B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.

An immunoinformatic approach towards development of a potent and effective multi-epitope vaccine against monkeypox virus (MPXV).

Monkeypox is a viral zoonotic disease, often transmitted to humans from animals. While the whole world is haggling with the COVID-19 pandemic, the emergence of the monkeypox virus (MPXV) arose as a new challenge to mankind. Till date, numerous cases related to the MPXV have been reported in several countries across the globe, but, its momentary distribution in the current time has left everyone in fright with increasing mortality and limited clinically approved treatments. Therefore, it is of immense importance to develop a potent and highly effective vaccine capable of inducing desired immunogenic responses against the highly contagious MPXV. Herein, using various immunoinformatic and computational biology tools, we made an attempt to develop a multi-epitope vaccine construct against the MPXV which is antigenic, non-allergen and non-toxic in nature and capable of exhibiting immunogenic behavior. The sequence of vaccine construct was designed using the proposed 4 MHC-I, 3 MHC-II and 4 B-cell epitopes linked with suitable adjuvant and linkers. The modeled structure of the vaccine construct was used to assess its interaction with the Toll-like Receptor 4 (TLR4) using ClusPro and HADDOCK. All-atoms molecular dynamics simulation of the MPXV vaccine construct-TLR4 complex followed by a high level of gene expression of the construct within the bacterial system affirmed its stability along with induction of immunogenic response within the host cell. Altogether, our immunoinformatic approach aid in the development of a stable chimeric vaccine construct against MPXV and needs further experimental validation for its immunological relevance and usefulness as a vaccine candidate.Communicated by Ramaswamy H. Sarma.

Cyclic constrained immunoreactive peptides from crucial P. falciparum proteins: potential implications in malaria diagnostics.

Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian, and Latin American regions. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by the administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs- insensitivity in scenarios of low transmission settings and deletion of one of the target proteins- Histidine rich protein 2/3 (HRP-2/3) are evident from multiple reports, thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides- C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5458 cyclic constrained peptides (in duplicate) against P. falciparum-infected sera. Peptides (C6, A8, B7- cyclic constrained) and (G11, DSQ, NQN- corresponding linear peptides) were fairly immunoreactive towards P. falciparum-infected sera in dot-blot assay. Using direct ELISA, cyclic constrained peptides (C6 and B7) were found to be specific to P. falciparum-infected sera. A substantial number of samples were tested and the peptides successfully differentiated the P. falciparum positive and negative samples with high confidence. In conclusion, the study identified 3 cyclic constrained immunoreactive peptides (C6, B7, and A8) from P. falciparum secretory/surface proteins and further validated for diagnostic potential of 2 peptides (C6 and B7) with field-collected P. falciparum-infected sera samples.

Naturally acquired antibody response to a Plasmodium falciparum chimeric vaccine candidate GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) in individuals living in Brazilian malaria-endemic areas.

BACKGROUND: The GMZ2.6c malaria vaccine candidate is a multi-stage Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated. METHODS: This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay. RESULTS: The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. CONCLUSIONS: The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.

Identification of Single-Nucleotide Polymorphisms in the Mitochondrial Genome and Kelch 13 Gene of Plasmodium falciparum in Different Geographical Populations.

The emergence of artemisinin-resistant Plasmodium falciparum parasites in Southeast Asia threatens malaria control and elimination. The interconnectedness of parasite populations may be essential to monitor the spread of resistance. Combining a published barcoding system of geographically restricted single-nucleotide polymorphisms (SNPs), mainly mitochondria of P. falciparum with SNPs in the K13 artemisinin resistance marker, could elucidate the parasite population structure and provide insight regarding the spread of drug resistance. We explored the diversity of mitochondrial SNPs (bp position 611-2825) and identified K13 SNPs from malaria patients in the districts of India (Ranchi), Tanzania (Korogwe), and Senegal (Podor, Richard Toll, Kaolack, and Ndoffane). DNA was amplified using a nested PCR and Sanger-sequenced. Overall, 199 K13 sequences (India: N = 92; Tanzania: N = 48; Senegal: N = 59) and 237 mitochondrial sequences (India: N = 93; Tanzania: N = 48; Senegal: N = 96) were generated. SNPs were identified by comparisons with reference genomes. We detected previously reported geographically restricted mitochondrial SNPs (T2175C and G1367A) as markers for parasites originating from the Indian subcontinent and several geographically unrestricted mitochondrial SNPs. Combining haplotypes with published P. falciparum mitochondrial genome data suggested possible regional differences within India. All three countries had G1692A, but Tanzanian and Senegalese SNPs were well-differentiated. Some mitochondrial SNPs are reported here for the first time. Four nonsynonymous K13 SNPs were detected: K189T (India, Tanzania, Senegal); A175T (Tanzania); and A174V and R255K (Senegal). This study supports the use of mitochondrial SNPs to determine the origin of the parasite and suggests that the P. falciparum populations studied were susceptible to artemisinin during sampling because all K13 SNPs observed were outside the propeller domain for artemisinin resistance.

Peripheral Merozoite Surface Proteins Are Targets of Naturally Acquired Immunity against Malaria in both India and Ghana.

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons. Here, we assessed antibody responses against intact merozoites and a panel of 24 recombinant merozoite antigens in longitudinal cohort studies of Ghanaian (n = 115) and Indian (n = 121) populations using the same reagents and statistical methods. Anti-merozoite antibodies were associated with NAI in both the Indian (hazard ratio [HR] = 0.41, P = 0.020) and the Ghanaian (HR = 0.17, P < 0.001) participants. Of the 24 antigen-specific antibodies quantified, 12 and 8 were found to be protective in India and Ghana, respectively. Using least absolute shrinkage and selection operator (LASSO) regression, a powerful variable subselection technique, we identified subsets of four (MSP6, MSP3.7, MSPDBL2, and Pf12) and five (cMSP33D7, MSP3.3, MSPDBL1, GLURP-R2, and RALP-1) antigens that explained NAI better than the individual antibodies in India (HR = 0.18, P < 0.001) and Ghana (HR = 0.31, P < 0.001), respectively. IgG1 and/or IgG3 subclasses against five antigens from these subsets were associated with protection. Through this comparative study, maintaining uniformity of reagents and methodology, we demonstrate that NAI across diverse geographic regions may result from antibodies to multiple antigenic targets that constitute the peripheral merozoite surface protein complexes.

Chorismate synthase from malaria parasites is bifunctional enzyme.

Chorismate synthase (Cs) is the last enzyme of the main trunk of shikimate pathway and catalyzes formation of chorismate, a major aromatic metabolite precursor. We have previously reported that Cs is highly conserved across different Plasmodium sp. Here we report that Cs from malaria parasites are bifunctional enzymes through expression and functional studies of two recombinant proteins rPfCs (Cs from P. falciparum) and rPvCs (Cs from P. vivax). We confirm bifunctional activity of both rPfCs and rPvCs based on their ability to catalyze formation of chorismate under aerobic conditions as well as their ability to catalyze generation of reduced flavin mononucleotide (FMN) as assessed through diaphorase assay.

Breadth of Functional Antibodies Is Associated With Plasmodium falciparum Merozoite Phagocytosis and Protection Against Febrile Malaria.

BACKGROUND: The specific targets of functional antibodies against Plasmodium falciparum merozoites remain largely unexplored and, more importantly, their relevance to naturally acquired immunity in longitudinal cohort studies (LCSs) is yet to be tested. METHODS: Functionality of immunoglobulin G (IgG) antibodies against 24 merozoite antigens was determined at the baseline of an LCS in Ghana using a bead-based opsonic phagocytosis assay (BPA). Antigen-specific IgG3 subclass antibodies were quantified in the same samples by the Luminex multiplex system. RESULTS: A wide range of BPA activity was observed across the different antigens. High BPA responses of nMSP3K1, GLURP-R2, MSP23D7, MSP119k, and PfRh2-2030 coupled beads were significantly associated with a higher probability of children not experiencing febrile malaria. Children with high breadth of functional antibodies against these antigens together with cMSP33D7 had a significantly reduced risk of febrile malaria (adjusted hazard ratio, 0.36 [95% confidence interval, .18-.72]; P = .004). Five of the 6 BPA activities significantly (likelihood ratio rest, P ≤ .05) contributed to the protective immunity observed with the IgG3 antibodies. CONCLUSIONS: The development of BPA allowed profiling of functional antibodies in an LCS. Identification of targets of opsonic phagocytosis may have implications in the development of a subunit malaria vaccine.

Biochemical characterization and spatio-temporal analysis of the putative L-DOPA pathway in Mucuna pruriens.

MAIN CONCLUSION: Transcriptome analysis and biochemical characterization of the putative l-3,4-dihydroxyphenylalanine (l-DOPA) pathway in Mucuna pruriens (L.) DC have been performed. Spatio-temporal quantification of the putative l-DOPA biosynthetic pathway genes and its correlation with respective metabolites was established. l-tyrosine, l-DOPA, and dopamine from all plant parts were quantified. The de novo transcriptome analysis was performed using leaves of the selected M. pruriens mutant T-IV-9 during maturity. The putative L-DOPA pathway and its regulatory genes were retrieved from transcriptome data and the L-DOPA pathway was biochemically characterized. The spatial and temporal gene expression for the L-DOPA pathway was identified with respect to the chemical constituents. L-tyrosine, L-DOPA, and dopamine contents were highest in leaves during maturity (about 170-day-old plants). The polyphenol oxidase (PPO) was highly expressed in tender stems (230-fold higher as compared to seeds) as well as a high L-DOPA content. The PPO gene was highly expressed in leaves (3367.93 in FPKM) with a 79-fold increase compared to control plants during maturity. L-DOPA was found in every part with varied levels. The highest L-DOPA content was found in mature dried seed (3.18-5.8%), whereas the lowest amount was recorded in mature and dried leaves. The reproductive parts of the plant had a higher amount of L-DOPA content (0.9-5.8%) compared to the vegetative parts (0.2-0.91%). Various amino acid transporters and permeases were expressed in M. pruriens. The transcripts of dopa decarboxylase (DDC) were found in almost all parts of the plant, but its higher content was limited to the leaf.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH2+) for CO2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway.

Analyzing trichomes and spatio-temporal expression of a cysteine protease gene Mucunain in Mucuna pruriens L. (DC).

Mucuna pruriens is a well-known legume for the itching attributes of the trichome and a valuable medicinal herb that is used for the treatment of Parkinson's disease, sexual debilities, etc. Its cultivation was deprived due to its itching behavior. The wild genotype of M. pruriens have the largest trichome length (2015 ± 29 µm) compared to other genotype and mutants. The white-seeded variety of M. pruriens was found to be the most suitable for large-scale cultivation due to the small trichome size and less trichome density on the pod. The external surface trichomes have protuberance with unknown function. The unicellular trichomes of Mucuna show the flowing fluid or cytoplasm inside the trichome. The unigenes regulating the differentiation and development of the trichome such as GLABRA-1, GLABRA-2, and cpr-5 have been identified in M. pruriens transcriptome of the leaf. The Mucunain shows a higher transcript abundance in the flower and pod cover compared to the seeds. The Mucunain was found in every stage of plant growth, but it was highly expressed during maturity (about 170 days) with a high fragment per kilobase per million value.

Naturally Acquired Antibodies Target the Glutamate-Rich Protein on Intact Merozoites and Predict Protection Against Febrile Malaria.

Background: Plasmodium species antigens accessible at the time of merozoite release are likely targets of biologically functional antibodies. Methods: Immunoglobulin G (IgG) antibodies against intact merozoites were quantified in the plasma of Ghanaian children from a longitudinal cohort using a novel flow cytometry-based immunofluorescence assay. Functionality of these antibodies, as well as glutamate-rich protein (GLURP)-specific affinity-purified IgG from malaria hyperimmune Liberian adults, was assessed by the opsonic phagocytosis (OP) assay. Results: Opsonic phagocytosis activity was strongly associated (hazard ratio [HR] = 0.46; 95% confidence interval [CI] = .30-.73; P = .0008) with protection against febrile malaria. Of the antimerozoite-specific antibodies, only IgG3 was significantly associated with both OP and protection (HR = 0.53; 95% CI = .34-.84; Pcorrected = .03) against febrile malaria. Similarly, GLURP-specific antibodies previously shown to be protective against febrile malaria in this same cohort were significantly associated with OP activity in this study. GLURP-specific antibodies recognized merozoites and also mediated OP activity. Conclusions: These findings support previous studies that found OP of merozoites to be associated with protection against malaria and further shows IgG3 and GLURP antibodies are key in the OP mechanism, thus giving further impetus for the development of malaria vaccines targeting GLURP.

A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites.

BACKGROUND: The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface. METHODS: Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-165 were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. RESULTS: Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-165 and GLURP proteins. CONCLUSIONS: Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PURPOSE: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. MATERIALS AND METHODS: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. RESULTS: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). CONCLUSION: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Phenotypic and genotypic characterization of Shigella spp. with reference to its virulence genes and antibiogram analysis from river Narmada.

Water samples of the river Narmada from origin to end were analyzed for the presence of shigellae. Analysis of 40 water samples by biotyping, serotyping, and molecular typing were done. Out of all 40 isolates, 23 were identified as Shigella flexneri, 10 as Shigella sonnei, and seven as Shigella dysenteriae. Serotyping was found to be a better identification method than biotyping since biotyping was not found to be very sharp. In the present investigation, amplified ribosomal DNA restriction analysis (ARDRA) with a probe complementary to 16S rRNA was performed. Repeated ARDRA analysis establishes the similarities between the isolates and thus suggested ARDRA as authentic and precise detection protocol. The isolates were also analyzed for the presence of virulence genes including ipaBCD, ipaH, and stx1 and all the 40 isolates of Shigella showed positive result for ipaH gene while the plasmid encoded invasion-associated genes ipaBCD was present only in S. flexneri and stx1 gene was present only in S. dysenteriae. This study demonstrated the existence of Shigella in the river Narmada and dispersion of different virulence genes among these isolates, which appear to constitute an environmental reservoir of Shigella-specific virulence genes.

Molecular epidemiological characteristics of Shigella spp. isolated from river Narmada during 2005-2006.

Shigellosis is an acute gastroenteritis caused by Shigella species. Forty isolates of Shigella spp. were obtained from the river Narmada during 2005-2006. Twenty-three isolates were identified as S. flexneri, 10 as S. sonnei, and seven as S. dysenteriae on the basis of biochemical tests and serotyping. All the isolates harbored at least one plasmid (range: 1-4) and exhibited 12 distinct plasmid profile patterns. Out of 40 isolates, 90% were found to be resistant against more than two antibiotics. All of the isolates were resistant to ciprofloxacin. It is noteworthy that all of the S. dysenteriae strains were resistant to chloramphenicol and trimethoprim, and that all of the S. flexneri and S. sonnei strains were resistant to cephotaxime, amikacin, and norfloxacin, which can be used for the treatment of shigellosis. Forty-two and a half percent of Shigella isolates were found to be Congo red positive. Since the Congo red binding test is cheap and simple, it can be used to determine the virulence properties of Shigella species. We could not find a specific correlation between serotype, antimicrobial resistance, and plasmid profile.

Phenotypic and genotypic characterization of Shigella spp. with reference to its virulence genes and antibiogram analysis from river Narmada.

Water samples of the river Narmada from the source to the mouth were analyzed for the presence of shigellae and the Shigella isolates from 180 water samples were characterized by biotyping, serotyping and molecular typing. Out of all the 40 isolates, 23 were identified as S. flexneri, 10 as S. sonnei and 7 as S. dysenteriae. Serotyping was found to be the better identification method than biotyping. In the present investigation, amplified ribosomal DNA restriction analysis (ARDRA) with a probe complementary to 16S rRNA was performed. Repeated ARDRA analysis established the similarities between the isolates and thus suggested ARDRA as authentic and precise detection protocol. The isolates were also analyzed for the presence of virulence genes including ipaBCD, ipaH and stx1. All the 40 isolates of Shigella were found to be positive for the ipaH gene. The plasmid encoded invasion-associated genes ipaBCD were present only in S. flexneri and the stx1 gene was found only in S. dysenteriae. This study demonstrated the existence of Shigella in the river Narmada and the dispersion of different virulence genes among the isolates, which appear to constitute an environmental reservoir of Shigella-specific virulence genes.