Matthew L. Workentine

Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.

Metabolomic Investigation of the Bacterial Response to a Metal Challenge

ABSTRACT Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a “poised” physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.

Differences in Metabolism between the Biofilm and Planktonic Response to Metal Stress

Bacterial biofilms are known to withstand the effects of toxic metals better than planktonic cultures of the same species. This phenomenon has been attributed to many features of the sessile lifestyle not present in free-swimming populations, but the contribution of intracellular metabolism has not been previously examined. Here, we use a combined GC-MS and (1)H NMR metabolomic approach to quantify whole-cell metabolism in biofilm and planktonic cultures of the multimetal resistant bacterium Pseudomonas fluorescens exposed to copper ions. Metabolic changes in response to metal exposure were found to be significantly different in biofilms compared to planktonic cultures. Planktonic metabolism indicated an oxidative stress response that was characterized by changes to the TCA cycle, glycolysis, pyruvate and nicotinate and niacotinamide metabolism. Similar metabolic changes were not observed in biofilms, which were instead dominated by shifts in exopolysaccharide related metabolism suggesting that metal stress in biofilms induces a protective response rather than the reactive changes observed for the planktonic cells. From these results, we conclude that differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance. An altered metabolic response to metal toxicity represents a novel addition to a growing list of biofilm-specific mechanisms to resist environmental stress.

Hydrogen Sulfide Protects from Colitis and Restores Intestinal Microbiota Biofilm and Mucus Production

Background:Microbiota dysbiosis and impaired barrier function are among the most prominent features of inflammatory bowel disease. In the gastrointestinal tract, hydrogen sulfide (H2S) is an important regulator of mucosal homeostasis. We hypothesized that H2S promotes resolution of colonic inflammation through actions on microbiota biofilm and the mucus barrier. Methods:We used mice genetically deficient for a key enzyme for H2S production (cystathionine &ggr;-lyase) and pharmacologically inhibited that enzyme during colitis in wild-type mice. We tested the effects of administering an H2S donor (diallyl disulfide) to rodents during hapten-induced colitis. Colonic microbiota biofilm was visualized by fluorescent in situ hybridization, and mucus granules were quantified with periodic acid–alcian blue staining. We exposed human microbiota biofilms and planktonic bacteria to H2S donors ex vivo to determine changes in their growth, viability, and biomass. Results:Intestinal microbiota formed linear biofilms in the colon of healthy rodents. During colitis, microbiota biofilms were fragmented and mucus granule production decreased. Endogenous production of H2S had beneficial effects on establishment of microbiota biofilms and colonic mucus production. Therapeutic delivery of H2S into the colon reduced inflammation, restored the microbiota biofilm, and increased the production of mucus granules. In ex vivo human microbiota, H2S not only promoted biofilm formation but also reduced growth of planktonic bacteria. Conclusions:Our results suggest that H2S donors could be used therapeutically during colitis, facilitating correction of microbiota biofilm dysbiosis and mucus layer reconstitution.

The GacS-GacA two-component regulatory system of Pseudomonas fluorescens: a bacterial two-hybrid analysis.

The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many other aspects of bacterial physiology. Although it is known that the sensor kinases RetS and LadS feed into the GacS/GacA system, the mechanism through which this occurs is unknown, as are the protein-protein interactions in this system. To characterize and define these interactions, we utilized a bacterial two-hybrid system to study the interactions of GacS and GacA from Pseudomonas fluorescens CHA0. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in vivo was investigated. We found that the entire GacA molecule is required for GacA to interact with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. In addition, homologs of Pseudomonas aeruginosa hybrid sensor kinases, RetS and LadS, were identified in P. fluorescens, and shown to interact with GacS, but not GacA.

Phenotypic and metabolic profiling of colony morphology variants evolved from Pseudomonas fluorescens biofilms

Colony morphology variants isolated from natural and laboratory-grown biofilms represent subpopulations of biofilm cells that may be important for multiple aspects of the sessile lifestyle, from surface colonization to stress resistance. There are many genetic and environmental factors that determine the frequency at which colony morphology variants are recovered from biofilms. One of these factors involves an increased selection for variants in biofilms of Pseudomonas species bearing inactivating mutations in the global activator of cyanide biosynthesis/regulator of secondary metabolism (gac/rsm) signal transduction pathway. Here we characterize two distinct colony morphology variants isolated from biofilms of Pseudomonas fluorescens missing the gacS sensor kinase. These variants produced more biofilm cell mass, and in one case, this was likely due to overproduction of the exopolysaccharide cellulose. Nuclear magnetic resonance (NMR) metabolomics revealed distinct metabolic changes for each of the two phenotypic variants, and these changes involved amino acids and metabolites produced through glutathione biochemistry. Some of these metabolites are hypothesized to play a role in redox and metal homeostasis, and corresponding to this, we show that biofilm populations grown from each of these variants had a different ability to survive when exposed to toxic doses of metal ions. These data suggest that colony morphology variants that evolve during growth of P. fluorescens as a biofilm may have distinct metabolic capacities that contribute to their individual abilities to withstand environmental stress.

Twenty-Five-Year Outbreak of Pseudomonas aeruginosa Infecting Individuals with Cystic Fibrosis: Identification of the Prairie Epidemic Strain

ABSTRACT Transmissible strains of Pseudomonas aeruginosa have been described for cystic fibrosis (CF) and may be associated with a worse prognosis. Using a comprehensive strain biobank spanning 3 decades, we sought to determine the prevalence and stability of chronic P. aeruginosa infection in an adult population. P. aeruginosa isolates from sputum samples collected at initial enrollment in our adult clinic and at the most recent clinic visit were examined by a combination of pulsed-field gel electrophoresis and multilocus sequence typing and compared against a collection of established transmissible and local non-CF bronchiectasis (nCFB) isolates. A total of 372 isolates from 107 patients, spanning 674 patient-years, including 66 patients with matched isolates from initial and final encounters, were screened. A novel clone with increased antibacterial resistance, termed the prairie epidemic strain (PES), was found in 29% (31/107 patients) of chronically infected patients referred from multiple prairie-based CF centers. This isolate was not found in those diagnosed with CF as adults or in a control population with nCFB. While 90% (60/66 patients) of patients had stable infection over a mean of 10.8 years, five patients experienced strain displacement of unique isolates, with PES occurring within 2 years of transitioning to adult care. PES has been present in our cohort since at least 1987, is unique to CF, generally establishes chronic infection during childhood, and has been found in patients at the time of transition of patients from multiple prairie-based CF clinics, suggesting broad endemicity. Studies are under way to evaluate the clinical implications of PES infection.

Hydrogen sulphide protects against NSAID-enteropathy through modulation of bile and the microbiota.

Hydrogen sulphide is an important mediator of gastrointestinal mucosal defence. The use of non‐steroidal anti‐inflammatory drugs (NSAIDs) is significantly limited by their toxicity in the gastrointestinal tract. Particularly concerning is the lack of effective preventative or curative treatments for NSAID‐induced intestinal damage and bleeding. We evaluated the ability of a hydrogen sulphide donor to protect against NSAID‐induced enteropathy.

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms

Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.

Evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40 days after arrival at a feedlot

Bovine respiratory disease complex (BRDc) is a major cause of morbidity and mortality in beef cattle. There is recent evidence suggesting that the nasopharyngeal microbiota has a key role in respiratory health and disease susceptibility in cattle. However, there is a paucity of knowledge regarding evolution of the nasopharyngeal microbiota when cattle are most likely to develop BRDc (i.e., from weaning to 40days after arrival at a feedlot). The objective was to describe the evolution of the nasopharyngeal microbiota of beef cattle from weaning to 40days after arrival at a feedlot. Deep nasal swabs (DNS) from 30 Angus-cross steers were collected at weaning, on arrival at a feedlot, and at day 40 after arrival. The DNA was extracted from DNS and the hypervariable region V3 of the 16S rRNA gene was amplified and sequenced (Illumina MiSeq platform). Nasopharyngeal microbiota underwent a profound evolution from weaning to arrival at the feedlot and from arrival to day 40, with the abundance of 92 Operational Taxonomic Units (OTUs) significantly changing over time. Mycoplasma (M. dispar and M. bovirhinis) was the most abundant genus in the nasopharynx, accounting for 53% of the total bacterial population. Because an evolving bacterial community may be less capable of resisting colonization by pathogenic bacteria, the instability of the nasopharyngeal microbiota documented in this study might explain why cattle are most likely to be affected with BRDc during the first weeks after weaning and arrival at a feedlot.