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

Abstract

We 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.

Importance: During 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.

FIG 1

The taxonomic composition of the gut microbiome at the phylum level determined by 16S rRNA analysis of stool samples collected from healthy volunteers (A), ICU patients dying with signs of severe sepsis (as indicated by black circles on the time line) (B), and ICU patients who had recovered (as indicated by green circles on the time line) (C). Dates of stool collection are displayed in numbered quadrants.

FIG 2

Chao1 diversity index of bacterial composition in fecal samples. Chao1 index values for stool samples of healthy volunteers (black circles) and ICU patients (colored circles) are ordered as follows (left to right): 1 to 5, healthy volunteers; 6 and 7, ICU1-1 and ICU1-2; 8 to 11, ICU2-1 to ICU2-4; 12 to 15, ICU3-1 to ICU3-4; 16 to 19, ICU4-1 to ICU4-4; 20, ICU5-1; 21 and 22, ICU6-1 and ICU6-2; 23, ICU8-1; 24, ICU9-1; 25, ICU10-1; 26 to 30, ICU11-1 to ICU11-5; 31, ICU12-1; 32, ICU13-1; 33, ICU14-1; and 34 and 35, ICU15-1 and ICU15-2. The horizontal dashed lines corresponding to Chao1 index values <50 and <10 represent arbitrary cutoffs. For the samples in this study, 14 had Chao1 index values of <10, 12 had Chao1 index values of 10 to 15, and 9 had Chao1 index values of >50. Mean Chao1 index value, 33; median, 25; range, 1.46 to 100.24.

FIG 3

Taxonomic and culturing composition of 2-member pathogen communities in correlation to antibiotic regimen, antibiotic resistance of stool isolates, and microbial data of cultured species from nonintestinal samples. (A) Patient ICU1. (B) Patient ICU6. (C) Patient ICU11. (D) Patient ICU4. Cultured species from sources other than the gut refer to species cultured from extraintestinal sources such as blood, urine, and lung. The arrows indicate the time points along the course of antibiotic exposure at which these organisms were cultured. Vanc, vancomycin; cipro, ciprofloxacin; difluca, fluconazole (Diflucan); AmphoB, amphotericin B; flagyl, metronidazole (Flagyl); Pip, piperacillin; TMP/SUX, trimethoprim/sulfamethoxazole; Quinupristin/dalf, quinupristin/dalfopristin; Staph, Staphylococcus.

FIG 3

Taxonomic and culturing composition of 2-member pathogen communities in correlation to antibiotic regimen, antibiotic resistance of stool isolates, and microbial data of cultured species from nonintestinal samples. (A) Patient ICU1. (B) Patient ICU6. (C) Patient ICU11. (D) Patient ICU4. Cultured species from sources other than the gut refer to species cultured from extraintestinal sources such as blood, urine, and lung. The arrows indicate the time points along the course of antibiotic exposure at which these organisms were cultured. Vanc, vancomycin; cipro, ciprofloxacin; difluca, fluconazole (Diflucan); AmphoB, amphotericin B; flagyl, metronidazole (Flagyl); Pip, piperacillin; TMP/SUX, trimethoprim/sulfamethoxazole; Quinupristin/dalf, quinupristin/dalfopristin; Staph, Staphylococcus.

FIG 3

Taxonomic and culturing composition of 2-member pathogen communities in correlation to antibiotic regimen, antibiotic resistance of stool isolates, and microbial data of cultured species from nonintestinal samples. (A) Patient ICU1. (B) Patient ICU6. (C) Patient ICU11. (D) Patient ICU4. Cultured species from sources other than the gut refer to species cultured from extraintestinal sources such as blood, urine, and lung. The arrows indicate the time points along the course of antibiotic exposure at which these organisms were cultured. Vanc, vancomycin; cipro, ciprofloxacin; difluca, fluconazole (Diflucan); AmphoB, amphotericin B; flagyl, metronidazole (Flagyl); Pip, piperacillin; TMP/SUX, trimethoprim/sulfamethoxazole; Quinupristin/dalf, quinupristin/dalfopristin; Staph, Staphylococcus.

FIG 3

Taxonomic and culturing composition of 2-member pathogen communities in correlation to antibiotic regimen, antibiotic resistance of stool isolates, and microbial data of cultured species from nonintestinal samples. (A) Patient ICU1. (B) Patient ICU6. (C) Patient ICU11. (D) Patient ICU4. Cultured species from sources other than the gut refer to species cultured from extraintestinal sources such as blood, urine, and lung. The arrows indicate the time points along the course of antibiotic exposure at which these organisms were cultured. Vanc, vancomycin; cipro, ciprofloxacin; difluca, fluconazole (Diflucan); AmphoB, amphotericin B; flagyl, metronidazole (Flagyl); Pip, piperacillin; TMP/SUX, trimethoprim/sulfamethoxazole; Quinupristin/dalf, quinupristin/dalfopristin; Staph, Staphylococcus.

FIG 4

The commensal behavior of the 2-member pathogen communities. Data represent Kaplan-Meier survival curves of C. elegans fed on 2-member pathogen communities and their individual members (n = 10/plate, 3 plates/group). Data represent the cumulative results of 2 experiments.

FIG 5

Replacement of bacterial comember switches the commensal behavior to pathogenic. (A) Kaplan-Meier survival curves of C. elegans fed on ICU1-1 and ICU1-2 pathogen communities (n = 10/plate, 3 plates/group). Data represent the cumulative results of 2 experiments. (B and C) Scanning electron microscopy images of ICU1-1 (B) and ICU1-2 (C) grown for 20 h in 0.1× TY.

FIG 6

Opioids can break the commensalism of 2-member pathogen communities. Kaplan-Meier survival curves of C. elegans fed on ICU6-2 (A), ICU11-2 (B), ICU11-4 (C), and ICU4-4 (D) 2-member communities in the presence of an opioid (50 µM U-50,488) (n = 10/plate, 3 plates/group; P < 0.01). Data represent the cumulative results of 2 experiments. Exposure to opioids broke the commensalism in the ICU6-2 and ICU11-2 communities.

FIG 7

Pi-PEG15-20 stabilizes the commensal behavior of 2-member communities. Kaplan-Meier survival curves of C. elegans fed on ICU6-2 (left panel) and ICU11-2 (right panel) 2-member communities in the presence of an opioid (50 µM U-50,488) with or without 10% Pi-PEG15-20 (n = 10/plate, 3 plates/group; P < 0.01). Data represent cumulative results of 2 experiments.