Biology subjects

Ryan, E. T.

Publications and source records attributed to Ryan, E. T..

14 recordsLinked to original sources

bioRxiv2025

Insights into global antimicrobial resistance dynamics through the sequencing of enteric bacteria from U.S. international travelers

Antimicrobial resistance (AMR) is an urgent threat to public health, but gaps in surveillance limit the detection of emergent novel threats and knowledge about the global distribution of AMR genes. International travelers frequently acquire AMR organisms, and thus may provide a window into AMR dynamics in otherwise poorly monitored regions and environments. To assess the utility of travelers as global AMR sentinels, we collected pre- and post-travel stool samples from 608 travelers, which were screened for the presence of extended-spectrum beta-lactamase producing Enterobacterales, carbapenem-resistant Enterobacterales, and mcr-mediated colistin-resistant Enterobacterales. A total of 307 distinct AMR organisms were sequenced in order to determine genotypic patterns and their association with travel region and behavior. Travel-associated AMR organisms were overwhelmingly E. coli, which exhibited considerable phylogenetic diversity regardless of travel region. However, the prevalence of resistance genes varied by region, with blaCTX-M-55 and blaCTX-M-27 significantly more common in travelers returning from South America and South-Eastern Asia, respectively. Hybrid assembly and plasmid reconstruction revealed the genomic neighborhood of blaCTX-M-55 frequently matched a motif previously linked to animal populations. Contact with animals was also associated with virulence factors in acquired AMR organisms, including carriage of the ColV plasmid, a driver of avian pathogenic E. coli. We identified novel variants of the mcr-1 gene in strains acquired from Western Africa, highlighting the potential for traveler surveillance to detect emerging clinical threats. Ongoing efforts to track travel-acquired organisms could complement existing global AMR surveillance frameworks. ImportanceWe collected pre- and post-travel stool samples from 608 U.S. international travelers, which were screened for the presence of three clinically relevant antimicrobial-resistant organisms, with the goal of understanding the utility of travelers as sentinels for emerging AMR and identifying regional resistance patterns. We sequenced 307 organisms and uncovered significant diversity and geographic heterogeneity in AMR organisms. We found significant differences in certain resistance gene alleles of travelers returning from South America and South-Eastern Asia, traveler contact with animals associated with virulence factors, including a plasmid that is a known driver of avian pathogenic E. coli, and novel variants of the colistin resistance mcr-1 gene in strains acquired from Western Africa. This work emphasizes the value of monitoring international travelers as a proxy for understanding AMR patterns globally and could be an important addition to existing global AMR surveillance frameworks.

genomics↗

bioRxiv2024

Limited O-specific polysaccharide (OSP)-specific functional antibody responses in young children with Shigella infection in Bangladesh

Shigellosis is the second leading cause of diarrheal death in children younger than five years of age globally. At present, there is no broadly licensed vaccine against shigella infection. Previous vaccine candidates have failed at providing protection for young children in endemic settings. Improved understanding of correlates of protection against Shigella infection and severe shigellosis in young children living in endemic settings is needed. Here, we applied a functional antibody profiling approach to define Shigella-specific antibody responses in young children versus older individuals with culture-confirmed shigellosis in Bangladesh, a Shigella endemic area. We analyzed Shigella-specific antibody isotypes, FcR binding and antibody-mediated innate immune cell activation in longitudinal serum samples collected at clinical presentation and up to 1 year later. We found that higher initial Shigella O-specific polysaccharide (OSP)-specific and protein-specific IgG and Fc{gamma}R binding levels correlated with less severe disease regardless of patient age, but that individuals under 5 years of age developed a less prominent class switched, FcR-binding, functional and durable antibody response against both OSP and protein Shigella antigens than older individuals. Focusing on the largest cohort, we found that functional S. flexneri 2a OSP-specific responses were significantly induced only in individuals over age 5 years, and that these responses promoted monocyte phagocytosis and activation. Our findings suggest that in a Shigella endemic region, young children with shigellosis harbor a functional antibody response that fails to maximally activate monocytes; such a response may be important in facilitating subsequent innate cell clearance of Shigella, especially via recruitment and activation of polymorphonuclear cells capable of directly killing Shigella.

immunology↗

bioRxiv2024

Prevalent chromosome fusion in Vibrio cholerae O1

Two circular chromosomes are a defining feature of the family Vibrionaceae, including the pathogen Vibrio cholerae, with rare reports of isolates with a single, fused chromosome. Here we report chromosome fusions in clinical V. cholerae O1 isolates, including several independent fusion events stable enough to be transmitted between patients within a household. Fusion occurs in a 12 kilobase-pair homologous sequence shared between the two chromosomes, which may lead to reversible chromosomal fusion.

microbiology↗

bioRxiv2024

Diversity of Vibrio cholerae O1 through the human gastrointestinal tract during cholera

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that the V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from ten cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than co-infection with divergent V. cholerae O1 lineages. The number of single nucleotide variants decreased between vomit and stool in four patients, increased in two, and remained unchanged in four. The number of genes encoded in the V. cholerae genome decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract. IMPORTANCEVibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes or genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution.

microbiology↗

bioRxiv2023

Shigella O-specific polysaccharide functional IgA responses mediate protection against shigella infection in an endemic high-burden setting

Shigella is the second leading cause of diarrheal disease-related death in young children in low and middle income countries. The mechanism of protection against shigella infection and disease in endemic areas is uncertain. While historically LPS-specific IgG titers have been associated with protection in endemic settings, emerging deeper immune approaches have recently elucidated a protective role for IpaB-specific antibody responses in a controlled human challenge model in North American volunteers. To deeply interrogate potential correlates of immunity in areas endemic for shigellosis, here we applied a systems approach to analyze the serological response to shigella across endemic and non-endemic populations. Additionally, we analyzed shigella-specific antibody responses over time in the context of endemic resistance or breakthrough infections in a high shigella burden location. Individuals with endemic exposure to shigella possessed broad and functional antibody responses across both glycolipid and protein antigens compared to individuals from non-endemic regions. In high shigella burden settings, elevated levels of OSP-specific FcR binding antibodies were associated with resistance to shigellosis. OSP-specific FcR binding IgA found in resistant individuals activated bactericidal neutrophil functions including phagocytosis, degranulation and reactive oxygen species production. Moreover, IgA depletion from resistant serum significantly reduced binding of OSP-specific antibodies to FcR and antibody mediated activation of neutrophils and monocytes. Overall, our findings suggest that OSP-specific functional IgA responses contribute to protective immunity against shigella infection in high-burden settings. These findings will assist in the development and evaluation of shigella vaccines.

immunology↗

bioRxiv2022

Use of a MAIT activating ligand, 5-OP-RU, as a mucosal adjuvant in a murine model of Vibrio cholerae O1 vaccination.

BackgroundMucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in the mucosa with capacity for B cell help. We hypothesize that targeting MAIT cells, using a MAIT activating ligand as an adjuvant, could improve mucosal vaccine responses to bacterial pathogens. MethodsWe utilized murine models of Vibrio cholerae vaccination to test the adjuvant potential of the MAIT activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). We measured V. cholerae-specific antibody and antibody-secreting cell responses, and used flow cytometry to examine MAIT cell and B cell phenotype, in blood, bronchoalveolar lavage fluid (BALF), and mucosal tissues, following intranasal vaccination with live V. cholerae O1 or a V. cholerae O1 polysaccharide conjugate vaccine. ResultsWe report significant expansion of MAIT cells in the lungs of 5-OP-RU treated mice, and increases in BALF V. cholerae O-specific-polysaccharide IgG responses in our conjugate vaccine model adjuvanted with low-dose 5-OP-RU. No significant differences in humoral responses were found in our live V. cholerae vaccination model. ConclusionsUsing a murine model, we demonstrate the potential, as well as the limitations, of targeting MAIT cells to improve antibody responses to a mucosal cholera vaccine. Our study highlights the need for future research optimizing MAIT cell targeting for improving mucosal vaccines. One Sentence SummaryTargeting mucosal-associated invariant T (MAIT) cells with a mucosal adjuvant in an intranasal cholera vaccine model resulted in significant expansion of lung MAIT cells, but limited improvements in cholera-specific antibody responses.

immunology↗

bioRxiv2021

A Co-abundant Gene Analysis for Identification of Gut Microbial Strains Associated with Immune Responses to Oral Cholera Vaccine

The degree of protection conferred after receiving an oral cholera vaccine (OCV) varies based on age, prior exposure to Vibrio cholerae, and unknown factors. Recent evidence suggests that the microbiota may mediate some of the unexplained differences in oral vaccine responses. We used metagenomic sequencing of the microbiota at the time of vaccination, and then related microbial features to immune responses after OCV using a reference-independent gene-level analysis. We found that the presence of sphingolipid-producing bacteria was associated with the development of protective immune responses after OCV. We experimentally tested these results by stimulating human macrophages with Bacteroides xylanisolvens metabolites and found that sphingolipid-containing extracts increased innate immune responses to OCV antigens. Our findings demonstrate a new analytic method for translating metagenomic sequencing data into strain-specific results associated with a biological outcome, and in validating this tool, we identified that microbe-derived sphingolipids impact in vitro immune responses to OCV antigens.

microbiology↗

bioRxiv2021

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

immunology↗

bioRxiv2020

A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients

Vibrio cholerae can cause a range of symptoms, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous in V. cholerae DNA repair genes in five out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome, and in one of three patients with isolate genomes sequenced. Second, mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

microbiology↗

bioRxiv2020

Humans surviving cholera develop antibodies against Vibrio cholerae O-specific polysaccharide that inhibit pathogen motility

The mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae correlate highly with protection against cholera. V. cholerae is highly motile and possesses a flagellum sheathed in O-specific polysaccharide (OSP), and motility of V. cholerae correlates with virulence. Using high speed video microscopy, and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block V. cholerae motility at both subagglutinating and agglutinating concentrations. This anti-motility effect is reversed by pre-adsorbing sera and polyclonal antibody fractions with purified OSP; and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. F[ab] fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated crosslinking in motility inhibition. We show that OSP-specific antibodies do not directly affect V. cholerae viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used in vivo competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of V. cholerae in intestinal tissues, and that this impact is motility-dependent. Our findings suggest that the impedance of motility by antibodies targeting V. cholerae OSP contributes to protection against cholera. IMPORTANCECholera is a severe dehydrating illness of humans caused by Vibrio cholerae. V. cholerae is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and V. cholerae motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since V. cholerae is a non-invasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit V. cholerae motility and are associated with protection against challenge in a motility-dependent manner.

microbiology↗

bioRxiv2020

Predicting Vibrio cholerae infection and disease severity using metagenomics in a prospective cohort study

BackgroundSusceptibility to Vibrio cholerae infection is impacted by blood group, age, and pre-existing immunity, but these factors only partially explain who becomes infected. A recent study used 16S rRNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution. MethodsTo achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera. ResultsUsing machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism also correlated with protection. ConclusionOur results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera. SUMMARYCholera infection and disease severity can be predicted using metagenomic sequencing of the gut microbiome pre-infection in a prospective cohort, and suggests potentially protective bacterial species and genes.

microbiology↗

bioRxiv2019

Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiVs immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera. Author summaryVaccines for cholera are gaining acceptance as public health tools for prevention of cholera and curtailing the spread of outbreaks. However, current killed vaccines provide minimal protection in young children, who are especially susceptible to this diarrheal disease, and do not stimulate immunity against antigens that may only be expressed by live bacteria during infection. We recently developed HaitiV, an extensively engineered live-attenuated oral cholera vaccine candidate, derived from a clinical isolate from the Haiti cholera outbreak. Here, we found that the HaitiV induces immunological correlates of protection against cholera in germ free mice and leads to protection against disease in their offspring. Protection in this model was dependent on passively acquired factors in the milk of immunized dams and not transmission or colonization of HaitiV. Coupling the immunogenicity data presented here with our previous observation that HaitiV can protect from cholera prior to the induction of adaptive immunity, suggests that HaitiV may provide both rapid-onset short-term protection from disease while eliciting stable and long-lasting immunity against cholera.

microbiology↗

bioRxiv2018

Cognate T and B cell interaction and association of Follicular helper T cells with B cell responses in Vibrio cholerae O1 infected Bangladeshi adults

Vibrio cholerae O1 can cause life threatening diarrheal disease if left untreated. A long lasting immune response, producing 3-5 years of protection from subsequent, symptomatic disease following natural infection, is mediated by B cell mediated humoral immunity. T cells can play critical roles in inducing such immunity. However, the mechanism of T cell dependent B cell maturation and whether a key sub-population of T cells are involved is not well established in cholera. We hypothesized that a specific population of T cells, follicular helper T (Tfh) cells, are involved in B cell maturation following cholera; we used flow cytometry, culture and colorimetric assays to address this question. We found that V. cholerae infection induces significant increase in circulating Tfh cells expressing B cell maturation associated protein CD40L early in disease. The increased Tfh cells expressing CD40L recognize cholera toxin most prominently, with lessened responses to two antigens tested, V. cholerae membrane preparation (MP) and Vibrio cholerae cytolysin (VCC). We further showed that early induction of Tfh cells and CD40L was associated with later memory B cell responses to same antigens. Lastly, we demonstrated in vitro that Tfh cells isolated after cholera can stimulate class switching of cocultured, isolated B cells from patients with cholera, leading to production of the more durable IgG antibody isotype. These studies were conducted on circulating Tfh cells; future studies will be directed at examining role of Tfh cells during cholera directly in the gut mucosa of biopsied samples, at the single cell level if feasible.

immunology↗

bioRxiv2017

Vibrio cholerae genomic diversity within and between patients

Cholera is a severe, waterborne diarrheal disease caused by toxin-producing strains of the bacterium Vibrio cholerae. Comparative genomics has revealed \"waves\" of cholera transmission and evolution, in which clones are successively replaced over decades and centuries. However, the extent of V. cholerae genetic diversity within an epidemic or even within an individual patient is poorly understood. Here, we characterized V. cholerae genomic diversity at a micro-epidemiological level within and between individual patients from Bangladesh and Haiti. To capture within-patient diversity, we isolated multiple (8 to 20) V. cholerae colonies from each of eight patients, sequenced their genomes and identified point mutations and gene gain/loss events. We found limited but detectable diversity at the level of point mutations within hosts (zero to three single nucleotide variants within each patient), and comparatively higher gene content variation within hosts (at least one gain/loss event per patient, and up to 103 events in one patient). Much of the gene content variation appeared to be due to gain and loss of phage and plasmids within the V. cholerae population, with occasional exchanges between V. cholerae and other members of the gut microbiota. We also show that certain intra-host variants have phenotypic consequences. For example, the acquisition of a Bacteroides plasmid and nonsynonymous mutations in a sensor histidine kinase gene both reduced biofilm formation, an important trait for environmental survival. Together, our results show that V. cholerae is measurably evolving within patients, with possible implications for disease outcomes and transmission dynamics.\n\nAuthor SummaryVibrio cholerae is the etiological agent of cholera, a severe diarrheal disease endemic to Bangladesh and responsible for global outbreaks, including one ongoing in Haiti. Certain bacterial pathogens can evolve and diversify within the human host, often altering virulence and antibiotic resistance. However, most examples of within-host evolution have come from chronic infections, in which the pathogen has sufficient time to mutate and diversify, and little attention has been paid to more acute infections such as the one caused by V. cholerae. The goal of this study was to measure the extent of within-host evolution of V. cholerae within individual infected patients. By sequencing multiple bacterial isolates_from each of eight patients from Bangladesh and Haiti, we found that cholera patients can harbor a diverse population of V. cholerae. As expected for an acute infection, this diversity is limited, ranging from zero to three point mutations (single nucleotide variants) per patient. However, gene gain/loss events are more prevalent than point mutations, occurring in every single patient, and sometimes involving the transfer of dozens of genes on plasmids. Even if rare, point mutations and gene gain/loss events may be maintained by natural selection, and can alter clinically-and environmentally-relevant phenotypes such as biofilm formation. Therefore, within-patient evolution has the potential to impact clinical and epidemiological outcomes. Together, our results demonstrate that within-patient evolution may be a general feature of both acute and chronic infections, and that gene gain/loss may be an important but under-appreciated feature of within-host evolution.

microbiology↗