Probiotics and Coronavirus disease 2019: think about the link

The pandemic of Coronavirus disease 2019 (COVID-19) is rapidly progressing, causing significant morbidity and mortality. Various antiviral drugs, anti-inflammatory drugs, and immunomodulators have been tried without substantial clinical benefits. The severe and critical cases of COVID-19 disease are characterised by gut microbiome dysbiosis, immune dysregulation, hyper-inflammation, and hypercytokinemia (cytokine storm). Therefore, the strategies which target these pathophysiological processes may be beneficial. Probiotics are one such strategy that exerts beneficial effects by manipulation of the gut microbiota, suppression of opportunistic pathogens in the gut, decrease translocation of opportunistic organisms, activate mucosal immunity, and modulation of the innate and adaptive immune response. Probiotics are the potential candidates to be tested in moderate and severe cases of COVID-19 due to several beneficial effects, including easy availability, easy to administer, and safe, and economical to use.

gut-lung interaction is bidirectional, and the gut plays a vital role in influencing lung disease. In diseased states, the gut microbiome is disturbed (dysbiosis), leading to translocation of endotoxins and microbial metabolites from the gut, which can then impact the lung through the bloodstream. Similarly, when inflammation occurs in the lungs (e.g. respiratory viral infections), it causes perturbations in the gut microbiome (gut-lung axis) (16,32,33) .
This bidirectional gut-lung interaction may be playing an important role in COVID-19, leading to increased disease severity. In extreme of ages and among those with underlying co-morbid conditions, the gut microbiota is less in diversity leading to gut dysbiosis (34)(35)(36)(37) . This could be one of the reasons for the increased disease severity of COVID-19 among cases with the extreme of ages and in those with underlying co-morbid medical conditions (38,39) . It has been demonstrated that the gut microbiome is altered in cases with COVID-19 in terms of a significant reduction in diversity and number of beneficial gut microbiota and an increase in the number of opportunistic pathogens (16,40,41) . This can ultimately lead to gut dysbiosis, translocation of pathogenic organisms across the gut mucosa, secondary bacterial infections, enhanced inflammatory response, multiple organ dysfunction and poor clinical outcome. Zuo et al. (41) performed metagenomic sequencing analyses on the faecal samples of COVID-19 patients (n 15) from the time of admission until discharge and compared them with fifteen healthy controls. The authors demonstrated that these patients had significant depletion of beneficial commensals and enrichment of opportunistic pathogens at the time of admission as well as at all time points during the hospital stay when compared with controls. Similarly, Gu et al. (40) , in a cross-sectional study involving COVID-19 patients (n 30), demonstrated that the gut microbiome (faecal sample collected at admission) of COVID-19 cases had significantly reduced bacterial diversity, a lower count of beneficial symbionts and a higher number of opportunistic pathogens (Streptococcus, Rothia, Veillonella and Actinomyces). Therefore, the heightened or dysregulated inflammation and gut dysbiosis may be the major pathophysiological processes in cases with COVID-19 leading to increased severity of illness and poor clinical outcomes.

Probiotics in Coronavirus disease 2019
In the absence of proven effective therapy for COVID-19, the treatment is mainly supportive. Therefore, the alternative strategies that target pathophysiological pathways in patients with COVID-19 need to be tested. One such approach is probiotics. The WHO and FAO defined probiotics as 'live microorganisms which, when administered in adequate amounts, confer a health benefit on the host.' Many different micro-organisms have been considered as probiotics, including Lactobacillus species (L. acidophilus, L. casei and L. rhamnosus); Bifidobacterium species (B. bifidum, B. longum and B. lactis); Enterococcus species (E. faecalis and E. faecium) and Saccharomyces (S. boulardii and S. cerevisiae). The most commonly used strains are Lactobacillus and Bifidobacterium (52) .
It is important to note that not all probiotic strains exert the same actions (29) . The combination of multiple strains may exert one or a few of these actions and thus act synergistically. Therefore, multistrain probiotics could be more effective than mono-strain probiotics (67) . A review comparing single strain and multistrain probiotics demonstrated that the multistrain probiotics had greater efficacy than individual strains, including single strains that were components of the mixtures themselves (68) .
Due to the beneficial effects of probiotics on gut microbiota, they have been tested to prevent various infective complications among hospitalised patients. Many strain of probiotics has been demonstrated to be safe and effective in the prevention of Clostridium difficile-associated diarrhoea among hospitalised children and adults (69,70) and secondary bacterial infections including ventilator-associated pneumonia in critically ill patients (71)(72)(73)(74) , postoperative infections (respiratory, urinary tract and wound infections) in patients undergoing gastrointestinal surgery (75) and candida colonisation in the gastrointestinal tract and invasive candidiasis in critically ill children (52)(53)(54) . Systematic reviews and meta-analysis involving critically ill adults demonstrated that probiotic supplementation has been associated with decreased risk of nosocomial infections, lesser gastrointestinal complications, lower mortality and shortened ICU stays (76)(77)(78) . Among preterm neonates, a meta-analysis (n 8000) demonstrated that supplementation with certain probiotics resulted in a reduction in necrotising enterocolitis, nosocomial sepsis and all-cause mortality (79) .

Modulation of inflammation by probiotics
The role of probiotics in the modulation of inflammation has been demonstrated in clinical studies involving children and adults in health and several diseased states (55,62,63,65,66,(82)(83)(84)(85) . Kazemi et al. (82) , in a recent systematic review and meta-analysis, analysed 167 clinical trials that investigated the effect of oral probiotics or synbiotic (for >1 week) on inflammatory markers (C-reactive protein, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, TNF-α, interferon-γ and transforming growth factor-β) among healthy individuals and in various disease conditions. Authors noted that some probiotics lead to decrease in C-reactive protein levels in healthy individuals, metabolic disorders, inflammatory bowel disease, arthritis and critically ill condition; decrease in TNF-α levels in healthy individuals, fatty liver, inflammatory bowel disease and hepatic cirrhosis; increase in IL-6 in cirrhosis and renal failure; and increase in IL-10 in arthritis. In another meta-analysis (eight clinical trials, 1337 patients with diabetes), it has been demonstrated that probiotics and synbiotic supplementation significantly decreased C-reactive protein and TNF-α levels but did not affect IL-6 levels (83) . These meta-analyses suggest that supplementation with probiotics leads to a decrease in proinflammatory markers (hs-C-reactive protein, TNF-a, IL-6, IL-12, IL-4 and TNF-α) and an increase in anti-inflammatory cytokines (IL-10) in healthy and several diseased states (82)(83)(84) .
In an RCT (n 100), Angurana et al. (55) demonstrated that administration of a multistrain probiotic (Lactobacillus paracasei, L. plantarum, L. acidophilus, L. delbrueckii, Bifidobacterium longum, B. infantis, B. breve and Streptococcus salivarius) to critically ill children with severe sepsis for 7 days resulted in a significant decrease in levels of pro-inflammatory cytokines (IL-6, IL-12p70, IL-17 and TNF-α) and increase in antiinflammatory cytokines (IL-10 and transforming growth factor-β 1) as compared with placebo. Other studies also demonstrated that certain probiotics were useful in the modulation of inflammation in critically ill patients (66,85) .
As cytokine storm occurs in patients with severe COVID-19, the effect of probiotics on pro-inflammatory and immunoregulatory cytokines allows viral clearance and minimise immune response-mediated tissue damage in the lungs and other organs. So, these immune-modulatory effects of probiotics might be relevant to prevent lung injury, acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome which are significant complications of COVID-19 (29) .

Antiviral effects of probiotics
The antiviral effects of probiotics have been demonstrated in experimental and clinical studies. Few experimental studies showed that various strains of probiotics exert antiviral effect by impairing virus entry into human cells and preventing viral replication (86)(87)(88)(89) . Ang et al. (86) , in an in vitro study involving human skeletal muscle and colon cell lines, demonstrated that Lactobacillus reuteri Protectis showed significant dosedependent antiviral activity against Coxsackievirus type A strains 6 and 16 and enterovirus 71. Lee et al. (87)  Viruses are the most frequent cause of upper respiratory tract infections. A Cochrane review (twelve RCT with 3720 participants, including children and adults) showed that probiotics were superior to placebo in decreasing the number of participants experiencing episodes of acute upper respiratory tract infections, mean duration of an episode of acute upper respiratory tract infections, antibiotic use and cold-related school absenteeism (95) . In influenza viral infection, probiotics have been shown to modulate respiratory immunity and had a beneficial impact on virus clearance and inflammatory-mediated lung injury (96) .
Recently, Anwar et al. (88) , in a computational docking study, demonstrated that three metabolites of Lactobacillus plantarum (Plantaricin W, Plantaricin JLA-9 and Plantaricin D) significantly blocked the binding of SARS-CoV-2 with angiotensin-converting enzyme 2 receptors (blocking the viral entry into the cells) and suggested antiviral property of Lactobacillus plantarum against SARS-CoV-2.
Therefore, probiotics may act as antiviral agents by interfering with the viral entry into cells and/or inhibit virus replication. This may lead to a reduction in the dissemination of SARS-CoV-2 in the respiratory tract and gut. Also, with the restoration of the gut and respiratory microbiota, immune function and gut-lung axis, the course of COVID-19 may be altered (29) .
It has been suggested to exercise caution while using probiotics in the presence of a one major risk factor (immunocompromised state and premature infants) or more than one minor risk factor (central venous catheter, cardiac valvular disease, impaired intestinal epithelial barrier, administration of probiotics by jejunostomy and probiotics with properties of high mucosal adhesion or known pathogenicity) because of the risk of probiotics-sepsis (80,105) .
Other safety concerns are the genetic transfer of antimicrobial resistance from probiotic strains to pathogenic organisms in the gut (particularly Staphylococcus aureus and Enterococcus) (106,107) , deleterious metabolic activities and excessive immune stimulation in susceptible individuals (52,80) . The use of probiotics may be limited in the presence of immunocompromised states or premature neonates because of safety concerns, as discussed above. The supplementation with probiotics may not be possible in whom enteral administration is contraindicated.
Therefore, probiotics, due to their mechanisms of action, are a potential strategy that need to be tested in moderate to severe cases with COVID-19 by conducting well-designed clinical trials. Few registered trials are going on to evaluate the possible beneficial effects of probiotics supplementation in cases with COVID-19 (NCT04366180, NCT04368351 and NCT04366089).
A better understanding of the pathophysiology of COVID-19 and the results of the clinical trials will guide further on the efficacy, benefits and safety of probiotics in COVID-19. Despite these limitations, considering various mechanisms of actions of probiotics on the regulation of gut dysbiosis and anti-inflammatory activity, and clinical evidence about the beneficial effects of probiotics in critically ill adults and children, probiotics are a potential therapeutic strategy that need to be tested in moderate to severe cases with COVID-19.

Conclusion
Some probiotics may have a beneficial role in the treatment of COVID-19 patients due to their antiviral activity, ability to restore gut microbiome, modulate inflammation, ready availability, ease of administration, relatively safe and economical. The recommendations regarding the strain, dose and duration of probiotic are lacking. However, given the large experience of clinical usage, and evidence for beneficial effects in the various clinical settings, Lactobacillus and Bifidobacterium can safely be used.