Papers by Kshipra Chandrashekhar
Plasmid, Jul 1, 2016
In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently ... more In Campylobacter spp., resistance to the antimicrobials kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095 nt) harboring tet(O) was identified in C. jejuni strain 11601MD, which was isolated from the jejunum of a turkey produced conventionally in North Carolina. Analysis of the p11601MD sequence revealed the presence of a high-GC content cassette with four genes that included tet(O) and a putative aminoglycoside transferase gene (aphA-3) highly similar to kanamycin resistance determinants. Several genes putatively involved in conjugative transfer were also identified on the plasmid. These findings will contribute to a better understanding of the distribution of potentially self-mobilizing plasmids harboring antibiotic resistance determinants in Campylobacter spp. from turkeys and other sources.
Veterinary World, 2010
Immunological memory is one of the lesser understood aspects of adaptive immunity which protects ... more Immunological memory is one of the lesser understood aspects of adaptive immunity which protects organisms from recurrent and persistent attack by pathogens. The central event in the generation of both humoral and cell mediated immune responses is the activation and clonal expansion of T cells. T cell activation is initiated by interaction of the TCR-CD3 complex with processed antigenic peptide bound to either a class I (CD8+cells) or class II (CD 4+cells) MHC molecule on the surface of antigen presenting cell (APC). On interaction of a naive T cell with the processed antigen initiates a cascade of events which activates the resting T cell to enter the cell cycle, proliferating and developing into a clone of progeny cells, which differentiate into memory or effector T cells. Memory T cells are generated by antigen interaction and remain long but quiescent in nature, however responding with greater reactivity to a subsequent challenge with the same antigen, generating a secondary res...
Microbiology Resource Announcements, 2020
Eubacterium callanderi AMC0717 was isolated from the mucosa of the transverse colon of an 11-year... more Eubacterium callanderi AMC0717 was isolated from the mucosa of the transverse colon of an 11-year-old organ donor. This strain contains genes putatively encoding short-chain fatty acids (SCFAs), exopolysaccharide (EPS), and several B vitamins. ABSTRACT Eubacterium callanderi AMC0717 was isolated from the mucosa of the transverse colon of an 11-year-old organ donor. This strain contains genes putatively encoding short-chain fatty acids (SCFAs), exopolysaccharide (EPS), and several B vitamins.
.................................................................................................... more ........................................................................................................................ ii Acknowledgments....................................................................................................... vii Vita ................................................................................................................................ x List of Tables ............................................................................................................. xiv List of Figures ............................................................................................................ xvi Chapter 1: Introduction and Literature Review ........................................................... 1 Chapter 2: Transducer Like Proteins of Campylobacter jejuni: Role in chemotaxis, virulence-associated traits, and colonization of the chicken gastrointestinal tract ..... 57 2.1 Abstract ............................................................................................................ 57 2.2 Introduction..............................................................................58 2.3 Materials and Methods.................................................................61 2.4 Results....................................................................................75 2.5 Discussion...............................................................................89 2.6 References...............................................................................95 Chapter 3: Transducer like Protein in Campylobacter jejuni with a role in mediating chemotaxis to iron and phosphate...........................................................100 xiii 3.1 Abstract.................................................................................100 3.2 Introduction............................................................................101 3.3 Materials and Methods...............................................................103 3.4 Results..................................................................................117 3.5 Discussion..............................................................................132 3.6 References..............................................................................139 Chapter 4: PPK1and PPK2-dependent Transcriptomes of the Foodborne Pathogen Campylobacter jejuni..................................................................................145 4.1 Abstract.................................................................................145 4.2 Introduction............................................................................146 4.3 Materials and Methods...............................................................150 4.4 Results and Discussion...............................................................156 4.5 Conclusions............................................................................170 4.6 References..............................................................................171 Chapter 5: Summary and Conclusions..................................................... 176 5.1 Future studies..........................................................................178 References ................................................................................................................. 180 Appendix A: Supplemental Figures .......................................................................... 225
Microbiology Resource Announcements, 2020
Citrobacter freundii AMC0703 was isolated from the intestinal mucosa of an 11-year-old organ dono... more Citrobacter freundii AMC0703 was isolated from the intestinal mucosa of an 11-year-old organ donor. Genome analysis revealed the presence of multiple factors potentially aiding in pathogenicity, including fimbriae, flagella, and genes encoding resistance to fluoroquinolones, cephamycin, fosfomycin, and aminocoumarin. ABSTRACT Citrobacter freundii AMC0703 was isolated from the intestinal mucosa of an 11-year-old organ donor. Genome analysis revealed the presence of multiple factors potentially aiding in pathogenicity, including fimbriae, flagella, and genes encoding resistance to fluoroquinolones, cephamycin, fosfomycin, and aminocoumarin.
Gut Microbiome
Knowledge of the intra-individual spatial and regional distribution of intestinal microbial popul... more Knowledge of the intra-individual spatial and regional distribution of intestinal microbial populations is essential to understand gut host–microbial interactions. In this study, we performed a compositional analysis of luminal and mucosal samples from the small and large intestine of four organ donors by 16S rRNA amplicon sequencing and high-throughput quantitative polymerase chain reaction. Since the human microbiota is subject to selection pressure at lower taxonomic levels, we isolated over 400 bacterial strains and investigated strain-level variation of 11 Lactobacillus rhamnosus from different intestinal regions. Results substantiate reported inter-individual variability as well as intra-individual differences along the gastrointestinal tract. Although the luminal and mucosal-associated communities were similar within individuals, relative abundance reflected the donors’ demographic and potential pathologies. The total bacterial load of all donors increased from small intestin...
The American Journal of Tropical Medicine and Hygiene
Rotavirus is the leading cause of childhood deaths due to diarrhea. Although existing oral rotavi... more Rotavirus is the leading cause of childhood deaths due to diarrhea. Although existing oral rotavirus vaccines are highly efficacious in high-income countries, these vaccines have been demonstrated to have decreased efficacy in low- and middle-income countries. A possible explanation for decreased efficacy is the impact of gut microbiota on the enteric immune system's response to vaccination. We analyzed the gut microbiome of 50 children enrolled in a prospective study evaluating response to oral pentavalent rotavirus vaccination (RV5) to assess associations between relative abundance of bacterial taxa and seroconversion following vaccination. Stool samples were taken before the first RV5 dose, and microbiome composition characterized using 16S rRNA amplicon sequencing and Quantitative Insights Into Microbial Ecology software. Relative abundance of bacterial taxa between seroconverters following the first RV5 dose, those with ≥ 4-fold increase in rotavirus-specific IgA titers, and nonseroconverters were compared using the Wilcoxon-Mann-Whitney test. We identified no significant differences in microbiome composition between infants who did and did not respond to vaccination. Infants who responded to vaccination tended to have higher abundance of Proteobacteria and Eggerthella, whereas those who did not respond had higher abundance of Fusobacteria and Enterobacteriaceae; however, these differences were not statistically significant following a multiple comparison correction. This study suggests a limited impact of gut microbial taxa on response to oral rotavirus vaccination among infants; however, additional research is needed to improve our understanding of the impact of gut microbiome on vaccine response, toward a goal of improving vaccine efficacy and rotavirus prevention.
Frontiers in Microbiology
Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environment... more Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducerlike proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The tlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO 4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (P tlp2) was induced by Pi and iron, both in the ferrous (Fe 2+) and ferric form (Fe 3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The tlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron.
Gut Microbes, 2017
Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (ch... more Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (chemoeffectors/ligands that can be attractants or repellents) in the environment, plays an important role in the adaptation of Campylobacter jejuni to disparate niches. The chemotaxis system consists of core signal transduction proteins and methyl-accepting-domain-containing Transducer like proteins (Tlps). Ligands binding to Tlps relay a signal to chemotaxis proteins in the cytoplasm which initiate a signal transduction cascade, culminating into a directional flagellar movement. Tlps facilitate substrate-specific chemotaxis in C. jejuni, which plays an important role in the pathogen's adaptation, pathobiology and colonization of the chicken gastrointestinal tract. However, the role of Tlps in C. jejuni's host tissue specific colonization, physiology and virulence remains not completely understood. Based on recent studies, it can be predicted that Tlps might be important targets for developing strategies to control C. jejuni via vaccines and antimicrobials.
Case Studies of Outbreaks in the Agri-Food Industries, 2016
Frontiers in Microbiology, 2013
Campylobacter jejuni is a Gram-negative food-borne bacterium that can cause mild to serious disea... more Campylobacter jejuni is a Gram-negative food-borne bacterium that can cause mild to serious diseases in humans. A variety of stress conditions including exposure to formic acid, a weak organic acid, can cause C. jejuni to form viable but non-culturable cells (VBNC), which was proposed as a potential survival mechanism. The inability to detect C. jejuni VBNC using standard culturing techniques may increase the risk of exposure to foods contaminated with this pathogen. However, little is known about the cellular mechanisms and triggers governing VBNC formation. Here, we discuss novel mechanisms that potentially affect VBNC formation in C. jejuni and emphasize the impact of formic acid on this process. Specifically, we highlight findings that show that impairing inorganic polyphosphate (poly-P) metabolism reduces the ability of C. jejuni to form VBNC in a medium containing formic acid. We also discuss the potential effect of poly-P and formate metabolism on energy homeostasis and cognate VBNC formation. The relationship between poly-P metabolism and VBNC formation under acid stress has only recently been identified and may represent a breakthrough in understanding this phenomenon and its impact on food safety.
The accuracy of the Content should not be relied upon and should be independently verified with p... more The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Frontiers in Cellular and Infection Microbiology, 2015
Transducer Like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable... more Transducer Like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable enteric pathogens to respond to changing nutrient levels in the environment by mediating taxis toward or away from specific chemoeffector molecules. Despite recent advances in the characterization of chemotaxis responses in Campylobacter jejuni, the impact of Tlps on the adaptation of this pathogen to disparate niches and hosts is not fully characterized. The latter is particularly evident in the case of C. jejuni 81-176, a strain that is known to be highly invasive. Furthermore, the cytoplasmic group C Tlps (Tlp5, 6, and 8) were not extensively evaluated. Here, we investigated the role of C. jejuni 81-176 Tlps in chemotaxis toward various substrates, biofilm formation, in vitro interaction with human intestinal cells, and chicken colonization. We found that the tlp6 and tlp10 mutants exhibited decreased chemotaxis toward aspartate, whereas the tlp6 mutant displayed a decreased chemotaxis toward Tri-Carboxylic Acid (TCA) cycle intermediates such as pyruvate, isocitrate, and succinate. Our findings also corroborated that more than one Tlp is involved in mediating chemotaxis toward the same nutrient. The deletion of tlps affected important phenotypes such as motility, biofilm formation, and invasion of human intestinal epithelial cells (INT-407). The tlp8 mutant displayed increased motility in soft agar and showed decreased biofilm formation. The tlp8 and tlp9 mutants were significantly defective in invasion in INT-407 cells. The tlp10 mutant was defective in colonization of the chicken proximal and distal gastrointestinal tract, while the tlp6 and tlp8 mutants showed reduced colonization of the duodenum and jejunum. Our results highlight the importance of Tlps in C. jejuni's adaptation and pathobiology.
Campylobacter jejuni is a foodborne bacterium and a leading cause of gastroenteritis worldwide. W... more Campylobacter jejuni is a foodborne bacterium and a leading cause of gastroenteritis worldwide. We previously showed that the polyphosphate kinases (PPK1 and PPK2) play key roles in inorganic polyphosphate (poly-P) homeostasis and the pathobiology of C. jejuni. To expand on these studies and better understand how these enzymes act on a system level, we performed RNA-seq analysis of the Δppk1 and Δppk2 mutants to directly measure the effect of deleting these genes on global gene expression. We observed differential expression of 15 and 53 genes in Δppk1 in comparison to the wild type (WT) during the exponential and stationary phases of growth, respectively. Differentially expressed genes (DEGs) in the Δppk1 included those encoding flagella associated components (FlaA, FlaG, FlgH, FlgE, FliD, PseA), heat shock response proteins, and proteins involved in glycosylation (PglI), chromosome partitioning (ParB) and chemotaxis (CheY). In the Δppk2, 14 and 12 genes were differentially expressed in comparison to the WT during the exponential and stationary phases of growth, respectively. Differentially expressed genes in the Δppk2 included genes encoding catalase (KatA), a protease, a protein (DcuB) that mediates fumarate respiration, and proteins that are associated with peptidoglycan biosynthesis (alanine racemase) and protein export (SecE). Taken together, this RNA-Seq analysis suggested that in the absence of ppk1 and ppk2 the transcription of several genes involved in core biological processes are dysregulated in C. jejuni; consistent with the pleiotropic effects associated with impairment of poly-P metabolism in this pathogen.
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Papers by Kshipra Chandrashekhar