key: cord-0780035-kzvd139d
authors: Prasad, Ram; Patton, Michael John; Floyd, Jason L; Vieira, Cristiano Pedrozo; Fortmann, Seth; DuPont, Mariana; Harbour, Angie; Jeremy, Chen See; Wright, Justin; Lamendella, Regina; Stevens, Bruce R.; Grant, Maria B.
title: Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis
date: 2021-04-06
journal: bioRxiv
DOI: 10.1101/2021.04.06.438634
sha: 8559fa08560af566e3c24e5de19f6e3919f9c4d9
doc_id: 780035
cord_uid: kzvd139d

The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282±199.6 vs 838.1±91.33; p=0.0757), PGN (34.64±3.178 vs 17.53±2.12; p<0.0001), and LPS (405.5±48.37 vs 249.6±17.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

metric 29 based on the normalized table and rooted tree. The resulting distance matrix was visualized as a Principal Coordinates Analysis plot in R.

The level of fatty acid-binding protein-2 (FABP2) 30 , a marker of intestinal barrier damage, was determined by ELISA in the plasma samples using a colorimetric assay kit (#DFBP20, R&D systems, Minneapolis, MN) following the manufacturer's protocol. The absorbance was measured at 450nm using a microplate reader, and the levels of FABP2 were calculated as per the standard curve and expressed as pg/mL.

The level of peptidoglycan (PGN) in plasma samples was measured using a colorimetric assay kit for mouse peptidoglycan (#MBS263268, MyBioSource Inc., San Diego, CA) following the manufacturer's protocol. The absorbance was measured at 450nm using a microplate reader and the levels of peptidoglycans were calculated as per the standard curve and expressed as ng/mL. The levels of lipopolysaccharides (LPS) were also measured by ELISA kit (#EKC34448, Biomatik, Wilmington, DE) following the manufacturer's instruction manual. The levels of LPS were calculated by standard curve and expressed as pg/mL.

Data were evaluated for presence of outliers and adherence to a normal distribution using GraphPad Prism, version 8.1 software. Statistical significance of normally and nonnormally distributed data were assessed via student's t-test and Mann-Whitney test, respectively, at p=0.05.

We enrolled 30 patients confirmed to have COVID-19 infection (Table 1) Table 2) .

Admission laboratories for the COVID-19 cohort included complete blood count (CBC) with differential and a metabolic panel. These results are detailed in Tables 3 and 4, respectively. CBC results indicated decreased abundance of lymphocytes and red blood cells (RBCs) in 47.8% and 64% of COVID-19 subjects, respectively. All COVID-19 subjects (n=23) exhibited abundance of monocytes above the normal range. Neutrophils, the first-responders of bacterial infection, were at an abundance above the normal range in 58.3% of male and 45.5% of female COVID-19 subjects. Total white blood cells counts were outside the normal range in 38.5% and 41.7% of COVID-19 subjects, respectively. Lastly, 16.7% of COVID-19 subjects exhibited platelets outside the normal range.

Biochemical evaluation of these patients was performed as seen in Table 4 . Of COVID-19 positive subjects, 100% of those with BNP out of range (n=3) were male of which one subject exhibited BNP between 100-200 pg/mL, likely indicative of compensated congestive heart failure (CHF), and two subjects with BNP >400 pg/mL, indicating likely moderate to severe CHF. All subjects exhibited CRP levels greater than normal of which four male subjects (44%) and two female subjects (33%) exhibited CRP >100 mg/L which is associated with severe inflammation such as sepsis. Six male (75%) and six female (100%) subjects exhibited D-dimer levels higher than the normal range, indicating activation of the procoagulant and fibrinolytic systems. Five male (71.4%) and three female (60%) exhibited ferritin levels greater than the normal range of which two male (40%) and two female subjects (67%) had ferritin levels >1000 ng/mL which can be associated acute or chronic inflammation. Twelve male (80%) and 11 female (91.7%) exhibited fasting glucose levels greater than the normal range. Nine male (64.3%) and 8 female (66.7%) subjects exhibited hemoglobin levels below the normal range. Six male (85.7%) and six female (100%) subjects exhibited LDH levels greater than normal. Of male subjects, four subjects (80%) exhibited troponin-I levels above the normal range and one subject (20%) had levels below the normal range. Of female subjects, one (16.7%) exhibited troponin-I levels greater than the normal range. Troponin-I levels greater than the normal range suggest myocardial injury. Three male (60%) and three female (60%) subjects exhibited procalcitonin levels greater than the normal range with one male (33.3%) and one female 

Plasma samples were obtained under sterile conditions and evaluated for the presence of bacteria. Specifically, the taxonomic units, distribution of abundances, and alpha diversity were measured. Alpha diversity, a representation of the total microbial population in the sample, was assessed using Pielou's Evenness, Faith's Phylogenic Diversity, Observed Features Metrics, and Shannon's Index, (Fig.1A-D) . A total of 152,536 sequencing reads were obtained from 14 COVID-19 plasma samples. 16S rRNA sequencing data suggests that 65% (9 out of 14 patient samples) yielded a strong bacterial signal. Alpha diversity revealed that the plasma microbiome for each patient exhibited unique evenness and richness. However, notable differences were observed in the Pielou's, Faith's, Observed, and Shannon index between samples. Beta diversity was determined using principal coordinate analysis (PCoA) (Fig.1E) .

Overall, the plasma microbiome community was not different between the COVID-19 samples.

The dysbiosis index is a PCR-based assay and was performed to quantify the abundance of bacterial groups in the given samples. As shown in Fig.2A , a dysbiosis index was determined in the plasma of all the COVID-19 samples. The relative abundance of microbial composition in the COVID-19 samples is shown in Fig.2B . Three major phyla (Proteobacteria, Firmicutes, and Actinobacteria) were identified in all 9 samples. Patient 6, however, exhibited abundance of unidentified bacteria greater than all other subjects. At the phylum level, the enrichment of Proteobacteria was highest in all samples ranging from 22%-91%, followed by Firmicutes (10%-71%), and Actinobacteria (6%-27%). Bacteroidetes was present in a very low percentage. Firmicutes abundance in P7 and P12, two of those which died during hospitalization, was low, suggesting plasma abundance of Firmicutes may be a prognostic marker of COVID-19 severity.

Next, the abundance of each microbial population was assessed and revealed that, at the genus level (Fig.3) , the prevalence of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 plasma samples. Notably, LPS, a major cell wall component of gram-negative bacteria which contributes to the activation of inflammatory signaling pathways, was significantly increased in COVID-19 subject plasma (p=0.0049), supporting the observed increase of gram-negative bacteria.

The plasma microbiome arises largely as a consequence of bacterial translocation from the gut into the systemic circulation 31-36 . Compromised intestinal barriers are an important pathogenic factor and contribute to promotion of inflammation. We measured gut permeability markers in the plasma of COVID-19 and control subjects. FABP2 is an intracellular protein which is expressed specifically in intestinal epithelial cells 37 and binds free fatty acids, cholesterol, and retinoids, and is involved in intracellular lipid transport. During mucosal damage, mature epithelial cells release this protein into the circulation 38 and higher levels of samples compared with non-COVID-19 patients (Fig.4C ).

Due to their role in regulating immune function and metabolism, gut microbes are key contributors in the maintenance of host health [51] [52] [53] [54] . The fecal microbiota and its translocation from the gastrointestinal tract into systemic circulation has been considered as a key driver of immune response and systemic inflammation [55] [56] [57] [58] . Abnormal presence of gut microbes in the plasma can initiate and intensify inflammatory cascades 59 . Although systemic and local tissue inflammation is paramount in the pathogenesis of COVID-19 infection, the clinical relevance of gut microbes in the plasma remains unclear. Therefore, in this study we sought to test the hypothesis that bacterial translocation from the intestine into the systemic circulation occurs and is associated with worsened outcomes in SARS-CoV-2 infection. Increased intestinal permeability due to mucosal barrier dysfunction could result in microbial translocation. Our results support that the COVID-19 patients exhibit gut barrier dysfunction as evidenced higher levels of FABP2, PGN, and LPS (Fig.4) and the presence of microbes in their plasma (Fig.1-3) .

The duration of fecal viral shedding ranged from 1 to 33 days after symptomatic recovery of lung pathology 60 61 . In children infected with SARS-CoV-2, rectal swabs were found positive for SARS-CoV-2 even after the nasopharynx was negative, suggesting that viral shedding from the digestive tract might be longer duration than that from the respiratory tract 62 .

During hospitalization, the fecal microbiome can be altered, thus, we selected to test the initial plasma samples of COVID-19 patients. In a small group of 9 patients, depletion of the commensal bacterium Lactobacillus was documented in 65% patients during COVID-19

infection. Commensal bacteria act on the host's immune system to induce a protective response and also inhibit the growth of respiratory pathogens 63 While most studies to date examine the blood metabolome, rather than the blood microbiome, we first sought to establish whether the plasma microbiome existed in COVID-19 subjects and then determine if the microbial diversity supported that the origin of these microbes was the intestine 68 69 . Results from numerous studies have linked the plasma metabolome to the gut microbiome and their implication for specific diseases 70 -P29  ----99  10.0  ----P30  ---------- 

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