**4. Efficacy of probiotics on respiratory diseases**

Studies report that probiotics can play a significant role in treatment and preventing or reducing the duration of respiratory system diseases [54–56] and it might be a practical alternative to promote recovery from these diseases [57, 58]. Besides, some evidence showed that probiotics may stimulate respiratory immunity by improving T regulatory response in the airway and enhance resistance to respiratory tract infections caused by bacteria and viruses [59–61]. Probiotics impact lung microbiota [62] and then exert anti-inflammatory activity in the lungs [63]. For instance, orally administered of *Lactobacillus helveticus* can modulated the immune response in a positive way [64–67]. And this probiotic strain by increasing the number of cells secreting IgA in the intestine and bronchial-associated lymphoid tissue has an immunoprotective impact on mucosal immunity [68]. Evidence indicates that lactic acid bacteria and their metabolites move from gut to lung and exert various immunomodulatory actions [69]. Different probiotic strains including *Bifidobacterium* and *Lactobacillus* were reported to be a great option in treating rotavirus infection in both animals and humans [70–72]. Besides, among chronic respiratory diseases, some probiotic strains have improved at least one symptom of allergic rhinitis (AR). In particular, *Lactobacillus paracasei* LP-33 can enhance the quality of life of patients with AR, according to a meta-analysis study carried out recently [73].

### **4.1 Influenza**

An animal study was done by Lu et al. [69] evaluated the effect of *Lactobacillus mucosae* 1025, *Bifidobacterium breve* CCFM1026, and their mixture on mice infected with the influenza virus for 19 days. The result shows that the clinical symptoms were improved by probiotic treatments. *L. mucosae* 1025 could directly decrease viral loading in the lung, and *B. breve* CCFM1026 might alter the immune responses. However, the mixture reduced viral loading and increased the antiviral protein MxA expression, which none of the single strains alone were not able to increase MxA expression. It is reported this is because of increasing the amount of butyrate production resulting from changing the gut microbiota composition [69]. In addition, other probiotic strains which are tested in mice with influenza infection and their effectiveness were determined are *Bacillus subtilis* 3 [74], *Lactobacillus rhamnosus* M21 [75], *Bacillus subtilis* PY79 [76]. Studies indicate *Bacillus subtilis* 3 is not only effective for the prevention and treatment of influenza but also helpful in the prevention and treatment of bacterial infections in both animal models [77, 78] and humans [78, 79]. The antibacterial property of this strain is due to its ability to produce an antibiotic named

aminocoumarin which can suppress a wide range of pathogens and also strengthen host resistance [80]. Furthermore, a double-blinded, placebo-controlled trial of the combination of different probiotic strains containing *Lacticaseibacillus paracasei* subsp*. paracasei, Lactobacillus casei* 431 and *Lactobacillus fermentum* PCC on patients with a common cold and influenza-like infections was conducted by Zhang et al. [81]. In this trial, probiotic mixture 50–60% compared to the placebo group decreased the outbreak of common cold and influenza-like symptoms [81].

### **4.2 Covid-19**

The evidence indicates that gut microbiota dysbiosis happened in COVID-19 patients even 6 months after recovery [82]. Restoring gut microbiota balance has been demonstrated to promote host resistance to viruses or invading pathogens at the respiratory mucosa level [83, 84]. Therefore, administration of prebiotics and probiotics are suggested to COVID-19 patients, to modulate the balance of gut microbiota and decrease the risk of secondary infection due to bacterial translocation [85].

In Covid-19 infected patients, reducing probiotic strains, especially *Lactobacillus* and *Bifidobacterium* may postpone recovery. Consequently, it is suggested that hostmicrobiota balance should be preserved in the gut and lung which can be beneficial in fighting against COVID-19 [86]. Besides, Mahooti et al. [87] recommend that because probiotics have antiviral properties against other viruses, so they can be a complementary treatment against SARS CoV-2.

In numerous human studies, it has been shown that probiotics, especially, *Lactobacillus rhamnosus* GG, has the ability to improve the barrier of intestinal and lung and homeostasis, by increasing regulatory T cells, enhancing anti-viral defense, and reduce pro-inflammatory cytokines in systemic and respiratory infections. These immunomodulatory agents may be helpful in individuals who have been infected or are at risk of developing, COVID-19 [88].

### **4.3 Pneumonia**

It seems probiotic therapy is a fascinating option as a nonantibiotic method for protection of the host microbiota balance and VAP prevention. Probiotics may probably decrease the incidence of VAP through diverse local and systemic effects that limit the colonization of pathogen species or improve host immune defenses [89]. Numerous studies confirmed the promising efficacy of probiotics on the prevention of VAP [90–95].

A RCT shows that the combination of four probiotic strains including *Lactobacillus acidophilus LA-5*, *Lactobacillus plantarum*, *Bifidobacterium lactis* BB-12, and *Saccharomyces boulardii* could reduce the incidence of VAP by 11.9%, the time of stay in the intensive care unit (ICU) and the length of hospital stay [96]. In addition, a clinical trial shows that using a mixture of probiotic supplements containing *Lactobacillus*, *Bifidobacterium*, and Streptococcus spp. for 14 days, can significantly reduce the length of ICU and hospital stays in VAP patients [97].

Despite a meta-analysis of including 15 randomized controlled trials involving 2039 patients which reported that probiotic therapy could prevent ventilator-associated pneumonia (VAP), and decrease the duration of antibiotic treatment of VAP, however, they had not shown any significant impact on the duration of mechanical ventilation, ICU length of stay and mortality [98]. Another meta-analysis also confirmed that there was no remarkable difference in ICU length of stay between probiotics and placebo groups [92].

Among the investigation we have done on reviewing probiotic meta-analysis studies until 2020, out of 6 studies on pneumonia, 5 of them related to VAP, and one study was on nosocomial pneumonia. The statistics show that probiotic supplementation could be 66.66% effective to prevent ventilator-associated pneumonia.
