Biology subjects

Pride, D. T.

Publications and source records attributed to Pride, D. T..

11 recordsLinked to original sources

bioRxiv2025

Enterocloster citroniae and related gut microbiome species modulate Vibrio cholerae biofilm formation through the production of bioactive small molecules

Cholera is a diarrheal disease that affects millions of people globally. Although the causative agent, Vibrio cholerae, has been extensively studied in isolation, investigation of its interactions with the gut microbiota started relatively recently. We and others previously showed that microbiota-derived metabolites significantly influence V. cholerae behavior. By investigating how an organic extract of human feces affects V. cholerae gene expression, we showed that gut metabolites strongly suppress swimming motility, a trait important for host colonization. Interestingly, extracts of pure cultures of a gut commensal, Enterocloster citroniae, recapitulated this inhibition. Here, we present a comprehensive examination of the effect of small molecules produced by E. citroniae and related species on V. cholerae behavior. We show that E. citroniae small molecules inhibit motility by various V. cholerae strains, and that several phylogenetically related species produce this activity, although the magnitude of the effect varies between strains. Using biofilm formation assays in static and flow conditions, we show that V. cholerae strongly induces biofilm formation in response to E. citroniae metabolites. Transcriptome and reporter analyses showed that several genes involved in synthesis of an extracellular polysaccharide are induced by E. citroniae metabolites. Lastly, we show that V. cholerae interactions with host cells are also modulated by this commensal. These findings advance our understanding of microbiome-pathogen interactions and how commensal bacteria influence V. cholerae virulence through the production of small molecules. In the future, this knowledge may be used to design novel microbiome-based therapeutic approaches to combat cholera and other infections. ImportanceThe human gut is home to a dense and rich community of microbes termed microbiota. This community has critical functions for host health, including protection against enteric pathogens. Despite this important role, we have only recently scratched the surface of the interactions that occur between members of the microbiota and pathogenic invaders. Cholerae is a disease that still causes significant morbidity and mortality worldwide. Studying how the causative agent, Vibrio cholerae, interacts with the microbiota will have implications not only for our understanding of this important microbial community, but may also lead to the development of new therapeutic strategies against cholera and potentially other infectious diseases.

microbiology↗

bioRxiv2024

Experimental Phage Evolution Results in Expanded Host Ranges Against MDR and XDR Klebsiella pneumoniae Isolates

Resistance to antibiotics is approaching crisis levels for organisms such as the ESKAPEE pathogens (includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) that often are acquired in hospitals. These organisms sometimes have acquired plasmids that confer resistance to most if not all beta-lactam antibiotics such as those produced by Carbapenem Resistant Enterobacterales (CREs). We have been developing alternative means for dealing with antibiotic resistant microbes that cause infections in humans by developing viruses (bacteriophages) that attack and kill them. We have been working with one of the ESKAPEE pathogens, K. pneumoniae, that has one of the highest propensities for antimicrobial resistance, to develop phages that target and kill it. We identified a number of phages that have lytic capacity against only a few clinical isolates, and through experimental evolution over the course of 30 days, were able to vastly expand the host ranges of these phages to kill a broader range of clinical K. pneumoniae isolates including MDR (multi-drug resistant) and XDR (extensively-drug resistant) isolates. Most interestingly, they were capable of inhibiting growth of clinical isolates both on solid and in liquid medium over extended periods. That we were able to extend the host ranges of multiple naive MDR and XDR K. pneumoniae through experimental phage evolution suggests that such a technique may be applicable to other antibiotic-resistant organisms to help stem the tide of antibiotic resistance and offer further options for medical treatments. ImportanceBacterial pathogens are becoming greater threats given the rise in antibiotic resistance, where traditional therapies may no longer work to cure some infections. Chief amongst these multidrug resistant infections (MDR) and extensively drug-resistant infections (XDR) is Klebsiella pneumoniae, which is known to sometimes harbor genetic elements that render it incredibly difficult to treat with conventional antibiotics. Treatments like bacteriophages have not had much success against such pathogens because resistance to the phages used often develops rapidly. We adapted a co-evolutionary technique to develop K. pneumoniae phages to be highly active longitudinally against K. pneumoniae clinical isolates. In as few as 30 days, we were able to vastly expand the host ranges of K. pneumoniae phages against MDR and XDR clinical isolates and that maintain their infectivity over clinically relevant time periods. By adapting these established techniques to clinical MDR and XDR K. pneumoniae isolates, we believe we can establish similar techniques for expanding phage host ranges against most antibiotic-resistant bacteria. As such, phages can be viable alternatives to antibiotics when antibiotic resistance exists in hospitals and communities.

microbiology↗

bioRxiv2024

Identification of a large cohort of Enterobacter jumbo phages with broad host ranges across pathogenic Gammaproteobacteria

ESKAPE pathogens cause most hospital-acquired infections globally and often carry antibiotic resistance. Many of them have been the target of bacteriophage therapies. Enterobacter is an ESKAPE pathogen but is less frequently a target for phage therapy due to a relative lack of available phages. We isolated eight jumbo phages with genomes ranging from 223 to 366 kbp targeting Enterobacter spp. and found that they belonged to separate phage clades. Six of them formed nucleus-like structures confirmed by DAPI-staining, and were phylogenetically related to Chimalliviridae. Two jumbo phages did not form nucleus-like structures and did not cluster with Chimalliviridae. Although these jumbo phages were found on Enterobacter, many were closely related to phages with non-Enterobacter hosts. To test whether these phages may have had expanded host ranges, we examined 14 pathogenic Gammaproteobacteria and found that these phages were capable of creating plaques on 8 of them. These species included Escherichia coli, Klebsiella aerogenes, Serratia marcescens, Salmonella spp., Shigella spp., Providencia spp., Citrobacter spp., and Cronobacter sakazakii. We verified that there was phage amplification in these microbes rather than lysis from without by performing qPCR to confirm DNA replication in each species. Phages typically have narrow host ranges, a benefit for microbiome-sparing compared to antibiotics. However, the broad host ranges of these Gammaproteobacteria jumbo phages suggests that not all phages have the same risk/benefit ratios. While this broad range could aid their development as antibiotic alternatives, further study is needed to assess potential microbiome disruption. SignificanceWith the growing threat of antibiotic resistant bacteria, alternative treatments like bacteriophages have emerged. Bacteriophages typically have narrow host ranges, a disadvantage compared to antibiotics. We discovered eight jumbo phages that kill antibiotic resistant Enterobacter, but were diverse phylogenetically. Six of them formed nucleus-like structures and were members of the Chimalliviridae family and the other two did not form nucleus-like structures. We confirmed that each phage had broad host ranges capable of lysing at least eight different human Gammaproteobacteria including pathogens such as E. coli, K. aerogenes, and S. marcescens. By identifying broad host range jumbo phages that attack pathogens, we may have identified phages with spectrums of activity more similar to antibiotics than have been traditionally attributed to phages.

microbiology↗

bioRxiv2024

Enhanced Suppression of Stenotrophomonas maltophilia by a Three-Phage Cocktail: Genomic Insights and Kinetic Profiling

In our era of rising antibiotic resistance, Stenotrophomonas maltophilia (STM) is an understudied, gram-negative, aerobic bacterium widespread in the environment and increasingly causing opportunistic infections. Treating STM infections remains difficult, leading to an increase in disease severity and higher hospitalization rates in people with Cystic Fibrosis (pwCF), cancer, and other immunocompromised health conditions. The lack of effective antibiotics has led to renewed interest in phage therapy; however, there is a need for well-characterized phages. In response to an oncology patient with a respiratory infection, we collected 18 phages from Southern California wastewater influent that exhibit different plaque morphology against STM host strain B28B, cultivated from a blood sample. Here, we characterize the genomes and life cycle kinetics of our STM phage collection. We hypothesize that genetically distinct phages give rise to unique lytic life cycles that can enhance bacterial killing when combined into a phage cocktail compared to the individual phages alone. We identified three genetically distinct clusters of phages, and a representative from each group was screened for potential therapeutic use and investigated for infection kinetics. The results demonstrated that the three-phage cocktail significantly suppressed bacterial growth compared to individual phages when observed for 48 hours. We also assessed the lytic impacts of our three-phage cocktail against a collection of 46 STM strains to determine if a multi-phage cocktail can expand the host range of individual phages. Our phages remained strain-specific and infect >50% of tested strains. The multi-phage cocktail maintains bacterial growth suppression and prevents the emergence of phage-resistant strains throughout our 40-hour assay. These findings suggest specialized phage cocktails may be an effective avenue of treatment for recalcitrant STM infections resistant to current antibiotics. IMPORTANCEPhage therapy could provide a vital strategy in the fight against antimicrobial resistance (AMR) bacterial infections; however, significant knowledge gaps remain. This study investigates phage cocktail development for the opportunistic pathogen Stenotrophomonas maltophilia (STM). Our findings contribute novel phages, their lytic characteristics, and limitations when exposed to an array of clinically relevant STM strains. Eighteen bacteriophages were isolated from wastewater influent from Escondido, California, and subjected to genomic analysis. We investigated genetically distinct phages to establish their infection kinetics and developed them into a phage cocktail. Our findings suggest that a genetically distinct STM phage cocktail provides an effective strategy for bacterial suppression of host strain B28B and five other clinically relevant STM strains. Phage therapy against STM remains poorly understood, as only 39 phages have been previously isolated. Future research into the underlying mechanism of how phage cocktails overwhelm the host bacteria will provide essential information that could aid in optimizing phage applications and impact alternative treatment options.

microbiology↗

bioRxiv2024

In-vitro resensitization of multidrug resistant clinical isolates of Enterococcus faecium and faecalis through phage-antibiotic synergy

Bacteriophages are an increasingly attractive option for the treatment of antibiotic resistant infections, but their efficacy is difficult to discern due to confounding effects of antibiotics. Phages are generally delivered in conjunction with antibiotics, and thus, when patients improve its unclear whether the phages, antibiotics or both are responsible. This question is particularly relevant for enterococcus infections, as limited data suggest phages might restore antibiotic efficacy against resistant strains. Enterococci can develop high-level resistance to vancomycin, a primary treatment. We assessed clinical and laboratory isolates of Enterococcus faecium and Enterococcus faecalis to determine whether we could observe synergistic interactions between phages and antibiotics. We identified synergy between multiple phages and antibiotics including linezolid, ampicillin, and vancomycin. Notably, antibiotic susceptibility did not predict synergistic interactions with phages. Vancomycin resistant isolates (n=6) were eradicated by the vancomycin-phage combination as effectively as vancomycin susceptible isolates (n=2). Transcriptome analysis revealed significant gene expression changes under antibiotic-phage conditions, especially for linezolid and vancomycin, with upregulated genes involved in nucleotide and protein biosynthesis and downregulated stress response and prophage-related genes. While our results do not conclusively determine the etiology of the observed synergistic interactions between antibiotics and phages, they do confirm and build upon previous research that observed these synergistic interactions. Our work highlights how using phages can restore the effectiveness of vancomycin against resistant isolates. This finding provides a promising, although unexpected, strategy for moving forward with phage treatments for Vancomycin Resistant Enterococcus infections.

microbiology↗

bioRxiv2023

Pangenome comparison of Bacteroides fragilis genomospecies unveil genetic diversity and ecological insights

Bacteroides fragilis is a Gram-negative commensal bacterium commonly found in the human colon that differentiates into two genomospecies termed division I and II. We leverage a comprehensive collection of 694 B. fragilis whole genome sequences and report differential gene abundance to further support the recent proposal that divisions I and II represent separate species. In division I strains, we identify an increased abundance of genes related to complex carbohydrate degradation, colonization, and host niche occupancy, confirming the role of division I strains as gut commensals. In contrast, division II strains display an increased prevalence of plant cell wall degradation genes and exhibit a distinct geographic distribution, primarily originating from Asian countries, suggesting dietary influences. Notably, division II strains have an increased abundance of genes linked to virulence, survival in toxic conditions, and antimicrobial resistance, consistent with a higher incidence of these strains in bloodstream infections. This study provides new evidence supporting a recent proposal for classifying divisions I and II B. fragilis strains as distinct species, and our comparative genomic analysis reveals their niche-specific roles.

genomics↗

bioRxiv2023

A simple solid media assay for detection of synergy between bacteriophages and antibiotics

The emergence of antibiotic resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. While phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates, and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat Vancomycin Resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.

microbiology↗

bioRxiv2023

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice

BackgroundThe gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mices gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. ResultsWe gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. ConclusionsThis study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

microbiology↗

bioRxiv2021

Targeting Biofilm of MDR Providencia stuartii by Phages Using a Catheter Model

Providencia spp. are emerging pathogens mainly in nosocomial infections. Providencia stuartii in particular is involved in urinary tract infections and contributes significantly to the high incidence of biofilm-formation in catheterized patients. Furthermore, recent reports suggested a role for multiple drug resistant (MDR) P. stuartii in hospital-associated outbreaks which leads to excessive complications resulting in challenging treatments. Phage therapy is currently one of the most promising solutions to combat antibiotic-resistant infections. However, the number of available phages targeting Providencia spp. is extremely limited, restricting the use of phage therapy in such cases. In the present study, we describe the isolation and characterization of 17 lytic and lysogenic bacteriophages targeting clinical isolates of Providencia spp. as part of the Israeli Phage Bank (IPB). These phages, isolated from sewage samples, were evaluated for host range activity and effectively eradicated 95% of the tested bacterial strains isolated from different geographic locations and displaying a wide range of antibiotic resistance. Their lytic activity is demonstrated on agar plates, planktonic cultures, and biofilm formed in a catheter model. The results suggest that these bacteriophages can potentially be used for treatment of antibiotic-resistant Providencia spp. infections in general and of urinary tract infections in particular.

microbiology↗

bioRxiv2021

Phage Cocktails can Prevent the Evolution of Phage-Resistant Enterococcus

Antibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy is often deployed as a cocktail of phages, but there is little understanding of how to most effectively combine phages. Here we utilized a collection of 20 Enterococcus phages to test principles of phage cocktail design and determine the phenotypic effects of evolving phage resistance in Enterococcus isolates that were susceptible or resistant to antibiotics (e.g., Vancomycin Resistant Enterococcus (VRE)). We tested the ability of each phage to clear Enterococcus host cultures and prevent the emergence of phage resistant Enterococcus. We found that some phages which were ineffective individually were effective at clearing the bacterial culture when used in cocktails. To understand the dynamics within phage cocktails, we used qPCR to track which phages increased in abundance in each cocktail, and saw dynamics ranging from one dominant phage to even phage growth. Further, we isolated several phage-resistant mutants to test for altered Vancomycin sensitivity. We found that mutants tended to have no change or slightly increased resistance to Vancomycin. By demonstrating the efficacy of phage cocktails in suppressing growth of antibiotic susceptible and VRE clinical isolates when exposed to phages, this work will help to inform cocktail design for future phage therapy applications. IMPORTANCEAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy in the form of cocktails is often suggested, with similar goals as the combination therapy that has been successful in the treatment of HIV infection, but there is little understanding about how to combine phages most effectively. Here we utilized a collection of 20 Enterococcus phages to test whether several phage cocktails could prevent the host from evolving resistance to therapy and to determine whether evolving resistance to phages affected host susceptibility to antibiotics. We showed that cocktails of two or three unrelated phages often prevented the growth of phage-resistant mutants, when the same phages applied individually were not able to.

microbiology↗

bioRxiv2019

Cervicovaginal microbiome composition drives metabolic profiles in healthy pregnancy

BackgroundMicrobes and their metabolic products influence early-life immune and microbiome development, yet remain understudied during pregnancy. Vaginal microbial communities are typically dominated by one or a few well adapted microbes, which are able to survive in a narrow pH range. In comparison to other human-associated microbes, vaginal microbes are adapted to live on host-derived carbon sources, likely sourced from glycogen and mucin present in the vaginal environment. MethodsUsing 16S rRNA and ITS amplicon sequencing, we characterized the cervicovaginal microbiomes of 18 healthy women throughout the three trimesters of pregnancy. Shotgun metagenomic sequencing permitted refinement of the taxonomy established by amplicon sequencing, and identification of functional genes. Additionally, we analyzed saliva and urine metabolomes using GC-TOF and LC-MS/MS lipidomics approaches for samples from mothers and their infants through the first year of life. ResultsAmplicon sequencing revealed most women had either a simple community with one highly abundant species of Lactobacillus or a more diverse community characterized by a high abundance of Gardnerella, as has also been previously described in several independent cohorts. Integrating GC-TOF and lipidomics data with amplicon sequencing, we found metabolites that distinctly associate with particular communities. For example, cervicovaginal microbial communities dominated by Lactobacillus crispatus also have high mannitol levels, which contradicts the basic characterization of L. crispatus as a homofermentative Lactobacillus species. It may be that fluctuations in which Lactobacillus dominate a particular vaginal microbiome are dictated by the availability of host sugars, such as fructose, which is the most likely substrate being converted to mannitol. Furthermore, indole-3-lactate (ILA) was also indicative of L. crispatus specifically. ILA has immunomodulatory properties through binding the human aryl hydrocarbon receptor (AhR), which may maintain the especially low diversity of L. crispatus dominated communities. ConclusionsOverall, using a multi- omic approach, we begin to address the genetic and molecular means by which a particular vaginal microbiome becomes vulnerable to large changes in composition.

microbiology↗