Membership and behavior of ultra-low-diversity pathogen communities present in the gut of humans during prolonged critical illness.

UnlabelledWe analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a "commensal lifestyle." However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style.ImportanceDuring critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis

Prevention of siderophore- mediated gut-derived sepsis due to P. aeruginosa can be achieved without iron provision by maintaining local phosphate abundance: role of pH

<p>Abstract</p> <p>Background</p> <p>During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal <it>P. aeruginosa </it>to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation.</p> <p>Results</p> <p>In this study we examined the role of pH in the protective effect of [Pi] supplementation as it has been shown to be increased in the distal gut following surgical injury. Surgically injured mice drinking 25 mM [Pi] at pH 7.5 and intestinally inoculated with <it>P. aeruginosa </it>had increased mortality compared to mice drinking 25 mM [Pi] at pH 6.0 (p < 0.05). This finding was confirmed in <it>C. elegans</it>. Transcriptional analysis of <it>P. aeruginosa </it>demonstrated enhanced expression of various genes involved in media alkalization at pH 6.0 and a global increase in the expression of all iron-related genes at pH 7.5. Maintaining the pH at 6.0 via phosphate supplementation led to significant attenuation of iron-related genes as demonstrated by microarray and confirmed by QRT-PCR analyses.</p> <p>Conclusion</p> <p>Taken together, these data demonstrate that increase in pH in distal intestine of physiologically stressed host colonized by <it>P. aeruginosa </it>can lead to the expression of siderophore-related virulence in bacteria that can be prevented without providing iron by maintaining local phosphate abundance at pH 6.0. This finding is particularly important as provision of exogenous iron has been shown to have untoward effects when administered to critically ill and septic patients. Given that phosphate, pH, and iron are near universal cues that dictate the virulence status of a broad range of microorganisms relevant to serious gut origin infection and sepsis in critically ill patients, the maintenance of phosphate and pH at appropriate physiologic levels to prevent virulence activation in a site specific manner can be considered as a novel anti-infective therapy in at risk patients.</p

Structure–Function Aspects of PstS in Multi-Drug–Resistant Pseudomonas aeruginosa

The increasing prevalence of multi-drug–resistant (MDR) strains of Pseudomonas aeruginosa among critically ill humans is of significant concern. In the current study, we show that MDR clinical isolates of P. aeruginosa representing three distinct genotypes that display high virulence against intestinal epithelial cells, form novel appendage-like structures on their cell surfaces. These appendages contain PstS, an extracellular phosphate binding protein. Using anti-PstS antibodies, we determined that the PstS-rich appendages in MDR strains are involved in adherence to and disruption of the integrity of cultured intestinal epithelial cell monolayers. The outer surface–expressed PstS protein was also identified to be present in P. aeruginosa MPAO1, although to a lesser degree, and its role in conferring an adhesive and barrier disruptive phenotype against intestinal epithelial cells was confirmed using an isogenic ΔPstS mutant. Formation of the PstS rich appendages was induced during phosphate limitation and completely suppressed in phosphate-rich media. Injection of MDR strains directly into the intestinal tract of surgically injured mice, a known model of phosphate limitation, caused high mortality rates (60%–100%). Repletion of intestinal phosphate in this model completely prevented mortality. Finally, significantly less outer surface PstS was observed in the MPAO1 mutant ΔHxcR thus establishing a role for the alternative type II secretion system Hxc in outer surface PstS expression. Gene expression analysis performed by RT-PCR confirmed this finding and further demonstrated abundant expression of pstS analogous to pa5369, pstS analogous to pa0688/pa14–55410, and hxcX in MDR strains. Taken together, these studies provide evidence that outer surface PstS expression confers a highly virulent phenotype of MDR isolates against the intestinal epithelium that alters their adhesive and barrier disrupting properties against the intestinal epithelium

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression

Mice Fed an Obesogenic Western Diet, Administered Antibiotics, and Subjected to a Sterile Surgical Procedure Develop Lethal Septicemia with Multidrug-Resistant Pathobionts

Despite antibiotics and sterile technique, postoperative infections remain a real and present danger to patients. Recent estimates suggest that 50% of the pathogens associated with postoperative infections have become resistant to the standard antibiotics used for prophylaxis. Risk factors identified in such cases include obesity and antibiotic exposure. To study the combined effect of obesity and antibiotic exposure on postoperative infection, mice were allowed to gain weight on an obesogenic Western-type diet (WD), administered antibiotics and then subjected to an otherwise recoverable sterile surgical injury (30% hepatectomy). The feeding of a WD alone resulted in a major imbalance of the cecal microbiota characterized by a decrease in diversity, loss of Bacteroidetes, a bloom in Proteobacteria, and the emergence of antibiotic-resistant organisms among the cecal microbiota. When WD-fed mice were administered antibiotics and subjected to 30% liver resection, lethal sepsis, characterized by multiple-organ damage, developed. Notable was the emergence and systemic dissemination of multidrug-resistant (MDR) pathobionts, including carbapenem-resistant, extended-spectrum β-lactamase-producing Serratia marcescens, which expressed a virulent and immunosuppressive phenotype. Analysis of the distribution of exact sequence variants belonging to the genus Serratia suggested that these strains originated from the cecal mucosa. No mortality or MDR pathogens were observed in identically treated mice fed a standard chow diet. Taken together, these results suggest that consumption of a Western diet and exposure to certain antibiotics may predispose to life-threating postoperative infection associated with MDR organisms present among the gut microbiota. IMPORTANCE Obesity remains a prevalent and independent risk factor for life-threatening infection following major surgery. Here, we demonstrate that when mice are fed an obesogenic Western diet (WD), they become susceptible to lethal sepsis with multiple organ damage after exposure to antibiotics and an otherwise-recoverable surgical injury. Analysis of the gut microbiota in this model demonstrates that WD alone leads to loss of Bacteroidetes, a bloom of Proteobacteria, and evidence of antibiotic resistance development even before antibiotics are administered. After antibiotics and surgery, lethal sepsis with organ damage developed in in mice fed a WD with the appearance of multidrug-resistant pathogens in the liver, spleen, and blood. The importance of these findings lies in exposing how the selective pressures of diet, antibiotic exposure, and surgical injury can converge on the microbiome, resulting in lethal sepsis and organ damage without the introduction of an exogenous pathogen

Pseudomonas aeruginosa Overrides the Virulence Inducing Effect of Opioids When It Senses an Abundance of Phosphate

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies

Fecal microbiota transplant rescues mice from sepsis due to multi-drug resistant healthcare pathogens by restoring systemic immunity

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression

Candida albicans Isolates from the Gut of Critically Ill Patients Respond to Phosphate Limitation by Expressing Filaments and a Lethal Phenotype

Candida albicans is an opportunistic pathogen that proliferates in the intestinal tract of critically ill patients where it continues to be a major cause of infectious-related mortality. The precise cues that shift intestinal C. albicans from its ubiquitous indolent colonizing yeast form to an invasive and lethal filamentous form remain unknown. We have previously shown that severe phosphate depletion develops in the intestinal tract during extreme physiologic stress and plays a major role in shifting intestinal Pseudomonas aeruginosa to express a lethal phenotype via conserved phosphosensory-phosphoregulatory systems. Here we studied whether phosphate dependent virulence expression could be similarly demonstrated for C. albicans. C. albicans isolates from the stool of critically ill patients and laboratory prototype strains (SC5314, BWP17, SN152) were evaluated for morphotype transformation and lethality against C. elegans and mice during exposure to phosphate limitation. Isolates ICU1 and ICU12 were able to filament and kill C. elegans in a phosphate dependent manner. In a mouse model of intestinal phosphate depletion (30% hepatectomy), direct intestinal inoculation of C. albicans caused mortality that was prevented by oral phosphate supplementation. Prototype strains displayed limited responses to phosphate limitation; however, the pho4Δ mutant displayed extensive filamentation during low phosphate conditions compared to its isogenic parent strain SN152, suggesting that mutation in the transcriptional factor Pho4p may sensitize C. albicans to phosphate limitation. Extensive filamentation was also observed in strain ICU12 suggesting that this strain is also sensitized to phosphate limitation. Analysis of the sequence of PHO4 in strain ICU12, its transcriptional response to phosphate limitation, and phosphatase assays confirmed that ICU12 demonstrates a profound response to phosphate limitation. The emergence of strains of C. albicans with marked responsiveness to phosphate limitation may represent a fitness adaptation to the complex and nutrient scarce environment typical of the gut of a critically ill patient