide, which is of much concern. The main goal of the present study is to determine the levels of antibiotic resistance in recent clinical isolates of Acinetobacter baumannii and to investigate the presence of diverse resistance determinants among these isolates and also to correlate these findings. A total of 133 A. baumannii clinical isolates from hospitalized patients were investigated for antibiotic resistance levels by determining their MIC by microbroth dilution method. The presence of β-lactamase-encoding genes (blaPER-1, blaNDM-1, blaIMP-1, blaADC, blaOXA51, blaOXA23, blaOXA58 and blaOXA24), aminoglycoside resistance-related genes (armA, aph3(III), aac3(I), aac6, ant3, and aph3(I) and genes associated with biofilm formation were checked by amplifying them by PCR using gene specific primers. The genotyping of isolates was carried out by REP-PCR. Coexistence of diverse intrinsic and acquired carbapenem-hydrolyzing β-lactamases (CHDLs) genes was observed in the isolates: Ambler class A: blaPER-1 (50.37

BACKGROUND:Microcrystalline cellulose (MCC) is a novel organic material developed by one of the authors in this study. When MCC was incorporated with conventionally available denture base resin, it demonstrated increased flexural strength and flexural modulus. However, it was speculated that because the material is organic, it can promote the growth of Candida. The purpose of this study is to evaluate the Candida albicans biofilm formation on polymethyl methacrylate (PMMA) denture base resin incorporated with MCC. MATERIALS AND METHODS:MCC is an organic material extracted from the oil palm empty fruit bunch (OPEFB). The growth of C. albicans and biofilm formation in three test groups were compared by biofilm assay and imaging techniques like microscopy (by safranin staining) and scanning electron microscopy. The three test groups were comprised of MCC-reinforced PMMA containing OPEFB fibers of 50-micrometer thickness at 5% weight reinforcement, conventionally and commercially available heat cure PMMA, and an empty well to assess any discrepancies from the environment. RESULTS:The test groups showed increased biofilm formation by C. albicans compared to commercially and conventionally available heat cure PMMA. Reinforcement with MCC showed higher biofilm formation of 1.43 times higher compared to conventional PMMA. Biofilms formed by Candida albicans on MCC-reinforced PMMA appeared heterogeneous in structure, comprised of yeast cells and hyphae, surrounded by a higher density of polysaccharide extracellular matrix material compared to that of conventionally available heat cure PMMA. CONCLUSION:Biofilm formation is increased in denture base resin incorporated with MCC. More investigation is warranted to study the antifungal efficacy of the addition of antifungal agents to the reinforced denture base resin.

Acinetobacter baumannii is an important Gram-negative nosocomial pathogen that causes opportunistic infections and employs different mechanisms to survive in the presence of antibiotics in the host. Nutrient limitation is one of the important defense mechanisms of the mammalian immune system to fight against the colonization of pathogens like A. baumannii. The present study describes an NtrC-type Response Regulator (RR) A1S_1978 involved in modulating the metabolism and cell morphology of A. baumannii via a two-component system. This RR was found to be highly conserved in the Acinetobacter and other important Gram-negative pathogens. Sequence analysis reveals that this RR contains an HTH_8 DNA-binding domain. It is also observed that deletion of this RR resulted in elongated cell phenotype and altered colony morphology of A. baumannii. We showed that the ability of A. baumannii to form biofilm and pellicle is partly abolished upon deletion of this response regulator. We showed that mutant strains lacking RR A1S_1978 have diminished growth in the absence of the nitrogen source. The transcriptome analysis of the A1S_1978 deletion mutant revealed that 253 genes were differentially expressed, including 80 genes that were upregulated by at least 2-fold and 173 genes that were down regulated in the ΔA1S_1978 strain. The transcriptome data showed an association between the A1S_1978 RR and key genes related to various nitrogen and amino acid metabolism processes, which was further confirmed by real time PCR analysis. The deletion of this RR leads to a reduction in persister cell formation against ciprofloxacin antibiotic. Taken together the results of this investigation provide significant evidence that the RR A1S_1978 is a global regulator in A. baumannii.

Clinical isolates of multi-drug resistant Acinetobacter baumannii are a major cause of nosocomial infections, often attributed to the highly adaptable genome that helps it to thrive under environmental selection pressure. Here, we aim to provide genotypic-based surveys and comparative whole genome sequencing (WGS) analysis to explore the genomics of the rare pyomelanin-forming clinical isolates of A. baumannii from India. A total of 54 clinical isolates of A. baumannii obtained from two tertiary care hospitals were genotyped using repetitive sequence-based PCR (REP-PCR) for elucidating their molecular epidemiology, followed by their resistance profiling through the determination of minimum inhibitory concentration using the micro broth dilution method. The isolates’ virulence and antibiotic-resistant determinants were detected by PCR screening, followed by biofilm quantification. Pyomelanin pigment produced by A. baumannii isolates was isolated and chemically characterized. Finally, WGS of three pigment-producing and one non-producing A. baumannii strains was performed to explore the factors contributing to their variability. REP-PCR genotyping identified around 8 clusters, with all isolates being multidrug-resistant (MDR). Pyomelanin-producing isolates were strong biofilm formers, characterized by the concurrent presence of ‘pgaB, BfmR, BfmS, ompA, and cusE’ biofilm-related genes. These pigmented strains belonged to ST2Pas and co-harbored blaOXA−23, blaADC−25, aph (3’)-VIa, armA, aph (6)-Id, tet(B) and msr(E) genes. Thirteen common IS elements and biosynthetic gene clusters of arylpolyene, NI-siderophore, and NRP-metallophore were identified. Notably, genomic islands containing aminoglycoside 3’-phosphotransferase, oxidative stress, two-component response regulators, efflux pump-related, toxin-antitoxin protein, and virulence-related genes were also mapped by WGS. The pyomelanin-forming isolates were MDR and virulent. The elucidation of WGS analysis provided critical insights for understanding the epidemiology, virulome, and mobilome of rare pigment-producing A. baumannii strains.

ESKAPE group are key opportunistic nosocomial pathogens singularized by their exceptional antibiotic resistance and intricate stress adaptation mechanisms. The Nucleoid Associated Proteins (NAPs), an enigmatic regulator that is central to ESKAPE adaptability, are known for binding and bending DNA to environmental nuances thus locally altering the nucleoid spatiotemporal organization, resulting in the cellular reprogramming of regulatory genes at transcriptional and post-transcriptional levels. Classically viewed as architectural proteins, NAPs are now showing emerging evidence of involvement in stress adaptation, virulence, and pathogenesis functions. Their growing classes of protein homologs and regulatory properties are now beginning to unravel.This mini-review embarks on the first cumulative effort of discussing key NAPs within the ESKAPE pathogen cohort. By spotlighting their influence on tailoring the chromosomal architecture, this review explores unveiling the intricacies of NAP’s function, offering insights that could potentially decipher new vulnerabilities within these formidable pathogens.

Introduction. Linezolid is an effective therapeutic option for treating severe infections caused by multidrug-resistant Gram-positive organisms. Several mechanisms have been reported to be responsible for resistance to this antibiotic.Hypothesis or Gap Statement. Although several mechanisms of linezolid resistance have been reported in Staphylococcus haemolyticus, the prevalence and potential for horizontal transfer of resistance genes have not been fully characterized, particularly among S. haemolyticus isolates from India.Aim. To perform whole-genome sequencing (WGS) of linezolid-resistant S. haemolyticus isolates to characterize the resistance mechanisms.Methodology. WGS was performed for 16 linezolid-resistant S. haemolyticus isolates to check for the presence of cfr, optrA and poxtA genes and mutations in 23S rRNA and ribosomal proteins (L3, L4 and L22) that are possible mechanisms implicated in linezolid resistance. Sequence types were identified using MLST finder. The minimum inhibitory concentration (MIC) of linezolid was determined using the E-test method. Polymerase chain reaction (PCR) was carried out for the detection of the cfr gene.Results. The study documented three different mechanisms of linezolid resistance in S. haemolyticus. Thirteen of the 16 isolates were phenotypically resistant to linezolid, of which 12 were positive for the cfr gene. The G2603T mutation in 23S rRNA was found in the majority of the isolates (n=13). Ten isolates had the R138V mutation in L3 ribosomal protein. Twelve isolates with the cfr gene in combination with either G2603T or R138V mutations displayed extremely high MIC values. Surprisingly, three phenotypically sensitive isolates were found to be positive for the cfr gene but negative for other resistance mechanisms. Importantly, in almost half of the isolates the cfr gene was present on a plasmid. ST3 and ST1 were found to be the predominant sequence types.Conclusion. All phenotypically resistant isolates exhibited two or three linezolid resistance mechanisms. The cfr gene was found on plasmids in many isolates, demonstrating its potential for horizontal transfer to more pathogenic organisms.

Multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens are a looming global health problem. Rapid acquisition of resistance genes by ESKAPE pathogens has severely hampered the treatment options for serious infections caused by them, which has increased the global burden of disease. Understanding the different resistance mechanisms of these pathogens is a key step in developing new antimicrobial drugs to combat this group of resistant bacteria. Development of biofilms is one of the important mechanisms that aid in building up resistance in these pathogens. This review focuses on research that tries to understand the genetic basis of biofilm formation, their exact role in antibiotic resistance, adhesion, and in development of persistent infections. Increasing fatality rates because of treatment failure in diseases caused biofilm bacteria to require a coordinated global response in developing novel alternative treatment approaches. Biofilm disruption and eradication could be one of the main strategies for the treatment of nosocomial infections caused by ESKAPE pathogens.

Many life-threatening infections are caused by Acinetobacter baumannii including ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound infection, and urinary tract infections (UTI). There is an increasing prevalence of multidrug resistant A. baumannii infections and several outbreaks caused by them worldwide. Since only a few antibiotics are efficient in treating infections caused by A. baumannii, to overcome this menace, it is imperative to have in-depth knowledge on the ways of this pathogen thrive in biofilms, how they manifest their infections and elaborate pathogenesis, and finally, how they resist their killing by developing various resistance mechanisms. In the present chapter, we aim to provide a current knowledge on different molecular mechanisms involved in biofilm development in A. baumannii and their impact on virulence, persistence, and pathogenesis.

ABSTRACT Among the species within Acb- complex, Acinetobacter lactucae has not been frequently isolated from clinical settings, unlike Acinetobacter baumannii , which is an important nosocomial pathogen. We report the genomic sequences of A. lactucae strains (PKAL1732 and 1828C) harboring multiple-resistance determinants including metallo-β-lactamase ( bla NDM-1 ) isolated from immunocompromised patients admitted to a referral hospital in India.

This study has investigated a total of 51 Acinetobacter baumannii isolates for the prevalence of resistant determinants in tigecycline susceptible and non-susceptible clinical isolates of A. baumannii. Antimicrobial susceptibility testing revealed 74% of isolates were tigecycline resistant. Mutations in RND-efflux pump regulatory genes and the expression of efflux pump genes were measured in tigecycline resistant isolates. There was a strong co-relation between the blaNDM-1 and armA wherein majority of the isolates that are positive for blaNDM-1 have also harbored armA. Compared with TSAB (tigecycline susceptible A. baumannii), TNAB (tigecycline non-susceptible A. baumannii) isolates show increased distribution of blaNDM-1 (P = 0.048), blaIMP-1 (P< 0.0001) and blaOXA-51 (P = 0.0029) carbapenemase genes. The variants of RND-efflux pump regulatory genes due to amino-acid mutations in adeS (F12S, K84E, W61R, N268H and Q299R) and adeL (G21R and Q262R) were identified in tigecycline resistant isolates as well as ISAba1 mediated disruption of adeN were observed causing overexpression of adeIJK efflux pump. Additionally, mutations in adeRS were also associated with increased expression of adeABC efflux pump. Besides, TNAB isolates showed significantly (P< 0.0001) higher ability of biofilm formation as compared to TSAB isolates. The tigecycline resistance due to mutations in contemporary A. baumannii isolates having a higher ability to form biofilm may pose therapeutic difficulties.

Introduction. Chronic persistent device-related infections (DRIs) often give culture-negative results in a microbiological investigation. In such cases, investigations on the device metagenome might have a diagnostic value. Materials and Methods. The 16SrRNA gene sequence analysis and next-generation sequencing (NGS) of clinical metagenome were performed to detect bacterial diversity on invasive medical devices possibly involved in culture-negative DRIs. Device samples were first subjected to microbiological investigation followed by metagenome analysis. Environmental DNA (e-DNA) isolated from device samples was subjected to 16SrRNA gene amplification followed by Sanger sequencing (n=14). In addition, NGS of the device metagenome was also performed (n=12). Five samples were only common in both methods. Results. Microbial growth was observed in only nine cases; among these, five cases were considered significant growth, and in the remaining four cases, growth was considered either insignificant or contaminated. Culture and sequencing analysis yielded identical results only in six cases. In culture-negative cases, Sanger sequencing of 16SrRNA gene and NGS of 16SrDNA microbiome was able to identify the presence of rarely described human pathogens, namely Streptococcus infantis, Gemella haemolysans, Meiothermus silvanus, Schlegelella aquatica, Rothia mucilaginosa, Serratia nematodiphila, and Enterobacter asburiae, along with some known common nosocomial pathogens. Bacterial species such as M. silvanus and S. nematodiphila that are never reported in human infection were also identified. Conclusions. Results of a small number of diverse samples of this pilot study might lead to a path to study a large number of device samples that may validate the diversity witnessed. The study shows that a culture free, a holistic metagenomic approach using NGS could help identify the pathogens in culture-negative chronic DRIs.

Antimicrobial resistance (AMR) is fast becoming a medical crisis affecting the entire global population. The bacterial membrane is the first layer of defense for the bacteria against antimicrobial agents (AMA), specifically transporters in the membrane efflux these AMA out of the bacteria and plays a significant role in the AMR development. Understanding the structure and the functions of these efflux transporters is essential to overcome AMR. This review discusses efflux transporters (primary, secondary, and tripartite), their domain architectures, substrate specificities, and efflux pump inhibitors (EPI). Special emphasis on nosocomial ESKAPEE (Enterococcus faecium., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli) pathogens, their multidrug efflux targets and inhibitors are discussed. Deep knowledge about the functioning of efflux pumps and their structural aspects will open up opportunities for developing new EPI, which could be used along with AMA as combination therapy to overcome the emerging AMR crisis.

Investigations on the selection pressure acting on point mutations in PmrAB two-component system may provide insights into the fate of colistin resistance in Acinetobacter baumannii. In this study, we performed Darwinian selection analysis of occurrence of non-synonymous mutations and synonymous mutations in pmrAB at each amino acid site. We analyzed PmrAB sequences in 3113 draft genomes of A. baumannii obtained from RefSeq database. Nearly all the amino acid substitutions previously reported in PmrAB occur at sites that are fairly conserved and under either neutral or purifying selection. Further, the sites with high levels of polymorphisms in PmrAB were found to be under neutral selection. Strong negative evolutionary selection pressure is also observed at sites throughout both PmrA and PmrB. Notably, there was no sign of positive selection. Mutations that cause colistin resistance are neither adaptive nor polymorphic. Some of them are rather deleterious. These conditions might be maintaining the incidence of colistin resistance in A. baumannii under check. In addition, because of the purifying selection on PmrAB, it is highly unlikely that these mutations will get fixed by random genetic drift. Therefore, in the context of colistin resistance, natural selection plays only a minor role and we argue that in future, A. baumannii may not be able to emerge as a colistin-resistant organism, since the documented mutations are not sustainable and hence, not able to successfully disseminate. Therefore, the concerns raised about continuing the usage of colistin for the treatment against A. baumannii infections are unnecessary

Introduction: Multidrug Resistance (MDR) in Klebsiella pneumoniae isolates is an increasingly recognised threat to hospital infection control. It is known to produce a wide array of cephalosporinase and carbapenemase enzymes. Aim: This study was done to determine the prevalence of MDR in K. pneumoniae with phenotypic and genotypic characterisation of Extended Spectrum Beta-Lactamase (ESBL), AmpC and carbapenemase mediated resistance mechanisms. Materials and Methods: Out of 562 K. pneumoniae isolates recovered during November 2014 to June 2015 in our tertiary care hospital in Pondicherry, 117 MDR strains were phenotypically analysed for presence of various types of beta lactamases and carbapenemases by ceftazidime-clavulanic acid combined disc test, AmpC disc test, Modified Hodge's test and meropenemEDTA combined disc test. These isolates were further screened for ESBL (blaCTX-M, blaSHV-1, blaTEM) and Carbapenemase genes (blaNDM-1, blaIMP-1, blaVIM-2, blaSIM-1 and blaKPC) by multiplex PCR. Results: Prevalence of MDR strains of K. pneumoniae was 20.8%. Out of 117 MDR K. pneumoniae, ESBL, AmpC and MBL mediated resistance was identified by phenotypic method in 91, 27 and 16 isolates respectively. Among the ESBL and MBL genes, blaCTX-M (60.6%), blaSHV-1 (69%), blaNDM-1 (33%) and blaIMP-1 (9%) genes were detected. Co-production of multiple enzymes was observed in 32% isolates. Conclusion: Beta-lactam hydrolysing enzymes are prevalent among MDR K. pneumoniae stains in our region. Co-expression of ESBL and MBL genes are found in a large proportion of clinical isolates of K. pneumoniae.

Investigations on the selection pressure acting on point mutations in PmrAB two-component system may provide insights into the future of colistin therapy in Acinetobacter baumannii , since mutations in pmrAB are implicated in colistin resistance. We performed adaptive selection analysis of pmrAB and compared with the available data on colistin resistant strains. We analysed PmrAB sequences in 3113 draft genomes of A. baumannii obtained from RefSeq database. Adaptive selection analysis was performed by two widely used programs namely, HyPhy and PAML. In addition, to examine the reliability of the approach, the same analysis was performed on gyrA of Escherichia coli and Salmonella enterica , since adaptive mutations on gyrA confer quinolone resistance. Mutations that had caused colistin resistance were found to be neither adaptive nor polymorphic, rather they occur at sites that are either under neutral or purifying selection. Strong negative evolutionary selection pressure is also observed at sites throughout both PmrA and PmrB. Sites with high levels of polymorphisms in PmrAB were found to be under neutral selection. Notably, there was no sign of positive selection. Some of them are rather deleterious. These conditions might be maintaining the incidence of colistin resistance in A. baumannii under check. Therefore, in the context of colistin resistance, natural selection plays only a minor role and we assert that in future, A. baumannii may not be able to sustain and successfully disseminate colistin resistance. Therefore, at present the concerns raised about continuing the usage of colistin for the treatment against A. baumannii infections appears to be unnecessary.

Coagulase-negative staphylococci (CoNS) have been increasingly recognized as a clinically important group of species that can cause several opportunistic nosocomial infections. There are at least 47 known species of Staphylococci and to differentiate all these species >40 biochemical tests need to be performed. The present study was able to refine the CoNS identification process by using only five tests to identify S. epidermidis from the rest and used six other tests to identify eleven other clinically significant CoNS species. A total of 242 CoNS isolates were collected from tertiary care hospitals and included in the study. The five-biochemical test scheme devised based on mathematical probability derived from a computer algorithm included fermentation of mannitol, maltose, mannose, trehalose and novobiocin susceptibility to differentiate S. epidermidis from other CoNS species. The remaining CoNS isolates other than S. epidermidis were further characterized with the help of six additional tests, which identified another eleven species. Species-specific PCR and 16SrDNA sequencing were used to confirm and validate the identification scheme. Species-specific PCR and 16SrDNA sequencing showed 100% agreement with non-divergent phenotypic test results, indicating that the five selected assays are highly specific for identifying S. epidermidis. In conclusion, this study used only 11 tests to identify most of the clinically significant CoNS that can reduce cost and time. This scheme is easy to perform in any laboratory with basic resources, the results of this study were validated using more accurate molecular methods such as PCR and 16S rDNA typing to confirm the utility of the proposed scheme.

Ultra-sensitive hybrid Silver/Zinc oxide/Gold (Ag/ZnO/Au) structure based three dimensional (3D) surface enhanced Raman scattering (SERS) substrates have been prepared by three step fabrication process using thermal evaporation, hydrothermal growth, and sputtering techniques. The size and inter-particle (IP) gap of decorated Au nanoparticles (NPs) on ZnO nanorods (NRs) in the sub-nanometer range have been achieved through varying the sputtering time of Au. The superhydrophobic nature, the formation of the Schottky barrier at ZnO/Au interface and the broad optical absorption spectrum facilitated towards higher SERS activity of Ag/ZnO/Au hybrid structures. The higher SERS activity of 3D SERS substrate as compared with two dimensional (2D) SERS substrate has been studied. The good SERS signal reproducibility of 3D hybrid structures have been explored through Raman mapping. Higher SERS enhancement factor (EF) of 1 x 10(10) has been achieved with a limit of detection (LOD) up to 10(-16) M and 10 ng/mu L for Rhodamine-6 G (Rh6G) and lambda DNA (lambda-DNA), respectively. The degradation of Rh6G and lambda-DNA molecules have been studied through photocatalytic degradation process to explore the reusability of the SERS substrates up to 10 and 4 times, respectively, with maintaining good SERS signal reproducibility. This metal/semiconductor/metal hybrid structure based SERS substrate with reusable capability indicates potential application towards biosensor for the detection of biologically important molecules at very low concentration level.

Nosocomial infections due to Methicillin Resistant Staphylococcus aureus (MRSA) has been an element of concern to the medical personnel for the past four decades. Community associated MRSA (CA-MRSA) which was initially considered as more sensitive than hospital acquired MRSA (HA-MRSA) is now presenting with increasing levels of drug resistance. Along with the mecA gene, pvl gene is characteristically present in most isolates of CA-MRSA. To study the varying drug resistant patterns of MRSA isolates with pvl gene. A total of 150 clinical isolates of MRSA analysed in the study were subjected to susceptibility testing to cefoxitin (30 µg) and growth on oxacillin screen agar containing 6 µg/mL of oxacillin for the detection of methicillin resistance. All the isolates which were included in the study, were checked by PCR for the presence of mecA gene, which codes for methicillin resistance and for pvl gene. Amplification of 540bp and 625bp gene fragments in the PCR reaction indicates the presence of mecA and pvl genes respectively. mecA gene was present in all the 150 isolates of MRSA and pvl gene was present only in 26 isolates. Of these 26 isolates that had pvl gene, 14 were in MRSA isolated from outpatient samples and were sensitive to most of the non- beta lactam antibiotics. Among the other 12 inpatient MRSA isolates which had the pvl gene, nine were sensitive to most of the non- beta lactam antibiotics, whereas remaining three were resistant to most of the antibiotics except vancomycin and linezolid. The presence of pvl gene can no longer be used to discriminate between CA-MRSA and HA-MRSA. Indiscriminate empirical treatment of MRSA infections with high end antibiotics like glycopeptides needs to avoided and therapy with non beta lactam antibiotics like lincosamides which have better soft tissue penetration should be used as very few new antimicrobial agents are in the pipeline.

Background: Streptococcus pneumoniae continues to cause morbidity and mortality across the globe, with developing countries bearing the brunt of the disease. It is mainly responsible for meningitis, pneumonia and septicaemia primarily in children, elderly and immunocompromised persons. Colonisation and persistence in the human nasopharynx occur during early childhood, and it appears to be prerequisite for invasive pneumococcal disease (IPD). Factors that help in persistent colonisation and subsequent invasion are ill understood. Several virulence factors have been incriminated for nasopharyngeal carriage (NC) as well as for the manifestation of the pathogenesis of IPD. Materials and Methods: This study attempts to characterise the S. pneumoniae isolates through analysing the distribution of different virulence markers such as lytA, ply, pbpA, eno, psaA, amiA, ciaR and wchA among the isolates obtained from disease and NC. A total of 37 isolates which include 14 invasive and 23 non-invasive isolates were investigated by polymerase chain reaction to detect the genes. Eight representative isolates were investigated for mutations in wchA by DNA sequencing that may responsible for capsular variation. Results: Ply, pbpA, amiA and eno were observed in a greater percentage of invasive isolates than non-invasive isolates though these differences are not statistically significant. Other two genes ciaH and psaA did not show any significant difference between two groups of isolates. Biofilm production was significantly higher in than non-invasive isolates when compared to invasive isolates. Sequence analysis of wchA revealed three significant point mutations or single-nucleotide polymorphisms (SNPs) among the isolates of one particular cluster (cluster III). These SNPs are responsible for a non-synonymous mutation in wchA bringing in an amino acid change in WchA protein, which is a part of the capsule of S. pneumoniae. Notably, all the three isolates present in cluster III had these SNPs and all of them were isolated from ocular infections. Conclusion: The results of our study implies a possible capsular variations among the isolates and this may have an impact on capsular typing.