Probiotics: An Alternative Therapeutic Strategy For COVID-19

The COVID19 pandemic has made the scientists today all around the globe to look for its specific prevention and treatment modalities. The disease presents itself as asymptomatic to mild to severe respiratory symptoms along with lesser common gastrointestinal symptoms of diarrhoea, nausea and vomiting. The situation worsens due to lack of precise treatment strategy. Healthcare system is being overwhelmed, emphasizing on the need to look for alternate supportive therapy which can not only enhance the immune status of people worldwidebut at the same time, ensure better prognosis. The relationship between the gut microbiota and upper and lower respiratory tract viral infections are well studied. Oral administration of probiotic microorganisms of genera Lactobacillus, Bifidobacterium and Bacillusin mice suffering from influenza infection have shown increased TNFá, IFN-ã and NK cell responses along with production of anti-Influenza IgG. At the same time they have shown immunomodulation by producing anti-inflammatory cytokines as well as cytotoxic T-cells and T-suppressorcells. Hence, probiotic strains of generaLactobacillus, Bifidobacterium and Bacillushave shown a probability to be used as preventive and therapeutic agents for SARSCoV-2.

The world today is facing the havoc of SARS-CoV-2 also known as COVID-19. The disease which reportedly was first seen in Wuhan city of China in December 2019 has now spread to nearly 190 countries of the world. It is a zoonotic disease primarily affecting a specie of bats but due to close proximity of animals and humans in the present world it has mutated and attacked humans. The spread of this disease has overwhelmed the healthcare systems of developing as well as developed countries. Various continuously evolving treatment strategies are being currently used ranging from the use of hydroxychloroqin to plasma therapy. Simultaneously several vaccine candidates are in I or II phase of clinical trials which means that currently no specific treatment or preventive vaccine is available. This has greatly emphasized on the need of some alternate therapy not only to combat the pathogen but also to enhance the immunity of the person to make him less prone to the infection as well as with better chances of recovery.
Various studies have shown that gastrointestinal tract is also involved in the spread of the virus as the virus has been isolated from stool samples of the patients [1][2][3][4][5][6] . Further, the study has revealed that corona virus including the novel one can invade enterocytes 7 causing severe distress in patients. Probiotic bacteria are well documented for their role in correcting gut dysbiosis 8,9 , reducing pro inflammatory reactions increasing defense against various pathogens 10 , and upgrading mucosal immunity 11 . They are useful in treating gut dysbiosis 12 , antibiotic associated diarrhea 13,14 , irritating bowel syndrome 15 etc. Latest studies have shown their impact in treating disorders caused due to gut dysbiosis such as obesity and atopic dermatitis 16 . This review tries to assess the potentiality of the probiotic strains as an alternate or supplementary therapeutic option for treating the COVID-19 disease.

Gut microbiota and Viral respiratory tract infections
Numerous studies have focussed on the importance of gut microbiota in defending or providing better immune response against various diseases [17][18][19][20][21] and respiratory viral infections are no exception. In a study, when mice were treated with antibiotics to deplete gut microbiota, a decrease in antibody production and reduction in influenza virus specific T-cells was observed 22 , along with increased morbidity and mortality 22,23 . This occurred due to decreased T-cell numbers and migration rate of dendritic cells. These mice were also unable to stimulate the response of CD4 + T-cell mediated to PR8 antigen. A decrease in number of influenza specific CD8 + T-cells was also observed.
Gut microbes also modulate the macrophage response during viral pulmonary infection. According to a study, the ability of macrophages to limit viral replication was impaired in mice treated with antibiotics along with reduced response to type I and type II IFNs 23 . The studyhighlighted the importance of gut microbiota in strengthening the immune system against viral infections.
Conversely, different studies have reported that viral respiratory infections, like influenza and Respiratory Syncytial Virus (RSV) causing common cold, have an impact on the gut microbiota 24-28 . These infections caused a disbalance of gut microbiota by increasing the phylum Bacteroidetes and leading to decrease in phylum Firmicutes 28 , especially Lactobacillus 24,29 . This change in gut microbiota composition after influenza infection is due to type-2 IFN which is produced by T-cells derived in the lung and recruited to the intestine 28 .

COVID-19 and Gut
Till 2019, there were six known species of coronavirus which caused human diseases. These include severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) causing severe respiratory tract infections and had high mortality rate. SARS-CoV-2, or COVID-19 as it is widely known, is the seventh to be added in this category 30 .
According to the document published by MoHFW 31 , Govt. of India in coherence with WHO guidelines, the disease presents itself in asymptomatic, mild to severe forms. The common symptoms range from flu like cough, cold, fever to non-specific flu like pneumonia to life threatening severe acute respiratory illness (SARI). It's a droplet borne infection spreading through direct contact with the patient or through fomites. The lesser common symptoms includes production of sputum, headache, GI symptoms like diarrhoea, nausea, vomiting 1,5 , thus exhibiting gut-lung crosstalk. Some studies have reported diarrhoea in the range of 1-3.8% patients 1,32-34 and other have observed a greater incidence of these symptoms i.e. nausea in 10.1% and vomiting in 3.6% 5 . These reports highlight the occurrence of gastrointestinal disorders in COVID-19 patients. This was proved by observing viral nucleic acid in anals swabs and fecal samples of COVID-19 patients 2,3,6 as well as in fecal samples of patients who had recovered from COVID-19 infections, 11 days ago 35 .
Earlier studies have reported angiotensinconverting enzyme (ACE-2) as the receptor for binding of SARS-CoV in lung epithelia 36,37 . This receptor is also found on the cholangiocytes in liver and patients of COVID-19 have shown presence of SARS-CoV-2 in liver 38 indicating that this virus is also using ACE-2 as receptor for binding host cell [39][40][41] . As this receptor is found in intestinal epithelia as well 2,3,42-46 ,thus increasing the possibility of its infection by COVID-19. ACE-2 mutants have shown a decrease in expression of antimicrobial peptides in gut and have shown dysbiosis. Thus, it can be inferred that COVID-19 infection may be related to gut microbiota and its dysbiosis 47 .
Yet another study found that cellular serine protease known as transmembrane protease serine 2 (TMPRSS-2) is also necessary for entry of COVID -19 in cells along with ACE-2 receptor 48 .
This expression of TMPRSS-2 was found in lung alveolar type 2 cells, ileum, oesophageal upper epithelial cells and colon. Therefore, COVID-19 can easily gain entry into these enterocytes and cause infection.

Probiotics and Viral Respiratory Tract Infections
According to FAO/WHO 49 , probiotics are defined as "live microorganisms, which when administered in adequate amounts confer a health benefit to the host". Probiotic microorganisms suppress pathogens in host and stimulate the proliferation of epithelial cells and hence provide health benefits to the host. They also regulate the gut microbiota and play an important role in immunomodulation 50 . Relation between gut microbiota dysbiosis and metabolic disorders and chronic low-grade inflammation has been established in different studies 51,52 . Beneficial effect of probiotics in alleviating gastrointestinal diseases has been reported by different randomized clinical trials 53,50 . These benefits may be due to alteration of diversity of gut microbiota by modulating the intestinal immunity, producing growth substrates and by competing for nutrients 54 .
Viral respiratory tract infections are a major cause of severe morbidity and mortality in human adults worldwide. Both innate 55,56 and adaptive 22 immune response play an important role for defence against influenza virus infection. Under in vivo conditions, the innate system recognizes the influenza virus through pattern recognition receptors (PRRs), and lead to generation of adaptive immune response. Ichinohe et al 57 reported reduced influenza virus-specific antibody titre and CD4 T-cell response in antibiotic treated mice along with diminished cytokine secretion and influenza virus specific CTLs. Synthesis of pro IL-1b and pro IL-18 and NLRPs was also found to be diminished.
Oral administration of probiotic microorganisms have been shown to reduce duration and severity of viral infections 58,59 . During influenza infection in mice L. plantarum has shown to stimulate type-I IFN responses and reduced viral titers in lung 60 . In another study, Lactobacillus spp. stimulated production of TNF-á and IFN-ã in nasal lymphocytes during influenza infection has been reported 61 . Ingestion of probiotic cocktail containing Lactobacillus has been shown to stimulate the signal pathways when infected with single-stranded RNA virus 62 .
Similarly,Bifidobacterium breve YIT 4064 stimulates production of anti-influenza IgG in children 63 . Bifidobacterium spp. has also lead to enhanced B-cell and T-cell immunity, IFN release and NK cells response 64 . Even L. rhamnosus GG (ATCC 53103) consumption in children decreased incidence of respiratory tract infections during winter season 65 . In a study by de Vrese et al 66  Amongst the elderly, the fourth most common cause of death is influenza and pneumonia 70,71 , while 77% deaths are due to infection in gastrointestinal tract 72 whereas as reportedly ninety percent due to respiratory tract infections 73 . This age-related diminution of innate and acquired immune system can be controlled by inclusion of functional foods like probiotics in daily diet. L. casei DN 114001 provided through a fermented dairy product reduced the duration of infection of gastrointestinal tract and respiratory tract, especially for upper respiratory tract infection like rhinopharyngitis. Others like L. paracasei and L. johnsonii reduced incidence of infection and improved systems of symptoms of respiratory tract infections 74,75 .
Probiotics can also regulate the immune system of the intestine as they produce many factors and metabolites which aid in the immunomodulation of the intestine 53 . Under in vitro conditions,L. reuteri 100-23 has been shown to reduce IL-2 production in bone marrow derived dendritic cells (BMDCs) and increase production of transforming growth factor beta (TGF-b) in mice 76 .L. reuteri 100-23 colonization has shown to increase number of FoxP3 positive cells in spleen and mesenteric lymph nodes. This suggests that L. reuteri 100-23 regulates inflammation of the gastrointestinal tract by recruiting immune cells and it also regulates recruitment of T-cells to gut epithelium.
Bacteria of genus Bacillus are one of the largest known producers of antimicrobials, out of which more than 795 have been identified 77 . These peptides have shown antibacteria 78 , antifungal 79 and antiviral propert 80 . Some species of this genus have also shown probiotic properties [81][82][83] . In one such study, probiotic B. subtilis 3 was able to inhibit influenza virus replication under in vitro conditions and saved 30% of mice from death due to the same virus 12 . It also produced a peptide,P18 which showed homology to Influenza A neutralizing antibody.It had the ability to inhibit the virus completely in vitro from concentrations ranging from 12.5-100 µg/ml.In mice, it proved as a better treatment by preventing 80% of mice from death due to Influenza as compared to oseltamivir phosphate treatment i.e. Tamiflu,which provided protection to 70% of mice.Overall, it was found more effective than oseltamivir phosphate for elimination of virus after its infection in mice.

Probiotics and COVID-19
Many different studies have shown that ventilator-associated pneumonia and enteritis can be reduced by modulating the gut microbiota 48. As of now there is absence of data on whether gut microbiota modulation can help in alleviating symptoms of COVID-19. However, according to guideline of China's National Health Commission and National Administration of Traditional Chinese Medicine, probiotics may prove useful in treating dysbiosis and preventing secondary bacterial infections and thus treating COVID-19 patients successfully 84 .
Lactobacillus rhamnosus GG, a probiotic bacterium, has shown its role in improving gut/ lung barrier and intestinal homeostasis. It has further shown to down-regulate pro-inflammatory cytokines, up-regulate regulatory T cells and also to better anti-viral defense in respiratory infections 85,86 . These immunomodulatory actions of Lactobacillus rhamnosus GG can certainly help individuals with COVID-19, or persons at risk of contracting the disease.

CONCLUSION
The review of literature done suggests that probiotic bacterial genera Lactobacillus, Bifidobacterium and Bacillus have shown great impact on the reinforcement of the immune system and at the same time have balanced the intestinal dysbiosis to ensure improved gut-lung crosstalk. The use of these probiotics for prevention and treatment of COVID-19 requires in-depth scientific study using metaanalysis and randomized clinical trials. At the same time, they can be used as supportive or alternative therapeutic agents that can build good immune responses for better prognosis of COVID-19. Groves H.T., Cuthbertson L., James P., Moffatt