Microscopic study on colonization and antimicrobial property of endophytic bacteria associated with ethnomedicinal plants of Meghalaya.
ABSTRACT: Microscopic visualization using transmission electron microscopy (TEM) can provide a better understanding of endophytic colonization within ethnomedicinal plants. Bacterial endophytes were found attached to the host cell wall colonizing the aerenchyma and intercellular spaces of the epidermis and outer cortex except the vascular system. Colonization was non-uniform as single cells, doublets or in the form of microcolonies. Analysis of in vivo antibacterial action of the methanolic extracts of the isolated endophytic bacteria against Gram-positive, Streptococcus pyogenes MTCC 1925 and Gram-negative, Salmonella enterica ser. paratyphi MTCC735 pathogens has revealed the morphological damages in the tested pathogens respectively, under scanning electron microscopy (SEM). Detached cell wall and cell burst were observed in Streptococcus pyogenes where as, cell blisters were shown in Salmonella enterica ser. paratyphi. The study on bacterial endophyte colonization process is important to better predict how endophytes interact with their host and establish themselves in the plant environment by procuring biocontrol activity.
Project description:In the present study, an attempt to evaluate the antimicrobial and antioxidant activity of fungal endophytes inhabiting Emblica officinalis has been made keeping in view the medicinal importance of the selected host plant in Indian traditional practices. A total of four endophytic fungi belonging to Phylum Ascomycetes were isolated from different parts of the plant which were characterized morphologically and by using rDNA-internal transcribed spacer. The most frequently isolated endophyte was Phomopsis sp. The antioxidant activity by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power assay, and total phenol were evaluated using ethanolic extract of endophytic fungi. DPPH activities in all the ethanolic extract increased with the increase in concentrations. Endophytes, Phomopsis sp. and Xylaria sp. showed highest antioxidant activity and also had the higher levels of phenolics. Antimicrobial activity of fungal extract were tested against four bacteria namely, Escherichia coli MTCC730, Enteroccocus faecalis MTCC2729, Salmonella enterica ser. paratyphi MTCC735 and Streptococcus pyogenes MTCC1925, and the fungus Candida albicans MTCC183. In general, the fungal extracts inhibited the growth of test organisms except E. coli.
Project description:Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).
Project description:Salmonella enterica serovar Paratyphi A is a human-specific serovar that, together with Salmonella enterica serovar Typhi and Salmonella enterica serovar Sendai, causes enteric fever. Unlike the nontyphoidal Salmonella enterica serovar Typhimurium, the genomes of S. Typhi and S. Paratyphi A are characterized by inactivation of multiple genes, including in the flagellum-chemotaxis pathway. Here, we explored the motility phenotype of S. Paratyphi A and the role of flagellin in key virulence-associated phenotypes. Motility studies established that the human-adapted typhoidal S. Typhi, S. Paratyphi A, and S. Sendai are all noticeably less motile than S. Typhimurium, and comparative transcriptome sequencing (RNA-Seq) showed that in S. Paratyphi A, the entire motility-chemotaxis regulon is expressed at significantly lowers levels than in S. Typhimurium. Nevertheless, S. Paratyphi A, like S. Typhimurium, requires a functional flagellum for epithelial cell invasion and macrophage uptake, probably in a motility-independent mechanism. In contrast, flagella were found to be dispensable for host cell adhesion. Moreover, we demonstrate that in S. Paratyphi A, but not in S. Typhimurium, the lack of flagellin results in increased transcription of the flagellar and the Salmonella pathogenicity island 1 (SPI-1) regulons in a FliZ-dependent manner and in oversecretion of SPI-1 effectors via type three secretion system 1. Collectively, these results suggest a novel regulatory linkage between flagellin and SPI-1 in S. Paratyphi A that does not occur in S. Typhimurium and demonstrate curious distinctions in motility and the expression of the flagellum-chemotaxis regulon between these clinically relevant pathogens.
Project description:The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.
Project description:Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.
Project description:Live oral Salmonella enterica serovar Typhi vaccine Ty21a induces specific antibodies that cross-react against Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Paratyphi B, although their functional role in clearance remains unknown. We utilized an in vitro assay with THP-1 macrophages to compare the phagocytosis and survival of Salmonella opsonized with heat-inactivated human sera obtained before and after vaccination with Ty21a or a live oral S. Typhi vaccine, CVD 909. Opsonization with postvaccination sera predominantly increased the phagocytosis of S. Typhi relative to the corresponding prevaccination sera, and increases were also observed with S. Paratyphi A and S. Paratyphi B, albeit of lower magnitudes. Relative to prevaccination sera, opsonization with the postvaccination sera reduced the survival inside macrophages of S. Typhi but not of S. Paratyphi A or S. Paratyphi B. Higher anti-S. Typhi O antigen (lipopolysaccharide [LPS]) IgG, but not IgA, antibody titers correlated significantly with postvaccination increases in opsonophagocytosis. No differences were observed between immunization with four doses of Ty21a or one dose of CVD 909. Ty21a and CVD 909 induced cross-reactive functional antibodies, predominantly against S. Typhi. IgG anti-LPS antibodies may be important in phagocytic clearance of these organisms. Therefore, measurement of functional antibodies might be important in assessing the immunogenicity of a new generation of typhoid and paratyphoid A vaccines. (The CVD 909 study has been registered at ClinicalTrials.gov under registration no. NCT00326443.).
Project description:Enteric fever remains a major public health problem in low resource settings and antibiotic resistance is increasing. In Asia, an increasing proportion of infections is caused by Salmonella enterica serovar Paratyphi A, which for a long time was assumed to cause a milder clinical syndrome compared to Salmonella enterica serovar Typhi.A retrospective chart review study was conducted of 254 unique cases of blood culture confirmed enteric fever who presented at a referral adult hospital in Phnom Penh, Cambodia between 2008 and 2015. Demographic, clinical and laboratory data were collected from clinical charts and antibiotic susceptibility testing was performed. Whole genome sequence analysis was performed on a subset of 121 isolates.One-hundred-and-ninety unique patients were diagnosed with Salmonella Paratyphi A and 64 with Salmonella Typhi. In the period 2008-2012, Salmonella Paratyphi A comprised 25.5% of 47 enteric fever cases compared to 86.0% of 207 cases during 2013-2015. Presenting symptoms were identical for both serovars but higher median leukocyte counts (6.8 x 109/L vs. 6.3 x 109/L; p = 0.035) and C-reactive protein (CRP) values (47.0 mg/L vs. 36 mg/L; p = 0.034) were observed for Salmonella Typhi infections. All but one of the Salmonella Typhi isolates belonged to haplotype H58 associated with multidrug resistance (MDR) (i.e. resistance to ampicillin, chloramphenicol and co-trimoxazole).;42.9% actually displayed MDR compared to none of the Salmonella Paratyphi A isolates. Decreased ciprofloxacin susceptibility (DCS) was observed in 96.9% (62/64) of Salmonella Typhi isolates versus 11.5% (21/183) of Salmonella Paratyphi A isolates (all but one from 2015). All isolates were susceptible to azithromycin and ceftriaxone.In Phnom Penh, Cambodia, Salmonella Paratyphi A now causes the majority of enteric fever cases and decreased susceptibility against ciprofloxacin is increasing. Overall, Salmonella Typhi was significantly more associated with MDR and DCS compared to Salmonella Paratyphi A.
Project description:Salmonella enterica serovars Typhi, Paratyphi A, and Sendai are human-adapted pathogens that cause typhoid (enteric) fever. The acute prevalence in some global regions and the disease severity of typhoidal Salmonella have necessitated the development of rapid and specific detection tests. Most of the methodologies currently used to detect serovar Typhi do not identify serovars Paratyphi A or Sendai. To assist in this aim, comparative sequence analyses were performed at the loci of core bacterial genetic determinants and Salmonella pathogenicity island 2 genes encoded by clinically significant S. enterica serovars. Genetic polymorphisms specific for serovar Typhi (at trpS), as well as polymorphisms unique to human-adapted typhoidal serovars (at sseC and sseF), were observed. Furthermore, entire coding sequences unique to human-adapted typhoidal Salmonella strains (i.e., serovar-specific genetic loci rather than polymorphisms) were observed in publicly available comparative genomic DNA microarray data sets. These polymorphisms and loci were developed into real-time PCR, standard PCR, and liquid microsphere suspension array-based molecular protocols and tested for with a panel of clinical and reference subspecies I S. enterica strains. A proportion of the nontyphoidal Salmonella strains hybridized with the allele-specific oligonucleotide probes for sseC and sseF; but the trpS allele was unique to serovar Typhi (with a singular serovar Paratyphi B strain as an exception), and the coding sequences STY4220 and STY4221 were unique among serovars Typhi, Paratyphi A, and Sendai. These determinants provided phylogenetic data on the genetic relatedness of serovars Typhi, Paratyphi A, and Sendai; and the protocols developed might allow the rapid identification of these Salmonella serovars that cause enteric fever.
Project description:Multidrug-resistant Salmonella enterica serovar Paratyphi B dT+ isolates from patients with gastroenteritis were identical with isolates from their home aquariums. Matched isolates had identical phage types, XbaI and IS200 profiles, and Salmonella genomic island 1 (SGI1). Ornamental fish tanks are reservoirs for SGI1-containing S. Paratyphi B dT+.
Project description:PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen "d," "a," or "b." Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity.