Vivek Sagar

Molecular heterogeneity among north Indian isolates of Group A Streptococcus

Aim:  To monitor molecular heterogeneity among the clinical isolates of group A Streptococcus (GAS) from north India by Vir and emm typing.

Comparative analysis of emm type pattern of Group A Streptococcus throat and skin isolates from India and their association with closely related SIC, a streptococcal virulence factor

BackgroundGroup A streptococcus (GAS) causes a wide variety of life threatening diseases in humans and the incidence of such infections is high in developing countries like India. Although distribution of emm types of GAS in India has been described, there is a lack of data describing either the comparative distribution of emm types in throat versus skin isolates, or the distribution of certain virulence factors amongst these isolates. Therefore in the present study we have monitored the emm type pattern of Group A streptococcus throat and skin isolates from India. Additionally, the association of these isolates with closely related sic (crs), a multifunctional compliment binding virulence factor, was also explored.ResultsOf the 94 (46 throat and 48 skin) isolates analyzed, 37 emm types were identified. The most frequently observed emm types were emm49 (8.5%) and emm112 (7.5%) followed by 6.5% each of emm1-2, emm75, emm77, and emm81. Out of 37 emm types, 27 have been previously reported and rest were isolated for the first time in the Indian Community. The predominant emm types of throat (emm49 and emm75) samples were different from those of skin (emm44, emm81 and emm112) samples. After screening all the 94 isolates, the crs gene was found in six emm1-2 (crs1-2) isolates, which was confirmed by DNA sequencing and expression analysis. Despite the polymorphic nature of crs, no intravariation was observed within crs1-2. However, insertions and deletions of highly variable sizes were noticed in comparison to CRS isolated from other emm types (emm1.0, emm57). CRS1-2 showed maximum homology with CRS57, but the genomic location of crs1-2 was found to be the same as that of sic1.0. Further, among crs positive isolates, speA was only present in skin samples thus suggesting possible role of speA in tissue tropism.ConclusionDespite the diversity in emm type pattern of throat and skin isolates, no significant association between emm type and source of isolation was observed. The finding that the crs gene is highly conserved even in two different variants of emm1-2 GAS (speA +ve and -ve) suggests a single allele of crs may be prevalent in the highly diverse throat and skin isolates of GAS in India.

Variability in the Distribution of Genes Encoding Virulence Factors and Putative Extracellular Proteins of Streptococcus pyogenes in India, a Region with High Streptococcal Disease Burden, and Implication for Development of a Regional Multisubunit Vaccine

ABSTRACT Streptococcus pyogenes causes a wide variety of human diseases and is a significant cause of morbidity and mortality. Attempts to develop a vaccine were hampered by the genetic diversity of S. pyogenes across different regions of the world. This study sought to identify streptococcal antigens suitable for a region-specific vaccine in India. We used a two-step approach, first performing epidemiological analysis to identify the conserved antigens among Indian isolates. The second step consisted of validating the identified antigens by serological analysis. The 201 streptococcal clinical isolates from India used in this study represented 69 different emm types, with emm12 being the most prevalent. Virulence profiling of the North and South Indian S. pyogenes isolates with a custom-designed streptococcal virulence microarray identified seven conserved putative vaccine candidates. Collagen-like surface protein (SCI), putative secreted 5′-nucleotidase (PSNT), and C5a peptidase were found in 100% of the isolates, while R28, a putative surface antigen (PSA), and a hypothetical protein (HYP) were found in 90% of the isolates. A fibronectin binding protein, SfbI, was present in only 78% of the isolates. In order to validate the identified potential vaccine candidates, 185 serum samples obtained from patients with different clinical manifestations were tested for antibodies. Irrespective of clinical manifestations, serum samples showed high antibody titers to all proteins except for SCI and R28. Thus, the data indicate that PSNT, C5a peptidase, PSA, HYP, and SfbI are promising candidates for a region-specific streptococcal vaccine for the different parts of India.

First Detection of Trimethoprim Resistance Determinant dfrG in Streptococcus pyogenes Clinical Isolates in India

Streptococcus pyogenes is capable of causing a wide spectrum of diseases, ranging from throat and skin infection to life-threatening invasive diseases ([2][1]). The sequelae of streptococcal infections, such as acute rheumatic fever, represent a big health hazard ([1][2]). Recently, the emergence of

DRS Is Far Less Divergent than Streptococcal Inhibitor of Complement of Group A Streptococcus

When 100 group A streptococcus isolates were screened, drs, a variant of sic, was identified in emm12 and emm55 isolates. Molecular characterization showed that the drs gene sequence is highly conserved, unlike the sic gene sequence. However, the variation in gene size observed was due to the presence of extra internal repeat sequences.

Association of rheumatic fever & rheumatic heart disease with plausible early & late-stage disease markers

Background & objectives: Rheumatic fever (RF) and rheumatic heart disease (RHD) are the autoimmune sequelae caused by Group A Streptococcus. RHD still remains a major concern in the developing countries due to its poor diagnosis, lack of vaccines and social awareness among population. This study was aimed to identify the plausible early- and late-stage disease markers associated with RF/RHD. Methods: A total of 84 patients with confirmed pharyngitis (n=18), RF (n=23) and RHD (n=43) were included in the comparative analysis of different factors involved in host-pathogen interaction during RF/RHD pathogenesis. Results: This study revealed high titre of serum antistreptolysin O (ASO) antibody in pharyngitis compared to RF and RHD patients, whereas procollagen type 1 C-peptide (PICP) level was elevated in RHD which showed an inverse correlation with serum ASO titre. The significant elevation of serum anti-peptide associated with RF (PARF) antibody in RF patients was correlated as a probable stage-specific determinant. In addition, pro-inflammatory cytokine profile revealed high levels of interleukin-12 (IL-12)/IL-23p40, IL-17A in RF, whereas IL-6 concentration was higher in RHD compared to healthy controls. Interpretation & conclusions: The overall assessment of the factors/disease markers involved in host-pathogen interaction in RF/RHD may be suggestive of plausible disease marker in different groups of patients. Further studies with larger sample need to be done to better understand RF/RHD pathogenesis.

emm 81, The Predominant Group a Streptococcus from North India in Year 2003 in Context to Adhesion, Invasion and Antimicrobial Susceptibility Pattern

Heterogeneity exists among Group a Streptococcus emm types worldwide. In 2003, we observed 11 circulating emm types in northern region of India, of which emm 81 was found to be the predominant type (17.5%). As emm 81 has been reported to be associated with invasive diseases in western countries, hence, in the present study, attempts were made to study the virulence potential of these isolates from skin and throat samples. Isolates were screened for nine fi bronectin binding protein (FBP) genes, evaluated for adherence & invasion potential along with drug resistance to various commonly prescribed antibiotics for treatment. Throat isolates showed higher distribution of FBP genes as compared to skin isolates. All the isolates were found to be positive for sciB and prtF15; 92.9% for sfb and fba, 78.6% for sciA; 35.2% for prtF1; 7.1% for prtF2 but none for sfb2 and pfbp. Isolates showed low (8.5%) to moderate (27.7%) adherence and negligible invasion potential in the experimental A549 cell line which was con firmed by immuno fluorescent confocal microscopy. Drug resistance pro fi ling showed isolates to be highly resistant to macrolides, tetracycline, co-trimaxazole but all susceptible to penicillin. The study shows emm 81 strains from northern India to be of less virulent nature with respect to adherence/invasion potential, highlighting that same emm type in different geographical regions may have a different clinical outcome, the latter being dependent on number of factors like ethnicity, geographical, socioeconomic factors besides its molecular type and source.

Evaluation of temperate carrot genotypes for quality attributes

Carrot roots having high dry matter content and rich in carotene are generally considered for dehydration purposes. High degree of β-carotene is very essential to retain better flavour in dehydrated products. Based on the different nutrient parameters such as β-carotene, lycopene, ascorbic acid, sensory score in respect of colour, flavour and texture of the dried product as well as drying and rehydration ratio, the genotype NS-16 followed by genotypes SH and Pusa Yamdagini were found to be superior for the preparation of dehydrated carrot slices, among the thirteen different genotypes of carrot analyzed.

Differences in virulence repertoire and cell invasive potential of Group A Streptococcus emm1-2 in comparison to emm1 genotype

Group A streptococcus (GAS, Streptococcus pyogenes) type emm1 is widely associated with streptococcal invasive disease. This type is prevalent worldwide but is rare in India. Instead, emm1-2 type which is closely related to emm1 but is a distinct type is more prevalent. Although emm1 has been well characterized, information available on emm1-2 is rare. In this study we present a comparative study of both types. DNA microarray analysis showed segregation of emm1 and emm1-2 isolates into two distinct clusters. Out of 229 arrayed genes, 83-87% were present, 6-9% absent and 4-8% genes were ambiguous in emm1 isolates. emm1-2 strains harboured only 68-77%, 11-13% were absent and 10-20% ambiguous genes. Fourteen genes, present in all emm1, were completely absent in the emm1-2 isolates. sfb1 is a gene which encodes for Streptococcal fibronectin binding adhesin and invasin which has restricted distribution among different emm types of GAS. A variant of sfb1 (sfb1-2) was the only gene which was present in all emm1-2 isolates, but absent from all emm1 strains. Sfb1 and Sfb1-2 differ in sequences in the aromatic domain and the proline rich repeat region, whereas the fibronectin binding region was conserved and exhibited similar fibronectin binding activity. The presence of Sfb1-2 in emm1-2 strains was concomitant with significantly higher fibronectin-binding and invasion efficiency of HEp-2 cells when compared to emm1 isolates. The role of Sfb1-2 in invasion was confirmed by latex bead assay. emm1-2 isolates follow membrane ruffling mechanism during invasion and intracellularly follow classical endocytic pathway. Further studies are required to understand the correlation between the presence of emm1-2 isolates and the disease pattern in North India.