Gut Microbiome Dysbiosis in Antibiotic-Treated COVID-19 Patients is Associated with Microbial Translocation and Bacteremia

Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut

3D reconstruction of a <i>T</i>. <i>muris</i> egg incubated with <i>E</i>. <i>coli</i> for 1.5 hours at 37°C from SBFSEM.

Video begins with slices through the egg and bacteria being shown. The 3D reconstruction of the larva (purple) and the polar plugs (green) is then revealed. One plug curves inward and the other plug appears rounded. Lastly, the reconstructed shell (yellow) and bacteria (blue) are shown. (MOV)</p

Bacteria display species-dependent effects on <i>T</i>. <i>muris</i> egg hatching.

(A) Representative light microscopy image of T. muris eggs induced to hatch after incubation with S. aureus at 37°C for 45 mins. White arrowhead denotes unhatched egg and black arrowhead denotes hatched egg. (B) Percent of T. muris eggs hatched after incubation in aerobic conditions with overnight cultures of E. coli and S. aureus, compared with their respective broth controls determined by light microscopy at indicated time points. Colony forming units (CFUs) of each bacterial species added to the eggs are indicated on the graphs. (C) Percent of T. muris eggs hatched at 1 hour after 37°C incubation with E. coli (~5 x 107CFU), S. aureus (~108 CFU) and E. faecalis (~5 X 107 CFU) grown overnight to saturation. (D) Percent of T. muris eggs hatched after incubation in aerobic conditions with overnight cultures of S. epidermidis, B. subtilis, E. faecalis and E. faecium compared with their respective broth controls determined by light microscopy at indicated time points. Colony forming units (CFUs) of each bacterial species added to the eggs are indicated on the graphs. (E) Percent of T. muris eggs hatched after incubation with S. aureus grown overnight in tryptic soy broth (TSB), Luria Bertani (LB) broth and Brain Heart Infusion (BHI) broth. (F) Percent of T. muris eggs hatched at 1 hour after 37°C incubation with an equal number (~108 CFU) of E. coli and E. faecalis. (G) Percent of T. muris eggs hatched after incubation with S. aureus (~3 x 108 CFU) and E. faecalis (~3 x 107 CFU) alone or together compared with broth controls. For the S. aureus + E. faecalis condition, bacterial cultures were grown separately overnight, and then were mixed the day of the experiment. (H) Percent of T. muris eggs hatched after incubation in anaerobic conditions with overnight cultures of E. coli (~5 x 107 CFU) and S. aureus (~2 x 108 CFU) compared with their respective broth controls determined by light microscopy at indicated time points. (I) Quantification of CFUs per gram of stool collected from mice monocolonized with S. aureus or E. faecalis. (J) Proportion of germ-free and S. aureus monocolonized mice that had harbored adult T. muris worms after double-dose infection. (K) Number of worms recovered from S. aureus monocolonized mice (n = 9–12 mice per group). (L) Number of worms recovered from E. faecalis monocolonized mice (n = 3 mice per group). Data points and error bars represent mean and SEM from 3 independent repeats for (B), (D), (E), (G), and (H). Dots represent a single well and bars show means and SEM from 3 independent repeats for (C) and (F). ~25 eggs per well were assed for (A)-(H). Dots represent a single mouse and bars show means and SEM from 3 independent repeats for (K) and 1 independent experiment for (L). Welch’s t test was used to compare area under the curve between each condition and its respective media control for (B), (D), and (H). Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used for (C). Ordinary one-way analysis of variance (ANOVA) test followed by a Tukey’s multiple comparisons test was used to compare AUC of hatching induced by different conditions to each other for (E) and (G). Mann Whitney test was used for (F), (K) and (L). Fisher’s exact test was used to determine whether there was a significant association between gut microbial composition of mice and the presence of adult worms in the cecum for (J).</p

Plugs on eggs from bacteria exposed samples show different stages of disintegration, related to Fig 4.

(A, B) Representative electron micrograph of a section of polar plugs (black asterisk) on eggs exposed to S. aureus (left) or E. coli (right). Images of Pole 1 (top) and Pole 2 (bottom) were collected from the same egg. Outer vitelline layer is denoted by white arrowheads and eggshell is denoted by black arrowheads. (C) Representative electron micrograph of a section of polar plug (black asterisk) on an egg exposed to S. aureus. Outer vitelline layer is denoted by the white arrowhead and point of contact between inner surface of the plug and the larva is denoted by the yellow arrowhead. (TIFF)</p

Live imaging of <i>T</i>. <i>muris</i> egg hatching.

T. muris eggs were incubated with 2 x 1010 cfu/ml of S. aureus and imaged continuously at 37°C for 45 mins. White arrows indicate eggs that hatch. Eggs were imaged using a 40x N.A. 1.3 objective. (MP4)</p

Degree of bacterial binding is directly proportional to the rate of <i>T</i>. <i>muris</i> egg hatching.

(A) Representative confocal microscopy image of T. muris eggs incubated with S. aureus GFP for 30 minutes. Regions defined as poles and sides are indicated. Scale bar represents 34μm. (B) Percent of GFP+ puncta present on the poles versus the sides of the T. muris eggs. (C) Percent of T. muris eggs hatched after incubation with 10-fold dilutions of overnight S. aureus culture ranging from approximately 105–108 CFU. (D) Percent of T. muris eggs associated with GFP+ puncta after incubation with 10-fold dilutions of overnight S. aureus-GFP culture ranging from approximately 105–108 CFU. (E) Correlation analysis comparing log10 CFUs of bacteria used and percent of T. muris eggs with GFP+ puncta present. (F) Number of GFP+ puncta bound per egg from (D). (G) Percent of GFP+ puncta present on the poles of the eggs versus the sides of the eggs from (D). (H) Percent of pole associated GFP+ puncta present on the higher bound pole versus the lower bound pole of eggs from (D). Bars and error bars show means and SEM from 3 independent repeats for (B), (D), (F),(G) and (H). Data points and error bars represent mean and SEM from 3 independent repeats for (C). ~25 eggs per well were assessed for (C). Dots represent percentage of 40 eggs that were GFP+ from a single experiment for (D) and (E) and number of GFP+ puncta found on a single egg for (F). Mann-Whitney test was used for (B). Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used for (D) and (F). Simple linear regression was performed for (E). Two-way ANOVA followed by a Sidak’s multiple comparisons test was used for (G) and (H).</p

Physical contact between the bacterial cell and egg is essential for <i>S</i>. <i>aureus</i> mediated hatching.

(A) Percent of T. muris eggs hatched after incubation with total overnight S. aureus culture, culture pellet resuspended in fresh media, culture supernatant filtered with a 0.22μm filter, or media control. (B) Experimental approach for determining whether a soluble hatching inducing factor is produced by S. aureus in response to exposure to eggs. Filtered supernatant from S. aureus grown with or without T. muris eggs for 4 h were transferred to a dish containing T. muris eggs. The ability of the supernatant to mediate egg hatching was evaluated over 4 hours. (C) Percent of T. muris eggs hatched after incubation with total overnight S. aureus culture or filtered supernatant obtained from S. aureus incubated with or without eggs as in (B) compared with media controls. (D) Percent of T. muris eggs hatched when placed in a 0.4μm transwell separated from bacteria or control media in the outer well compared with eggs incubated bacteria without transwell separation. Data points and error bars represent the mean and SEM from 3 independent experiments for (A) and (C). ~25 eggs per well were assessed for (A) and (C). Dots represent a single well containing ~200 eggs and bars show means and SEM from 3 independent experiments for (D). Ordinary one-way ANOVA test followed by a Tukey’s multiple comparisons test was used to compare resuspended pellet and supernatant conditions with total O/N culture for (A) and (C). Two-way ANOVA followed by a Tukey’s multiple comparisons test was used for (D). (B) was created using BioRender.com.</p

Untreated <i>T</i>. <i>muris</i> eggs harbor endogenous bacteria, related to Fig 3.

(A) Representative low (top) and high magnification (bottom) SEM images of T. muris eggs (clear arrowhead) that were untreated with bacteria. White arrowheads correspond to bacteria on polar plug regions of the eggs denoted by black arrowheads. Red arrow corresponds to debris present on egg suface. (B) Image of LB plate with overnight bacterial growth (yellow arrow) from 3 different batches of eggs (batch 14, 15, and 16). (TIF)</p

Collapse of the polar plug precedes hatching mediated by bacteria.

(A, B, C, D) Representative low (left) and high magnification (right) SEM images of T. muris eggs (clear arrowhead) that were exposed to S. aureus for 1 hour (A), E. coli for 1.5 hours (B) and E. faecalis for 1 hour (C) or untreated (D). White arrowheads correspond to bacteria on polar plug regions of the eggs denoted by black arrowheads. Yellow arrow in right panel of (A) and (B) indicates woolly substance present among bacteria. (E) Representative low (left) and high magnification (right) SEM images of hatching T. muris eggs (clear arrowhead) that were exposed to S. aureus for 1 hour (top) and E. coli for 1.5 hours (bottom). White arrowheads correspond to bacteria on polar plug regions of the eggs. Emerging larvae are denoted by white diamonds. (F) Number of bacterial cells visible on polar plugs of T. muris eggs incubated with E. coli, S. aureus, or E. faecalis. Bars showing mean from 2 eggs per condition. (G) Width of collar openings on T. muris eggs that were treated with either E. coli or S. aureus and were either unhatched or in the process of hatching. For low magnification images, scale bar represents 2μm for unhatched egg in (A), (B), (C) and (D) and 10μm for hatched egg in (A) and (B). For high magnification images, scale bar represents 1μm for (A), (B) and (D) and 2μm for (C). Dots represent a single plug and bars show mean and SEM of collar sizes from 4–7 eggs per condition for (G). Two-way ANOVA followed by a Tukey’s multiple comparisons test was used for (G). 4–10 eggs were imaged per condition.</p