Is Gut Microbiota Dysbiosis a Predictor of Increased Susceptibility to Poor Outcome of COVID-19 Patients? An Update

The scientific knowledge already attained regarding the way severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects human cells and the clinical manifestations and consequences for Coronavirus Disease 2019 (COVID-19) patients, especially the most severe cases, brought gut microbiota into the discussion. It has been suggested that intestinal microflora composition plays a role in this disease because of the following: (i) its relevance to an efficient immune system response; (ii) the fact that 5–10% of the patients present gastrointestinal symptoms; and (iii) because it is modulated by intestinal angiotensin-converting enzyme 2 (ACE2) (which is the virus receptor). In addition, it is known that the most severely affected patients (those who stay longer in hospital, who require intensive care, and who eventually die) are older people with pre-existing cardiovascular, metabolic, renal, and pulmonary diseases, the same people in which the prevalence of gut microflora dysbiosis is higher. The COVID-19 patients presenting poor outcomes are also those in which the immune system’s hyperresponsiveness and a severe inflammatory condition (collectively referred as “cytokine storm”) are particularly evident, and have been associated with impaired microbiota phenotype. In this article, we present the evidence existing thus far that may suggest an association between intestinal microbiota composition and the susceptibility of some patients to progress to severe stages of the disease.

system, featured by an exacerbated systemic inflammatory response and the massive release of cytokines, collectively described as a "cytokine storm" [40]. The resulting multiorgan failure fueled by a self-sustaining loop of ongoing age-related immunosenescence and inflammaging can additionally contribute to the poor outcomes in elderly patients with chronic comorbidities [15,41,42].
Among other relevant metabolic and structural protective functions, gut microbiota plays a major role in the host immune system education and ability to respond to insults, including to infections [1]. Disruption of gut microbiota influences the host's immune response, worsening SARS-CoV-2-induced injury, owing to an excessive reactivity of the immune system and a strong inflammatory state [43][44][45]. In addition, different lines of evidence show that respiratory viral infections may originate alterations in the intestinal microbiome composition, which predispose patients to secondary infections and aggravate their clinical status [11,12,[43][44][45]. We and others have recently proposed that the triad of gut microbiota dysbiosis, immune hyperresponse, and inflammation could eventually explain why some COVID-19 patients are more resilient, while others are more fragile when infected with SARS-CoV-2, recovering faster or progressing to more severe clinical condition, respectively [25,[46][47][48][49]. As the ongoing studies reveal new evidence, this hypothesis has been gaining more and more consistency, in such a way that gut microbiota composition might eventually be viewed as a putative predictor of COVID-19 susceptibility and severity. In the next paragraphs, we report the data already known that may contribute to validating this possibility.

What Is the Evidence So Far that Links Gut Microbiota Composition to COVID-19 Severity?
Gut microbiota is crucial to the process of development and function of the immune system [1,[50][51][52], as it modulates immune cells towards anti-or pro-inflammatory responses. Different studies have described significant changes regarding the innate and adaptive immune systems in COVID-19 patients [53][54][55][56]. The cytokine storm, in particular, clearly reflects an uncontrolled dysregulation of the host's immune function. Several pieces of evidence point to the occurrence of lymphocytopenia in individuals with SARS-CoV-2 [57][58][59][60][61]. In a study involving 452 severe COVID-19 patients in Wuhan, a significant decrease in the number of T lymphocytes, including helper and suppressor T cells, was observed [62]. Particularly, among helper T cells, the researchers reported a decrease in regulatory and memory T cells counts. However, naïve T cells percentage was increased in COVID-19 patients relative to healthy individuals, which might contribute to hyperinflammation events, as there is an imbalance between the activity of naïve T cells and that of regulatory and memory T cells [62]. Furthermore, a reduced number of memory T cells might be implicated in COVID-19 relapses, which are particularly evident when recurrences in recovered patients arise [62].
Kalfaoglu et al., by analyzing CD4+ T lymphocytes' transcriptomes from bronchoalveolar lavage fluid (BALF) belonging to moderate and severe COVID-19 patients, observed that SARS-CoV-2 is capable of inducing activation and differentiation processes in these cells, accelerating both their activation and death [55]. These authors proposed a hypothesis stating that the abnormally activated CD4+ T cells might be able to promote the viral entry through Furin production in critically ill patients. When compared with moderate patients, CD4+ T cells from severe patients present an increased expression of the genes fos, fosb, and jun; of the activation marker MKI67; of Th2-related genes maf and il4r; and of chemokines CCL2, CCL3, CCL4, CCL7, CCL8, and CXCL8. These results suggest that CD4+ T cells in severe COVID-19 patients' lungs are highly activated and recruit other immune cells. In contrast, these patients display decreased expression of interferon-induced genes, such as ifit1, ifit2, ifit3, and ifim1, as well as genes associated with interferon downstream pathways, suggesting that interferons might be suppressed in severe COVID-19 cases [55]. In addition, Huang et al. discovered that the plasmatic concentrations of interleukin (IL)-1β, IL-1ra, IL-7, IL-8, IL-9, IL-10, basic FGF, GM-CSF, G-CSF, VEGF, IP-10, MCP-1, IFN-γ, IFN-α, MIP-1α, and MIP-1β were higher in COVID-19 patients present in ICU, as well as non-ICU patients, when compared with healthy individuals [63]. A possible explanation for the potential contradictory evidence of a decrease in the expression of interferon-induced genes' and interferon downstream pathways and increased plasmatic IFNs levels might be associated with the fact that plasma levels are defined by the IFNs' input from several tissues, including the gut [64]. This study raises the premise that the cytokine storm observed in COVID-19 cases might be correlated with disease severity, as IL-2, IL-7, IL-10, G-CSF, MCP-1, IP-10, TNF-α, and MIP-1α levels were higher in ICU patients compared with non-ICU ones [63]. Moreover, a study evaluating a cohort of 44 hospitalized COVID-19 patients reported the existence of higher median fecal levels of IL-8 and lower levels of fecal IL-10 in COVID-19 patients compared with control individuals [65]. Furthermore, IL-23 fecal levels were increased in severe patients, suggesting the involvement of the GI tract in the SARS-CoV-2 infection in an immunological manner [65]. More severe cases were also associated with higher serum levels of IL-6, IL-8, tumor necrosis factor α (TNF-α), C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer, ferritin, and procalcitonin [65]. Furthermore, a study performed by Li et al. allowed to observe that the lower the counts on admission of total T cell, CD4+ T cell, and CD8+ T cell, the more serious the disease and the worse the prognosis of the patients [66]. A recent study also showed that lymphopenia and an increase in cytokine levels were significantly correlated with disease severity, with the IL-2R/lymphocyte ration being a potential biomarker for COVID-19 disease severity and progression identification [67].
Several studies have already demonstrated that, when compared with healthy individuals, COVID-19 patients present a significantly reduced bacterial diversity [31,68]; higher abundancy of opportunistic bacteria such as Streptococcus, Rothia, Veilonella, and Actinomyces [31,34,35]; and decreased levels of benefic symbionts, including Agathobacter, Fusicatenibacter, Roseburia, and Ruminococcaceae UCG-013 [31,35]. A study performed by Zuo et al. reported that most patients' gut microbiota composition alterations persisted even after viral clearance, suggesting that the infection or/and hospitalization might be associated with a long-lasting adverse effect regarding the composition of intestinal microflora community [35], which might be potentially associated with recovery delays. Remarkably, the existence of a correlation between the COVID-19 severity grade and the basal fecal microbiome has been established [35,68]. In the study performed by Zuo et al., twenty-three bacterial taxa showed a significant positive correlation with disease severity; with the main bacteria presenting a positive association with COVID-19 severity belong to the filo Firmicutes and the genus Coprobacillus, as well as the Clostridium ramosum and Clostridium hathewayi species [35]. Interestingly, the fact that Firmicutes presented this positive association with disease severity is in accordance with evidence showing that these bacteria possess a specific role in regulating ACE2 expression in the murine gut [69]. On the other hand, two beneficial bacterial species-Alistipes ondedonkii (important for the maintenance of intestinal homeostais) and Faecalibacterium prausnitzii (anti-inflammatory properties detainer)-showed a negative correlation with COVID-19 severity [35].
It is now acknowledged that gut microbiota is responsible for regulating several hosts' physiological functions [70][71][72]. Particularly, numerous studies have reported that intestinal microflora affects lung health through a bidirectional pathway designated as the "gut-lung axis" [73][74][75]. One of the main complications associated with COVID-19 is acute respiratory distress syndrome (ARDS) [76,77], in which microbiota composition and function might play an important part. An enrichment of lung microbiota with intestinal Bacteroides species is observed in many COVID-19 cases [78], an event linked to increased plasmatic inflammatory markers levels [78]. Another study reported an increase in Enterobacteiaceae and Lachnospiraceae levels in severely ill patients with ARDS, when compared with patients that did not present this condition [32,79]. These results suggest that the microbiota could be seen as potential marker to predict ARDS and other possible scenarios associated with COVID-19 pathology. Therefore, gut microbiota might provide information about the individual susceptibility to COVID-19. Gou et al. recently reported that changes in the normal composition and function of intestinal microbiota might predispose healthy individuals to an atypical inflammatory response, such as the one observed in COVID-19 cases [80]. Additionally, a study performed in Wuhan, China confirmed the existence of this relationship between gut microflora composition and the predisposition of healthy individuals to SARS-CoV-2 infection [78]. The researchers observed that individuals that display increased numbers of Lactobacillus present higher levels of IL-10, an anti-inflammatory cytokine, and generally a more favorable prognosis. In contrast, individuals displaying higher numbers of pro-inflammatory bacterial species, such as Klebsiella, Streptococcus, and Ruminococcus gnavus, showed increased levels of pro-inflammatory cytokines as well as more pronounced disease severity [78]. Furthermore, through the evaluation of the metabolomic and proteomic profile of COVID-19 patients' serum, a study performed by Shen et al. revealed specific alterations in severely ill patients [33]. In fact, increased serum concentrations of inflammatory markers, such as IL-1β, IL-6, TNF-α, and CRP, were associated with a higher prognostic risk score (PRS) in patients over 58 years old [33]. By investigating the potential role of gut microbiota on the susceptibility of healthy individuals to COVID-19, the investigators demonstrated that the observed alterations regarding blood proteomic markers would be preceded by intestinal microflora changes, suggesting a potential causal relationship in the case of older patients [33] Specifically, the genus Bacteroides and Streptococcus, as well as the order Clostridiales, showed a negative correlation with the majority of the tested cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α, and IFN-γ), while the genus Lactobacillus, Ruminococcus, and Blautia displayed positive associations with the referred cytokines [33]. Another study has reported that abundant bacteria in COVID-19 patients, including Rothia, Streptococcus, Veilonella, and Actinomyces, are positively correlated with high levels of CRP and D-dimer, once more evidencing the influence of gut microbiota composition in the host's inflammatory profile [31]. However, no significant alterations were observed in gut microbiota composition between patients with different disease severity stages [65]. These results suggest that, in this complex scenario of interactions between different systems (namely intestinal microbiota-immune system-inflammatory response), there may be additional factors playing a relevant role for disease severity. On the other hand, it also suggests that other elements able to shape microbiota should be carefully considered, including age; comorbidities; and especially the impact of drugs, particularly antibiotics, as highlighted in the study of Britton et al. [65].
Collectively, there is evidence suggesting that microbiota characteristics and related metabolites should be more profoundly investigated as potential prediction markers of individual susceptibility of COVID-19 patients to develop a more severe phenotype. Table 1 summarizes the major findings regarding gut microbiota, inflammation, and immune system changes in COVID-19 patients, and the suggested associations with disease severity. However, only the publication of more results from the different clinical trials related to gut microbiota with patients affected with distinct levels of severity could open up the possibility to clarify the existence of causality in this association. (i) Compared with healthy controls, COVID-19 patients displayed a depletion of Agathobacter, Fusicatenibacter, Roseburia, and Ruminocaccaceae UCG-013, whose abundancy was negatively correlated, mainly, with the levels of CRP, PCT, and D-dimer; (ii) CRP and D-dimer levels were positively correlated with bacteria abundance in COVID-19 cases, including Streptococcus, Rothia, Veilonella, and Actinomyces.
(i) In a sample of seven antibiotic-naïve patients, 23 bacterial taxa were discovered to correlate with disease severity; (ii) the main bacteria positively correlated with disease severity belonged to the phylum Firmicutes, Coprobacillus genus, and Clostrididum ramosum and Clostridium hathewayi species; (iii) the main bacteria negatively correlated with disease severity were Alistipes ondedonkii and Faecalibacterium prausnitzii. [55] 12 COVID-19 death cases (moderate vs. severe).
(i) Among all parameters studied, blood lymphocyte percentage (LYM%) showed the most significant and consistent trend, suggesting it as an indicator of disease progression; (ii) LYM% of severe patients decreased initially and then increased to >10% until being discharged; (iii) LYM% of moderate patients fluctuated very little after disease onset and was >20% when discharged. [59] 522 COVID-19 patients, 43 of which were admitted in the ICU; vs. 40 healthy controls.
(i) In the severe and critical disease patients, as well as the perished ones, total T cells, CD4 + , and CD8 + T cells were significantly lower than in the mild/moderate patients; (ii) total T cells, CD8 + T, or CD4 + T cells lower than 800, 300, and 400/µL, respectively, were negatively correlated with patient survival; (iii) T cell numbers were negatively correlated with serum IL-6, IL-10, and TNF-α levels, which were significantly higher in ICU patients than in non-ICU ones; (iv) increasing PD-1 and Tim-3 expression on T cells in patients progressed to symptomatic stages. [61] 39 COVID-19 patients.
(i) The more serious the disease and the worse the prognosis, the lower the T cell, CD4+ T cell, and CD8+ T cell counts on admission. [66] 452 COVID-19 patients: 166 non-severe; 286 severe.
(i) With increased severity of illness, leukocytes, neutrophils, infection biomarkers (CRP, PCT, and ferritin) and cytokines (IL-2R, IL-6, IL-8, IL-10, and TNF-α) were significantly increased, while lymphocytes were significantly decreased; (ii) the ratio of IL-2R to lymphocytes was found to be remarkably increased in severe and critical patients. IL-2R/lymphocytes ratio was superior vs. other markers for the identification of critical illness COVID-19; (iii) cytokine profile and IL-2R/lymphocytes were significantly decreased in recovered patients.
[67] (i) ARDS patients display an increased number of Lachnospiraceae and Enterobacteriaceae, which predicted fewer ventilator-free days; (ii) enrichment of the lungs with gut-associated bacteria was positively correlated with high levels of inflammatory markers, particularly TNF-α; (iii) increased Lachnospiraceae was a strong predictor of reduced survival in ARDS patients. [79] 31 COVID-19 patients (18 non-severe cases and 13 severe cases) and 990 non-COVID-19 individuals.

Concluding Remarks and Future Directions
COVID-19 patients display immune response deregulation and increased levels of specific inflammatory cytokines and chemokines, with these alterations being particularly intense in severe patients, in a condition often referred as cytokine storm [32,33,53,55]. Other studies also report that the blood lymphocyte percentage might reflect disease progression and severity [59], as well as the number of leukocytes and B and natural killer (NK) cells [81,82]. Gut microbiota plays major functions in the host, including immune system education and strengthening [1,51,52]. Several studies have reported major impairment of innate and adaptive immune systems in COVID-19 patients [53][54][55][56], accompanied by changes in gut microbiota composition [31][32][33][34][35]. It has been suggested that intestinal microflora composition could be correlated with the predisposition of healthy individuals to COVID-19 and with disease severity [31,[33][34][35]78]. In particular, some data suggest that certain microbiota characteristics allow the prediction of the occurrence of ARDS and other disease-associated scenarios [32,78]. Moreover, COVID-19 patients' microbial composition correlates with altered levels of inflammatory markers when compared with healthy individuals [31,33,78], reinforcing the potential relevance for the disease. These data have been leading researchers to refer to gut microbiota composition, inflammatory markers' levels, and immune cells' number and activity as potential predictors of susceptibility of healthy individuals to COVID-19, as well as of disease severity (Figure 1), as these parameters differ significantly between healthy and infected individuals, as well as between moderate and severe COVID-19 patients [31][32][33]35,59,78,80]. However, with the current knowledge, it is impossible to ensure a causal relationship, which remains an open hypothesis that deserves to be better dissected. inflammatory markers' levels, and immune cells' number and activity as potential predictors of susceptibility of healthy individuals to COVID-19, as well as of disease severity (Figure 1), as these parameters differ significantly between healthy and infected individuals, as well as between moderate and severe COVID-19 patients [31][32][33]35,59,78,80]. However, with the current knowledge, it is impossible to ensure a causal relationship, which remains an open hypothesis that deserves to be better dissected. The evidence collected thus far suggests that modifications in the characteristics of the intestinal microbial community and the relationship it establishes with the immune system, which leads to changes in inflammatory markers' levels and in the number and function of several immune cells, should be more profoundly investigated as potential predictors of individual susceptibility to a more severe COVID-19 phenotype. Additionally, these parameters might be used to support the implementation of therapeutic The evidence collected thus far suggests that modifications in the characteristics of the intestinal microbial community and the relationship it establishes with the immune system, which leads to changes in inflammatory markers' levels and in the number and function of several immune cells, should be more profoundly investigated as potential predictors of individual susceptibility to a more severe COVID-19 phenotype. Additionally, these parameters might be used to support the implementation of therapeutic measures to prevent disease evolution in populations with higher susceptibility. Critically ill patients on mechanical ventilation who were given probiotics, specifically Lactobacillus rhamnosus GG, live Bacillus subtilis, and Enterococcus faecalis, presented improvement of pneumonia when compared with placebo, in two randomized controlled trials [83,84]. However, the efficacy of probiotics use in COVID-19 patients remains to be proved and the issue is under debate [85,86], deserving more attention by the scientific-medical community.
Author Contributions: C.F. wrote the first draft, which was revised and completed by S.D.V. and F.R., who wrote other parts. S.D.V. drew the figure. All the authors (C.F., S.D.V., and F.R.) significantly contributed to the writing and have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.