Use of bacterial compositions in the treatment and / or prevention of respiratory tract diseases

Abstract

The present invention relates to the use of a bacterial composition having an arginine deaminase activity (inhibition of nitric oxide synthase by converting L-arginine to L-citrulline) of 11,000 μmol L-citrulline / h / g of composition and a sphingomyelinase (ceramide production) activity of 0.01-1,000 nanomoles ceramide / h / g of composition in the treatment and / or prevention of respiratory tract diseases caused by viruses such as influenza viruses, coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, avian viruses, respiratory syncytial virus (RSV), rhinoviruses, pneumoviruses, or respiratory failure induced or exacerbated by viral infection, angina pectoris, cor pulmonale, dyspnea, pulmonary edema, neonatal asphyxia, myocardial infarction, heart failure, respiratory failure, Pickwickian syndrome, pulmonary hypertension, pneumonia, or even cystic fibrosis (CF) exacerbated by viral infection.

Description

[Technical Field]

[0001] The present invention relates to the use of a bacterial composition having an arginine deaminase activity (inhibition of nitric oxide synthase by converting L-arginine to L-citrulline) of 11,000 μmol L-citrulline / h / g of composition and a sphingomyelinase (ceramide production) activity of 0.01-1,000 nanomoles ceramide / h / g of composition in the treatment and / or prevention of respiratory tract diseases caused by viruses such as influenza viruses, coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, avian viruses, respiratory syncytial virus (RSV), rhinoviruses, pneumoviruses, or respiratory failure induced or exacerbated by viral infection, angina pectoris, cor pulmonale, dyspnea, pulmonary edema, neonatal asphyxia, myocardial infarction, heart failure, respiratory failure, Pickwickian syndrome, pulmonary hypertension, pneumonia, or even cystic fibrosis (CF) exacerbated by viral infection. [Background technology]

[0002] The recently observed coronavirus disease 2019 (COVID-19), similar to that caused by SARS-CoV, is characterized by an exaggerated inflammatory response induced by a cytokine storm that correlates with disease severity (Huang, C. et al., 2020; Peiris, J.S.M. et al., 2003). Patients requiring intensive care units (ICUs) have higher plasma levels of many cytokines, including IP-10, MCP-1, MIP-1A, and TNF-α, than non-ICU subjects (Prompetchara, E. et al., 2020), suggesting that a proinflammatory state may play a role in disease progression and severity. Furthermore, high infiltration of inflammatory cells has been observed in the lungs of severely affected COVID-19 patients (Xu, Z. et al., 2020; Tian, ​​S. et al., 2020). These abnormal pathogenic cells enter the pulmonary circulation along with inflammatory monocytes and play a detrimental role in the immune response, causing pulmonary dysfunction, hypoxemia, organ damage, and rapid death.

[0003] The activity of enzymes and metabolites produced by bacteria belonging to the genera Lactobacillus, Bifidobacterium, and Streptococcus is the basis for the action these microorganisms can play against viral infections. In this context, considerable interest has been aroused in the production of the bacterial enzymes sphingomyelinase (SMase) (Lew, L.C. and Liong, M.T., 2013) and arginine deaminase (ADI) (Cunin, R. et al., 1986), which can catalyze the formation of ceramides similar to those present in eukaryotic membranes and regulate the production of nitrate, respectively, through competitive inhibition of nitric oxide synthase 2 (NOS2).

[0004] Regarding SMase activity, ceramides generated by the catalytic activity of this enzyme play important roles in different physiological processes, such as cell membrane remodeling, migration, proliferation, differentiation, and cell death, through a series of signaling cascades. Several studies have highlighted that ceramides can inhibit the replication of various viruses, including those that cause influenza (Tian, ​​Y. et al., 2019), suggesting that manipulating the metabolism of such biopolymers may represent a therapeutic approach to combat viral infections (Dai, L. et al., 2015; Finnegan, C. M. et al., 2004; Darwiche, N. et al., 2005; Pritzl, C. J. et al., 2015). Recently, sphingomyelinase enzyme activity has been reported to be associated with an increased ability of the SARS-CoV-2 virus to infect and replicate in susceptible human cells. Mechanistically, acid sphingomyelinase is involved in the formation of ceramide-rich membranous platforms susceptible to viral infection mediated by the spike protein (Carpinteiro et al., 2020; Schloer et al., 2020). Inhibition of this enzyme is associated with a reduced risk of SARS-CoV-2 infection.

[0005] Nitric oxide (NO) is a potent signaling molecule synthesized by many human cell types. It also regulates the functional activity, proliferation, and death of many immune and inflammatory cell types, including macrophages, T lymphocytes, and neutrophils. Inducible nitroxide synthase 2 (NOS2) constitutes the primary enzyme responsible for high-level NO synthesis (Green, SJ. et al., 1994; Bhat, N.R. et al., 1999). Systemic inhibition of NO and subsequent modulation of inflammatory responses have been reported as a protective factor against adverse events associated with viral infections (Perrone, L.A. et al., 2012; Akaike, T. et al., 1996). Although conflicting results have been reported for various diseases of viral origin, treatment with nitric oxide (NO) has been shown to have clear beneficial effects against SARS-CoV infection (Chen, R. et al., 2004; Darwish, I. et al., 2009). Recently, it has been proposed that NO administration could be effectively used to treat COVID-19 patients based on the antiviral activity exhibited by this molecule, as well as the central role that NO plays in regulating the immune response and preventing the cytokine storm characteristic of SARS-CoV-2 infection (Adusumilli, N.C. et al., 2020). Summary of the Invention [Problem to be solved by the invention]

[0006] In contrast to hypotheses pointing to inhibition of sphingomyelinase activity and NO delivery as a possible treatment for COVID-19 patients, Applicants have surprisingly found that direct administration to the airways or rectal administration of specific bacterial compositions comprising bacteria rich in sphingomyelinase and arginine deiminase that inhibit nitroxide production can improve oxygenation, antiviral and immune responses in subjects with hypoxia and / or hypoxemia, whether or not associated with an infection. [Means for solving the problem]

[0007] Summary of the invention The object of the present invention is the use of a bacterial composition having an arginine deaminase activity of 11,000 μmol L-citrulline / h / g of composition (inhibition of nitric oxide synthase by conversion of L-arginine to L-citrulline) and a sphingomyelinase (ceramide production) activity of 0.01 to 1,000 nanomoles ceramide / h / g of composition in the treatment and / or prevention of respiratory tract diseases caused by viruses such as influenza viruses, coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, avian viruses, respiratory syncytial virus (RSV), rhinoviruses, pneumoviruses, or respiratory failure induced or exacerbated by viral infection, angina pectoris, cor pulmonale, dyspnea, pulmonary edema, neonatal asphyxia, myocardial infarction, heart failure, respiratory failure, Pickwickian syndrome, pulmonary hypertension, pneumonia or even cystic fibrosis (CF) exacerbated by viral infection.

[0008] A further object of the present invention relates to the use as described above, wherein the bacterial composition comprises one or more strains of the species Lactobacillus brevis and Lactobacillus plantarum.

[0009] A further object of the present invention relates to the above use, wherein the bacterial composition comprises 99.99% to 0.01% of one or more strains of the species Lactobacillus brevis and Lactobacillus plantarum, and optionally at least one pharmaceutically acceptable excipient.

[0010] A further object of the present invention relates to the above use, wherein in addition to Lactobacillus brevis and Lactobacillus plantarum, the bacterial composition comprises one or more strains of species selected from Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus and / or Bifidobacterium animalis subsp. lactis, and optionally at least one pharmaceutically acceptable excipient.

[0011] A further object of the present invention relates to the above use, wherein the bacterial composition comprises 1-70% by weight of Lactobacillus brevis, 1-30% by weight of Lactobacillus plantarum, 1-20% by weight of Lactobacillus paracasei, 1-20% by weight of Lactobacillus acidophilus, 1-20% by weight of Lactobacillus helveticus, 1-70% by weight of Streptococcus thermophilus, and 1-40% by weight of Bifidobacterium animalis subsp. lactis, and optionally at least one pharmaceutically acceptable excipient.

[0012] A further object of the present invention relates to the above mentioned uses, wherein the bacterial composition is suitable for oral administration, such as in the form of a powder, capsule, granules or lipogel.

[0013] A further object of the present invention relates to the above use, wherein the oral composition has a high concentration of bacteria in the range of 50 to 800 billion.

[0014] A further object of the present invention relates to the above-mentioned use, wherein the bacterial composition is suitable for insertion into and use in conjunction with a facial mask or a non-invasive ventilation (NIV) device.

[0015] A further object of the present invention relates to the above use, wherein the bacterial composition is suitable for administration via the respiratory tract, such as by inhalation, insufflation, or in the form of a powder, solution, suspension or dispersion spray or aerosol fluid, or nasal drops, preferably aerosol fluid.

[0016] A further object of the present invention relates to the above use, wherein the administration is in the form of a fluid or powder by aerosol or insufflation, comprising at least 0.001% by weight of Lactobacillus brevis and at least 0.001% by weight of Lactobacillus plantarum and saline or purified water.

[0017] A further object of the present invention relates to the above use, wherein the administration is in the form of a fluid or powder by aerosol or insufflation, comprising 1-70% by weight of Lactobacillus brevis, 1-30% by weight of Lactobacillus plantarum, 1-20% by weight of Lactobacillus paracasei, 1-20% by weight of Lactobacillus acidophilus, 1-20% by weight of Lactobacillus helveticus, 1-70% by weight of Streptococcus thermophilus, and 1-40% by weight of Bifidobacterium animalis subsp. lactis, and saline or purified water.

[0018] A further object of the present invention relates to the above use, wherein the aerosol or insufflation fluid or powder composition contains a high concentration of bacteria ranging from 100,000 to 50 billion. .

[0019] A further object of the present invention relates to the above use in the form of an oral, buccal or rectal, preferably rectal, lipogel, wherein the composition contains a high concentration of bacteria ranging from 1 to 800 billion per gram.

[0020] A further object of the present invention relates to the above-mentioned uses, wherein the composition is used in combination with a further treatment, in particular oxygen therapy or ozone therapy.

[0021] A further object of the present invention relates to the above-mentioned use, wherein the bacteria used in the composition of the invention are viable, non-viable, sonicated, Tyndallized or freeze-dried.

[0022] The compositions of the present invention allow for achieving an increase in anti-inflammatory activity of at least 30% compared to an equivalent control composition, as measured by determining respiratory or systemic inflammation values ​​(erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), D-dimer, or ferritin). The compositions of the present invention also allow for antagonizing the decrease in oxygen detectable in arterial blood or capillaries, thereby improving hypoxemia, hypoxia, and / or organ and tissue function.

[0023] The compositions of the present invention can be prepared according to methods known in the art. [Brief explanation of the drawings]

[0024] [Figure 1] 1 shows the formation of NO catalyzed by NOS. [Figure 2] We show how some commensal gut bacteria produce nitrate reductase and nitrite reductase, thereby generating NO. [Figure 3] Possible mechanisms by which NO levels increase under hypoxic conditions are presented. [Figure 4] 1 shows the L-arginine deiminase pathway. [Figure 5] Trends in oxygenation parameters 6 hours after the start of treatment are shown: a) PaO2 / FiO2 ratio, b) blood oxygen tension (pO2), c) inspired oxygen fraction (FiO2), d) oxygenated hemoglobin (O2Hb), and e) oxygen saturation (sO2) for a representative group of patients treated with best available therapy (BAT) and subjects additionally supplemented with oral bacteriotherapy. [Figure 6] Boxplots are shown for the distribution of (a) PaO2 / FiO2 ratio, (b) blood oxygen tension (pO2), and (c) fraction of inspired oxygen (FiO2) values ​​within the groups treated with oral bacteriotherapy for COVID-19 and the group treated with best available therapy (BAT) at the start of treatment and 24 hours later. Statistical significance between the two groups at each time point considered, and between consecutive time points within each group, was reported, if present. [Figure 7] Boxplots depicting the distribution of values ​​for (a) oxygenated hemoglobin (O2Hb) and (b) percentage of oxygenated hemoglobin and oxygen-saturated hemoglobin (sO2) within the groups treated with oral bacteriotherapy for COVID-19 and the groups treated with best available therapy (BAT) at the start of treatment and 24 hours later are shown. Statistical significance between the two groups at each time point considered, and between consecutive time points within each group, was reported, if present. [Figure 8] Boxplots are shown for the distribution of (a) blood glucose, (b) lactate, and (c) hematocrit values ​​within the groups treated with oral bacteriotherapy for COVID-19 and the groups treated with best available therapy (BAT) at the start of treatment and after 24 hours. Statistical significance between the two groups at each time point considered, and for each group between consecutive time points, was reported, if present. [Figure 9] 1 shows bar graphs of the disappearance of diarrhea (a) and other symptoms (b) at different successive time intervals in subjects treated with the bacteria of the present invention and in untreated control subjects. The presence of statistically significant differences between the two groups of subjects was reported at each time interval. p≦0.05 was considered statistically significant. [Figure 10] Longitudinal analysis of data on the occurrence of respiratory failure in relation to the control and treatment groups, performed by generalized linear mixed model (GLIMMIX), is shown. Odds ratios (ORs), relative 95% confidence intervals (95% CIs), and statistical significance (p) were reported for each time point. p ≤ 0.05 was considered statistically significant. [Figure 11] Mortality in the ICU ward in the two groups (BAT vs. BAT treatment + oral bacteriotherapy) is shown, where BAT stands for "best available therapy." [Figure 12] Probability of death according to Kaplan-Meier curves showing BAT treatment and BAT + oral bacteriotherapy, where BAT means "best available treatment." [Figure 13] Multivariate forest plot analysis with 95% confidence intervals is shown. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention Experimental Part Coronavirus disease studies are being conducted in humans. Human Research - Introduction Understanding the invasive process of SARS-CoV-2 is essential to understanding this disease. It is known that viral entry routes into the body, such as the ACE2 receptor, are enzymes expressed by intestinal cells. Coronaviruses constantly change their binding patterns as they evolve, altering their potential targets in the lungs. In contrast, these targets tend to remain constant at the intestinal level. Therefore, intestinal mucosal cells (enterocytes) may be a reservoir for coronaviruses (Feng, Z. et al., 2020). COVID-19 affects both lungs. The alveoli become fluid-filled, reducing their ability to absorb oxygen, leading to shortness of breath, coughing, and other symptoms. In some individuals, respiratory problems can become so severe that they require supplemental oxygen or external respiratory support. In such cases, oxygen therapy, a method used by medical professionals to increase the partial pressure of oxygen in the blood (pO2), often requires invasive medical intervention.

[0026] The severity of hypoxemia (lack of oxygen in the blood) was strongly correlated with high levels of immune cells and markers of inflammation. The vicious cycle between hyperinflammation and gut dysbiosis appears to be associated with an increased risk of organ damage and respiratory distress, which can lead to a fatal outcome. During the acute phase, viral DNA is detectable in the blood in only 10% of patients, but in the feces of 50% of analyzed subjects. Intestinal lesions may also explain the wide variation in viral load determined by different tests performed on the same individual, suggesting that the gut environment could serve as a reservoir where the virus can hide and remain.

[0027] Surprisingly, we found that administering specific bacterial mixtures significantly increased oxygenation levels in severely ill SARS-CoV-2-infected patients, even in a very short period of time, compared with subjects treated with conventional drug therapy alone. These mixtures also reduced the rate of disease progression and mortality. Notably, improvement in hypoxemia has never been reported before, even for other diseases affecting humans. The rapid improvement in oxygen levels in subjects treated with oral bacteriotherapy has many clear advantages, first of all, the contrast with the complications typical of acute respiratory distress syndrome (ARDS) associated with COVID-19. Furthermore, increasing the amount of oxygen available to organs and body tissues in a short period of time can postpone the deleterious / injurious effects associated with acute hypoxia, thereby extending the time window during which COVID-19-infected patients can be effectively treated with pharmacological therapy and benefit from the antiviral and protective effects of the formulations of this invention. The bacterial strains present in the present formulations are rich in sphingomyelinase (SMase), which hydrolyzes sphingomyelin, resulting in the production of ceramide (Leyer, GJ. et al., 2009; Lew, L.C. and Liong, M.T., 2013). Ceramide plays an important role in various physiological processes, such as cell membrane remodeling, migration, proliferation, differentiation, and cell death, through a series of signaling cascades. Several studies have highlighted that ceramide can inhibit the replication of various viruses, including those that cause influenza (Tian, ​​Y. et al., 2019), suggesting that manipulating the metabolism of such biopolymers may represent an effective therapeutic approach to combat viral infections (Dai, L. et al., 2015; Finnegan, C.M. et al., 2004; Darwiche, N. et al., 2005; Pritzl, C.J. et al., 2015).

[0028] This dual mechanism of action makes the specific bacterial formulations claimed herein an option for the treatment of SARS-CoV-2 infection.

[0029] The estimated surface area of ​​the digestive tract is approximately 250–400 m 2 This body region is the site of physiological inflammation generated by luminal contents, which exerts a protective effect against many pathogens. The balance controlling physiological inflammation is disrupted by dysbiosis and acute infections. Under normal conditions, the intestinal mucosa receives 10% to 35% of the total cardiac output. The arteries supplying the intestine are subject to vasoconstriction or vasodilation. These processes regulate blood flow to the viscera and, when necessary, increase blood supply, as occurs during the digestive process. Conversely, in the presence of hypoxia, the processes of vasoconstriction and vasodilation reduce blood supply to the intestine, allowing more oxygen to be available to priority organs for survival, such as the heart and brain.

[0030] Metabolic regulation of mucosal vasodilation is typically attributed to nitric oxide (NO). NO is a gas signaling molecule with autocrine and paracrine activity. It acts by diffusing into vascular smooth muscle, inducing vasodilation. NO is produced by the activity of enzymes called nitric oxide synthases (NOS). These enzymes are classified as constitutive NOS, which is dependent on calcium and calmodulin, and inducible NOS (iNOS, also known as NOS2), which is expressed by macrophages and other cells upon proinflammatory cytokine activation. As shown in Figure 1, NOS enzymes use L-arginine and oxygen as substrates to catalyze NO formation. This reaction consumes 1.5 moles of NADPH and 2 moles of oxygen. As shown in Figure 2, luminal bacteria can also contribute to NO production near the intestinal epithelium via the inorganic nitrate reduction pathway.

[0031] When arterial oxygen levels decrease, increased blood flow (hypoxic vasodilation) occurs to restore oxygen supply. COVID-19-associated hypoxic vasodilation is an adaptive response involving increased local NO concentrations in response to the sudden drop in arterial partial pressure of oxygen caused by extensive lung injury. This phenomenon involves various mechanisms, including increased synthesis of NO by NOS, increased reduction of NO to NO by heme- or pterin-based enzymes, increased release of NO from the latter storage form, and reduced inactivation by mitochondrial cytochrome C oxidase (CcO).

[0032] Figure 3 shows possible mechanisms for increased NO levels under hypoxic conditions, including [1] increased production from NOS, [2] increased NO release from storage forms such as RSNOs, [3] increased reduction from NO2, and [4] decreased metabolism from CcO.

[0033] The mixture of the present invention has the ability to inhibit NOS2 enzyme activity in an in vitro model by producing the enzyme L-arginine deiminase (ADI). L-arginine deiminase (ADI) is an enzyme widely distributed within the lactic acid bacteria group (Cunin, R. et al., 1986). Organisms utilizing this pathway convert L-arginine into L-citrulline and ammonia. Figure 4 shows the biosynthetic pathway catalyzed by the enzyme L-arginine deiminase.

[0034] Due to its high binding affinity for L-arginine, this enzyme is able to reduce the amount of this amino acid available to NOS2 synthesized by intestinal epithelial cells, thereby reducing the amount of NO produced by them. This evidence allows the hypothesis that the bacterial strain contained in Sivomixx® interferes with NO production in the intestinal lumen.

[0035] Results from a study conducted on severe COVID-19 patients treated with conventional drug therapy alone or supplemented with Sivomixx® unexpectedly showed that the latter experienced significant improvements in blood oxygenation levels 4 to 6 hours after the first administration of probiotics. This improvement in blood oxygenation parameters over such a short period is surprising, especially considering the degree of pulmonary dysfunction exhibited by the patients at the start of treatment and the extremely low likelihood that the lungs' effectiveness in transporting oxygen from the atmosphere into the bloodstream will recover. In this context, the observed improvement in oxygenation parameters, present as early as a few hours after the initial ingestion of the bacterial strains included in this invention, suggests a metabolic mechanism of action associated with probiotic administration. Figure 5 shows the changes in oxygenation parameters 6 hours after the first treatment with the bacterial strains in a representative group of patients. Notably, the inhibition of NO production to improve the prognosis of such patients stands in stark contrast to a very recent article reiterating the usefulness of treating COVID-19 patients with NO (Adusumilli, N.C. et al., 2020).

[0036] First human studies In severe cases, COVID-19 affects both lungs, infecting alveolar type II cells, which express ACE2, and occasionally type I cells. In addition to damage to type I and type II cells, extensive endothelial damage occurs, accompanied by plasma protein permeability, hyaline membrane formation, and inflammatory exudates, impairing alveolar function. As the virus rapidly grows and spreads, it becomes increasingly difficult for the body to absorb oxygen. When the lungs alone cannot provide the body's necessary oxygen supply, treatment options such as high-flow nasal cannula (HFNC), noninvasive positive pressure ventilation (NIPPV), intubation and invasive mechanical ventilation, or extracorporeal membrane ventilation (ECMO) are commonly applied. The goal of these treatment strategies is to provide external respiratory support to ensure sufficient oxygen to the organs while waiting for lung function to recover.

[0037] The total membrane surface area of ​​the alveoli is approximately 100 m 2, representing a highly efficient gas exchange interface. However, in accordance with both the physical laws governing surface tension and Laplace's law, during acute respiratory distress syndrome (ARDS) and pneumonia, alveoli tend to become completely overflowing with exudate, lose their function, or fail to flow at all, without any change in activity. No matter how useful, oxygen therapy cannot restore the function of a significant number of non-functioning alveoli. It is also important to consider that the amount of oxygen administered to an individual cannot exceed a maximum threshold due to the toxicity of this gas and the pressure it exerts on the alveoli. This, combined with the impossibility of increasing the respiratory surface area and restoring damaged areas in a short time, poses serious problems for the oxygenation of the patient's organs and tissues.

[0038] Organs require sufficient amounts of oxygen to function efficiently, and when a patient becomes infected with COVID-19 or experiences severe respiratory distress for various reasons, the only solution is mechanical ventilation, with all the risks that this entails.

[0039] The effect of the inventive formulation on the production of nitric oxide (NO) in the intestinal lumen has been demonstrated, since the administered probiotic product has the associated ability to inhibit the activity of the enzyme nitric oxide synthase 2 (NOS2) due to the presence in the formulation of bacteria that produce L-arginine deiminase (ADI). The reduction in NO production leads to a decrease in the oxygen consumption required for such production, resulting in an increase in the bioavailability of oxygen itself in the circulatory system. This is because the estimated surface area of ​​the gastrointestinal tract is approximately 250-400 m. 2 This is even more important considering that under normal conditions, the intestinal mucosa receives 10%–35% of the total cardiac output. Because the arteries supplying the intestine are susceptible to NO-mediated vasoconstriction or vasodilation, local inhibition of such vasodilation and oxygen sparing affects peripheral blood levels by reducing hypoxemia.

[0040] patient Enrolled patients (n=69; mean age 63.2±16.3 years) tested positive for COVID-19. COVID-19 was diagnosed by two oropharyngeal and nasopharyngeal swabs for detection of SARS-CoV2 E and S genes using a polymerase chain reaction-based molecular method. All patients studied had the following characteristics: fever >37.5°C, need for noninvasive oxygen therapy, and pulmonary involvement of 50% or more as determined by CT scan. No enrolled patients required intensive care upon admission.

[0041] Before the start of treatment and at the end of the next 24 hours, each enrolled subject underwent a blood sample useful for blood gas analysis using standard methods. The parameters considered in this study were partial pressure of oxygen (pO2), fraction of inspired oxygen (FiO2), oxygenated hemoglobin (O2Hb), reduced hemoglobin (HHb), lactate, blood glucose, and hematocrit (HCT). According to the 2012 Guidelines for the Management of Severe Sepsis and Septic Shock, patients were considered to have respiratory failure if their ratio of arterial partial pressure to fraction of inspired oxygen (PaO2 / FiO2) was determined to be less than 300 (Dellinger et al., 2012).

[0042] CT scan High-resolution CT scans were used to identify lung lesions according to the official diagnostic and treatment protocols (6th edition) defined by the National Health Commission of China. Typical CT findings of COVID-19 are considered to be 1) ground-glass opacities, 2) consolidation, 3) reticular patterns, and 4) "crazy-paving" patterns (Ye, Z. et al., 2020).

[0043] Oral bacteriotherapy The formulation administered in this study contains the following bacteria: Streptococcus thermophilus DSM 32245, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, Lactobacillus brevis DSM 27961, Bifidobacterium animalis subsp. lactis DSM 32246, and Bifidobacterium animalis subsp. lactis DSM 32247. The levels of arginine deaminase (ADI) and sphingomyelinase (SMASI), as well as the ratio of the two enzymes, are important parts of formulation control. The levels were 120 μmol L-citrulline / h / g sphingomyelinase and 90 nmol ceramide / h / g arginine deaminase, respectively, per gram of composition. Patients were treated with a total of 10 grams of bacteria per day. Sphingomyelinase activity was assessed in bacterial extracts (from lyophilized preparations) using a fluorometric assay that assesses ceramide production after hydrolysis of C12-NBD sphingomyelin (N-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}sphingosine-1-phosphocholine) (Ala-baster, Alabama, USA) and is expressed as nanomoles ceramide / hour / g of initial bacterial lyophilizate. Enzymatic activity for arginine deaminase was determined by incubating bacterial extracts (from lyophilized preparations) with L-arginine and then colorimetrically measuring L-citrulline production. Enzyme activity is expressed as μmol L-citrulline / h / g of initial bacterial lyophilisate.

[0044] statistical methods Sample analysis was primarily performed using tables and graphs corresponding to the type of qualitative or quantitative variable. The chi-square test was applied to qualitative clinical variables to assess the presence of statistically significant differences between groups. The two-tailed Mann-Whitney U test was used to assess the presence of statistically significant differences between groups for continuous variables and Charlson index scores. For each group, the presence of significant differences between consecutive time points was assessed by the Wilcoxon signed-rank test. In all cases, a p value of ≤0.05 was considered statistically significant. Where necessary, p values ​​were corrected with the Benjamini-Hochberg procedure to account for multiple comparisons.

[0045] result Currently, there is no structured treatment for the medical treatment of COVID-19 patients, so hospitalized patients were treated with best available therapy (BAT), consisting of antibiotics and / or the antiviral drug remdesivir, possibly supplemented with oxygen. Data were collected and outcomes were compared between COVID-19-positive patients who received such treatment (29 patients; 42%) and those who were additionally treated with oral bacteriotherapy (40 patients; 58%). The main characteristics of patients in both groups are summarized in Table 1. [Table 1]

[0046] At the start of treatment, the two groups, determined based on the administration of Sivomixx® containing the bacterial strain of the present invention, were homogeneous with respect to all parameters considered, except for gender. No significant differences in the distribution of pharmacological treatment or mortality were found between the two groups, although the only death recorded was in the cohort of individuals treated with standard therapy alone.

[0047] Regarding the PaO2 / FiO2 ratio, which indicates the severity of pulmonary dysfunction, no significant difference was observed between the two groups at the beginning of treatment, whereas a significantly higher value of this ratio (a trend toward improvement) was observed in the group receiving Sivomixx® 24 hours after the start of treatment (Figure 6a).

[0048] Observation of the results obtained for the pO2 variable (Figure 6b) showed that at the beginning of treatment, no significant differences were observed between the two groups, while the values ​​of this parameter were significantly higher after 24 hours in the group treated with Sivomixx® compared to the group receiving only BAT. Both groups also showed a significant increase in pO2 24 hours after the start of treatment. The FiO2 values ​​observed at the beginning of the study were not significantly different between the two groups after 24 hours, but the group treated with BAT alone had significantly higher levels of this parameter than the group treated with probiotics (Figure 6c). As shown in Figure 5, the same trend was observed at 6 hours for a limited number of subjects representing the two groups of the study.

[0049] Analysis of oxygenated hemoglobin (O2Hb) levels yielded results completely consistent with those previously described for oxygen. Indeed, at the start of treatment, the two groups were homogeneous for this parameter, but significantly higher levels of O2Hb were found in the group treated with probiotics over the following 24 hours (Fig. 7a). This difference can be explained by the significant temporal increase in O2Hb values ​​in the group treated with Sivomixx® compared to the start of treatment, while subjects treated with BAT alone did not show any significant changes in this parameter. sO2 represents the proportion of hemoglobin in arterial blood that can bind oxygen and is saturated with this gas. This parameter is obtained according to the following formula:

number

[0050] Consistent with that shown for oxygenated hemoglobin, sO2 was not significantly different between the two groups at the start of treatment. Two hours after the start of treatment, significantly higher levels of sO2 were observed in the group administered Sivomixx® than in the group receiving BAT alone, although both groups showed a significant temporal increase in this variable (Figure 7b).

[0051] No significant differences were shown between the groups for the variables blood glucose, lactate, and hematocrit at the start of treatment and 24 hours later (Fig. 8a, b, and c).

[0052] conclusion This study surprisingly showed that a significant increase in blood oxygenation levels was recorded in the group of subjects treated with Sivomixx®, a product containing the bacterial strain of the present invention, which was already evident 24 hours after the first intake of the product. However, this unexpected increase in oxygenation was not related to an increase in the proportion of inhaled oxygen or due to a significant recovery of lung function, which is highly unlikely in the short period considered in this study. In light of the reported evidence and considerations, it is reasonable to assume that the increase in blood oxygenation observed in subjects treated with Sivomixx® does not result from gas exchange in the lungs but from an alternative source related to the administration of the probiotic mixture. In this regard, it is believed that at least part of the increase in blood oxygenation is due to the action of arginine deaminase (ADI) produced by the bacterial strain contained in Sivomixx®. This enzyme can regulate the production of nitric oxide by competitively inhibiting the enzyme responsible for its formation, thereby maintaining a reserve of available oxygen, which is inevitably used in the NO biosynthesis pathway.

[0053] Observational study This study aimed to evaluate the improvement in symptoms and the risk of developing respiratory failure in a representative group of hospitalized patients with a positive diagnosis of COVID-19 who were treated with the probiotic mixture Sivomixx® in addition to conventional drug therapy (hydroxychloroquine (HCQ), antibiotics (ABX), and / or tocilizumab (TCZ)). COVID-19-positive subjects (n=42) admitted to the same ward during the same period, from March 14, 2020, to April 4, 2020, constituted the control group treated with conventional therapy alone. The obtained results provide evidence of the importance of the gut-lung axis in COVID-19 infection, and its modulation induces a rapid and significant improvement in the clinical picture and a significant reduction in the risk of developing pulmonary dysfunction and / or the occurrence of a fatal event (no deaths were recorded in the probiotic-treated group compared to four deaths in the control group). With all necessary precautions taken, the data demonstrate that effective therapeutic action at the gut level induces beneficial responses at the lung level.

[0054] patient A total of 70 patients (mean age 59.9 ± 14.2 years) who tested positive for COVID-19 were enrolled in this study. COVID-19 diagnosis was determined by two repeated oropharyngeal and nasopharyngeal swabs for detection of SARS-CoV2 E and S genes using a polymerase chain reaction-based molecular method. Patients presented with dyspnea (44 patients, 62.9%), fever (66 patients, 94.3%), cough (54 patients, 77.1%), fatigue (15 patients, 21.4%), myalgia (4 patients, 5.7%), and diarrhea (33 patients, 47.1%). Fifty-six patients (80.0%) were characterized by the presence of 1 to 6 comorbidities.

[0055] Dyspnea was defined as "a subjective experience of respiratory illness consisting of qualitatively distinct sensations of varying intensity" (Laviolette, L. et al., 2014). Patients were considered to have positive respiratory failure if the PaO2 / FiO2 ratio, determined according to the 2012 Guidelines for the Management of Severe Sepsis and Septic Shock (Dellinger et al., 2012), was less than 300. Acute diarrhea was defined as stools with an increase in water content, volume, or frequency, lasting less than 14 days (Barr, W. and Smith, A., 2014).

[0056] All patients studied did not require ICU support upon admission.

[0057] For each patient, body mass index (BMI), Charlson comorbidity index, oxygen support requirements, and laboratory values, including alanine aminotransferase (ALT), aspartate aminotransferase (ALT), hemoglobin (Hb), pH, bicarbonate (HCO3), lactate, and arterial carbon dioxide pressure (PaCO2), were measured at the start of treatment. Observed arterial oxygen tension (PaO2), fractional inspired oxygen (FiO2), resolution of COVID-19-related symptoms, adverse events, and the number of patients transferred to the ICU were collected 24 hours, 48 ​​hours, 72 hours, and 7 days after the start of oral bacteriotherapy and hospitalization for all patients, regardless of the treatment used. Patients were considered to have positive respiratory failure if the measured PaO2 / FiO2 ratio was less than 300. Because this was a retrospective collection of real-world emergency data, some laboratory data were unavailable. In particular, when significant clinical and respiratory gas exchange improvement was observed, physicians sometimes chose not to repeat follow-up blood gas analysis, viewing it as an unnecessary invasive pain procedure.

[0058] CT scan High-resolution CT scans were used to identify lung lesions according to the official diagnostic and treatment protocols (6th edition) defined by the National Health Commission of China. Typical CT findings of COVID-19 are considered to be 1) ground-glass opacities, 2) consolidation, 3) reticular patterns, and 4) "crazy-paving" patterns (Ye, Z. et al., 2020).

[0059] Oral bacteriotherapy The formulation administered in this study contains the following bacteria: Streptococcus thermophilus DSM 32245, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, Lactobacillus brevis DSM 27961, Bifidobacterium animalis subsp. lactis DSM 32246, and Bifidobacterium animalis subsp. lactis DSM 32247. The levels of arginine deaminase (ADI) and sphingomyelinase (SMASI), as well as the ratio of the two enzymes, are important parts of formulation control. The levels were 120 μmol L-citrulline / h / g sphingomyelinase and 90 nmol ceramide / h / g arginine deaminase per gram of composition. Patients received a total of 2.4 billion bacteria per day in three equal doses. Sphingomyelinase activity was assessed in bacterial extracts (from lyophilized products) using a fluorometric assay that evaluates ceramide production after hydrolysis of C12-NBD sphingomyelin (N-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}sphingosine-1-phosphocholine) (Ala-baster, Alabama, USA) and is expressed as nanomoles ceramide / hour / g of initial bacterial lyophilizate. The enzyme activity for arginine deaminase was determined by incubating bacterial extracts (from lyophilized preparations) with L-arginine and then measuring L-citrulline production colorimetrically. The enzyme activity is expressed as μmol L-citrulline / h / g of initial bacterial lyophilizate.

[0060] statistical methods Sample analysis was performed using tables and graphs corresponding to the type of variable, primarily qualitative or quantitative. To assess the presence of statistically significant differences between groups, the chi-square test was applied to qualitative clinical variables. For continuous variables and Charlson index scores, the two-tailed Mann-Whitney U test was used.

[0061] Longitudinal data analysis for the variable incidence of respiratory failure associated with control versus treatment comparisons was performed using generalized linear mixed models (GLIMMIX), considering a binary model as the distribution and a logit as the connectivity function (GLIMMIX Procedure. SAS / STAT User's Guide. 2009, Version 9.2). All statistical analyses were performed using SAS v. 9.4 (SAS Institute Inc., Cary, NC, USA). A p-value ≤ 0.05 was considered statistically significant.

[0062] result Because there are currently no systematic treatments or guidelines for treating COVID-19 patients, hospitalized patients were treated on a case-by-case basis with hydroxychloroquine, antibiotics, and / or tocilizumab, possibly with oxygen. Data collected were used to compare COVID-19 patients who received such treatment with those who additionally received oral bacteriotherapy.

[0063] The two patient groups determined based on the administration of oral bacteriotherapy were homogeneous with respect to characteristics such as age, sex, BMI, ALS, AST, Hb, Charlson comorbidity index, and the prevalence of COVID-19-related symptoms, such as diarrhea, fever, cough, dyspnea, asthenia, and myalgia. Table 2 provides the characteristics of the patient groups obtained by the administration of bacteriotherapy. p-values ​​are relative to chi-square and Mann-Whitney U tests performed on individual and continuous clinical variables, respectively. A p-value ≤ 0.05 was considered statistically significant. [Table 2]

[0064] Although there was variability in the medications administered, there were no significant differences between groups regarding the number, type, and combination of medications used during the observation period. Table 3 shows the treatments administered to the two groups of patients. As reported in the statistical methods, p values ​​are for nonparametric tests. A p value ≤ 0.05 was considered statistically significant. [Table 3]

[0065] Table 4 shows the respiratory parameters presented by the two groups of subjects at the start of the study. Statistical analysis revealed no significant differences between the two groups in terms of PaO2 / FiO2, indicating the severity of pulmonary parenchymal dysfunction. No significant differences were observed between the two groups for other parameters considered, such as the prevalence of subjects requiring ventilatory support. [Table 4]

[0066] Regarding the resolution of COVID-19-associated symptoms, the results showed that 24 hours after the first administration of Sivomixx®, a product containing the bacterial strain of the present invention, a significantly higher proportion of subjects in the group receiving additional oral bacteriotherapy experienced resolution of diarrhea than the group treated with conventional drug therapy, as shown in Figure 9a. This significant difference persisted for up to seven days, with the difference between the two groups increasing over time. It is noteworthy that within three days of initiating treatment, more than 90% of patients receiving Sivomixx® experienced resolution of diarrhea, and by seven days, this condition had spread to all subjects in the group. Differently, after seven days of treatment, the proportion of subjects treated with conventional drug therapy characterized by resolution of diarrhea was lower than that of the group treated with probiotics 24 hours after initiating treatment.

[0067] Other symptoms considered cumulatively showed similar trends, although the effects within 24 hours of bacterial administration were more limited (Fig. 9b).

[0068] Regarding respiratory outcomes, at admission, there was a significantly higher proportion of patients with respiratory failure in the treatment group than in the control group, as determined by chi-square test (control group 11 / 42, 26.2%; treatment group 14 / 28, 50%; p-value=0.042).

[0069] By applying a generalized linear mixed model, it was possible to show a significant difference in the evolution of respiratory outcomes between the treatment and control groups (p=0.0002). Specifically, as shown in Table 5 and Figure 10, the calculated model showed that after 7 days of treatment, patients receiving Sivomixx® had an eighth-fold lower risk of developing respiratory failure compared to patients receiving conventional therapy. [Table 5]

[0070] Parameters useful for assessing the goodness of fit of the calculated models, such as inferential statistics, estimates of covariance parameters, and Type III tests of fixed effects, are reported in Tables 6-8 below. [Table 6] [Table 7] [Table 8]

[0071] The mortality rate and number of patients transferred from sub-intensive care units to intensive care units are shown in Table 9 below. [Table 9]

[0072] The mortality rate in the control group was consistent with that reported in Italy during the same period of the study (9.9% ± 1.7% deaths / total cases), and all 28 patients treated with oral bacteriotherapy were alive at the end of the observation period.

[0073] conclusion The results from the presented study demonstrate an association between the intake of probiotics Sivomixx® containing the bacterial strain according to the present invention and the rapid resolution of symptoms commonly associated with COVID-19. Such resolution appears to be limited to subjects not receiving treatment over the same time frame. Furthermore, a protective effect of Sivomixx® administration against the development of respiratory failure in SARS-CoV2-infected individuals was demonstrated.

[0074] Retrospective study Below are the results of a larger "real-world" study on the complementary use of a bacterial composition formed by Streptococcus thermophilus DSM 32245, Bifidobacterium animalis subsp. lactis DSM 32246, Bifidobacterium animalis subsp. lactis DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, and Lactobacillus brevis DSM 27961 (Sivomixx®) in a cohort of individuals infected with SARS-CoV2 and receiving best available treatment (BAT).

[0075] material and method The reported study represents a retrospective, "real-world," observational cohort study aimed to compare patients with severe COVID-19-associated pneumonia treated with best available therapy (BAT) with those additionally supplemented with oral bacteriotherapy in terms of crude mortality, need for intensive care unit (ICU) admission, and length of hospital stay. To this end, the study included subjects admitted to the Infectious Diseases Unit of the Policlinico Umberto I, Sapienza University of Rome, between March 6 and April 26, 2020.

[0076] patient A diagnosis of COVID-19 was defined by positive oropharyngeal and nasopharyngeal swabs performed twice in repeat for detection of SARS-CoV2 E and S genes by polymerase chain reaction-based molecular methods.

[0077] The target population included subjects aged 18 years and older.

[0078] All hospitalized patients received hydroxychloroquine (200 mg twice daily for 7 days), azithromycin (500 mg once daily for 7 days), lopinavir-ritonavir (400 / 100 mg twice daily) or darunavir-cobicistat (800 / 150 mg once daily) for 14 days, and low-molecular-weight heparin for deep venous thrombosis prophylaxis, as recommended by the Italian Society of Infectious Diseases at the time. Tocilizumab (8 mg / kg, up to a maximum of 800 mg / dose, twice 12 hours apart) was administered if serum IL-6 levels were elevated or if IL-6 administration was unavailable and respiratory patterns were significantly worse. Patients admitted to the ward with intestinal symptoms received oral bacteriotherapy in addition to BAT. This preparation was administered three times daily in equal doses for a total of 2.4 trillion bacteria per day.

[0079] Variables considered in the study included age, sex, admission and discharge dates, length of stay (LOS); cardiovascular (CV) disease, chronic lung disease, chronic kidney disease (CKD), hypertension, asthma, chronic obstructive pulmonary disease (COPD), diabetes mellitus, immunodeficiency, and cancer (defined as active or past / resolved).

[0080] For each patient, the Charlson comorbidity index was determined to assess the risk of 1-year mortality in the presence of various comorbidities.

[0081] The following parameters were used to define the severity of pneumonia: impaired consciousness-blood urea-respiratory rate-blood pressure score (CURB), impaired consciousness-blood urea-respiratory rate-blood pressure-65 years (CURB-65), impaired consciousness-blood urea-respiratory rate-blood pressure-65 years-lactate dehydrogenase, platelets and albumin (CURB-65 extended), and Pneumonia Severity Index (PSI) score. Comorbidities-age-lymphocyte-lactate dehydrogenase (CALL) were considered to predict the progression of COVID-19.

[0082] statistical analysis Statistical analyses were performed using Statistical Program for the Social Sciences (SPSS), version 22 (IBM SPSS, Chicago, IL). Continuous variables were presented as medians and interquartile ranges (IQR, 25°-75°), while the presence of statistically significant differences between groups was assessed using the nonparametric Mann-Whitney U test. Dichotomous variables were expressed as simple frequencies and percentages (%) and compared using the chi-square test. Multivariate stepwise regression analysis included age ≥65 years, lymphocytes <1,000 per μL of blood, and platelets 150-103 / mm3. 3Several potential confounders, including age < 32 g / dL, albumin < 32 g / dL, CV events, BAT therapy, and oral antimicrobial therapy, were considered as dependent variables. Standard survival analyses were performed by tracking participants from clinic admission to discharge or death. Event-free survival at follow-up was graphically represented by Kaplan-Meier survival curves using multivariable Cox regression analysis, including confounders with fixed baseline covariates. Treatment effects were presented using unadjusted odds ratios (ORs) with adjusted 95% confidence intervals (95% CIs). Major sources of uncertainty were identified for age, C-reactive protein (CRP), Charlson Comorbidity Index, CURB, CURB-65, PSI, CALL, lymphocyte count, and platelet count, likely contributing to both treatment assignment and outcome risk. In all cases, a two-sided p value ≤ 0.05 was considered statistically significant.

[0083] result The data used were from patients with positive SARS-CoV-2 infection who were admitted to the infectious disease ward between March 6 and April 26, 2020. A group of 200 patients, whose demographic and clinical characteristics are listed in Table 10 below, were included in the study. [Table 10]

[0084] Although the duration of symptoms before hospitalization could not be determined for the sample of patients in the study, given the efficient and free medical services in Lazio, it is reasonable to assume that hospitalization occurred within a few days of the onset of respiratory symptoms. The median duration of hospitalization was 15 days [IQR, (10-27)]. Of the 200 patients, 112 received BAT without oral bacteriotherapy, whereas 88 subjects received BAT with oral bacteriotherapy administration. The characteristics of the two groups are shown in Table 11. Oral bacteriotherapy was initiated a median of 1 day (min 0, max 2) after admission. [Table 11] The two groups were homogeneous with respect to all clinical parameters considered, except for C-reactive protein, LDH, and albumin.

[0085] The primary objective of this study was to evaluate the crude in-hospital mortality rate of patients in each treatment group. The crude mortality rate for the total subject population was 22% (44 patients). Comparing the two groups, the mortality rate in the group receiving oral bacteriotherapy was significantly lower than in the group receiving BAT alone (Table 11, Figure 11).

[0086] After adjusting for age, Charlson, CURB, CURB-65, PSI, and CALL scores, with an OR of 0.28, the significant reduction in risk of death present in patients treated with both BAT and oral bacteriotherapy was further confirmed (95% confidence interval, 0.13-0.6, p=0.001). The unweighted Kaplan-Meier curves shown in Figure 12 highlight the beneficial effect of combined BAT and oral bacteriotherapy on the probability of death parameters.

[0087] Based on the multivariate analysis performed, age >65 years, C-reactive protein >41.8 mg / L, platelets <150,000 mm 3 , and cardiovascular events were associated with an increased risk of death, whereas administration of oral bacteriotherapy was an independent variable associated with a decreased risk of death (Figure 13).

[0088] conclusion The analysis of a retrospective cohort clearly showed that in COVID-19 patients, characteristics such as older age, high levels of C-reactive protein, thrombocytopenia, and a history of cardiovascular events are risk factors for determining adverse events, and that administration of one of the formulations of the present invention constitutes an independent variable associated with a significant reduction in the risk of mortality. [Example]

[0089] Formulation Example 1 Lipogel capable of releasing bacterial compositions for use according to the present invention into the oral cavity, nasal cavity and intestine Various oily mixtures gelled with appropriate excipients and via suitable delivery devices have been evaluated for the facile administration of bacteria in the oral cavity, nasal cavity, and intestine.9 The main technical challenges are ensuring the biological stability of the bacteria within the lipogels even after long-term storage and their persistence and survival in the oral cavity after application. Composition of selected lipogels after preformulation testing (wt%): Medium Chain Triglycerides 46% Vitamin E Acetate 50% Microcrystalline Silica 4% Bacterial Loading: In 1 gram of selected lipogel, 0.1 g of bacterial composition can be dispersed for use in the present invention resulting in a concentration of 13.00±0.25 LogCFU / g. Viscosity of formulation: 400 mPa*s. Viability: Viability is maintained for up to 45 days.

[0090] List of References Adusumilli NC, Zhang D, Friedman JM, Friedman AJ. Harnessing nitric oxide for preventing, limiting and treating the severe pulmonary consequences of COVID-19. Nitric Oxide. 2020 Oct 1; 103:4-8; Bhat, NR., Zhang, P., Bhat, AN., "Cytokine induction of inducible nitric oxide synthase in an oligodendrocyte cell line: role of p38 mitogen-activated protein kinase activation", feb 1999, J. Neurochem., 72(2):472-8; Barr, W., Smith, A., "Acute diarrhea," 1 Feb. 2014, Am. Fam. Physician 89(3):180-9; Carpinteiro A, Edwards MJ, Hoffmann M, Kochs G, Gripp B, Weigang S, Adams C, Carpinteiro E, Gulbins A, Keitsch S, Sehl C, Soddemann M, Wilker B, Kamler M, Bertsch T, Lang KS, Patel S, Wilson GC, Walter S, Hengel H, Poehlmann S, Lang PA, Kornhuber J, Becker KA, Ahmad SA, Fassbender K, Gulbins E. Pharmacological Inhibition of Acid Sphingomyelinase Prevents Uptake of SARS-CoV-2 by Epithelial Cells. Cell Rep Med. 2020 Nov 17;1(8):100142; Chen L, Liu P, Gao H, Sun B, Chao D, Wang F, Zhu Y, Hedenstierna G, Wang CG. Inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a rescue trial in Beijing. Clin. Infect. This. 2004 Nov 15; 39(10):1531-5; Cunin R, Gransdorff N, Pierard A, Stalon V. Biosynthesis and metabolism of arginine in bacteria. Rev. Microbiol. 1987 Mar;51(1):178. Erratum for: Rev. Microbiol. 50:3 PMID: 16350242; PMCID: PMC373097; Dai、L.、Trillo-Tinoco、J.、Bai、A.、Chen、Y.、Bielawski、J.、Del Valle、L.、Smith、CD.、Ochoa、AC.、Qin、Z.、Parsons、C.、"Ceramide-induced apoptosis in renal tubular cells: a role of mitochondria and sphingosine-1-phoshate"、2015、Oncotarget 6:24246-24260; Darwiche、N.、Abou-Lteif、G.、Najdi、T.、Kozhaya、L.、Tayoun、AA.、Abou、Tayyoun、Bazarbachi、A.、Dbaibo、GS.、"Human T-cell lymphotropic virus type I-transformed T-cells have a partial defect in ceramide synthesis in response to N-(4-hydroxy-phenyl)retinamide"、2005、Biochem. J. 392:231-239; Darwish I、Miller C、Kain KC、Liles WC. Inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza. Int J. Med. Sci. 2012; 9(2):157-62; Dellinger RP、Levy MM、Rhodes A、Annane D、Gerlach H、Opal SM、Sevransky JE、Sprung CL、Douglas IS、Jaeschke R、Osborn TM、Nunnally ME、Townsend SR、Reinhart K、Kleinpell RM、Angus DC、Deutschman CS、Machado FR、Rubenfeld GD、Webb SA、Beale RJ、Vincent JL、Moreno R; Surviving Sepsis Campaign Guidelines Committee including the Pediatric Subgroup. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit. Care Med. 2013 Feb; 41(2):580-637; Feng、Z.、Wang、Y.、Qi、W.、"The small intestine、an underestimated site of SARS-CoV-2 infection: from Red Queen Effect to probiotics"、Preprints 2020、2020030161; Finnegan、CM.、Rawat、SS.、Puri、A.、Wang、JM.、Ruscetti、FW.、Blumenthal、R.、"Ceramide、a target for antiretroviral therapy"、2004、Proc. Natl. Acad. Sci. USA 101:15452-15457; Green、SJ.、Scheller、LF.、Marletta、MA.、Seguin、MC.、Klotz、FW.、Slayter、M.、Nelson、BJ. e Nacy、CA.、"Nitric oxide: cytokine regulation of nitric oxide in host resistance to intracellular pathogens"、1994、Immunol. Lett. 43:87-94; Huang、C.、Wang、Y.、Li、X et al.、"Clinical features of patients infected with 2019 novel coronavirus in Wuhan、China"、2020、Lancet 395:497-506; Laviolette、L.、Laveneziana、P、ERS Research Seminar Faculty、"Dyspnoea: a multidimensional and multidisciplinary approach"、giu. 2014、Eur. Respir. J. 43(6):1750-62; Lew、LC.、Liong、MT.、"Bioactives from probiotics for dermal health: functions and benefits"、2013、J. Appl. Microbiol. 114(5):1241-1253; Leyer、GJ.、Shuguang、Li、Mohamed E. Mubasher、Cheryl Reifer、Arthur C. Ouwehand、"Probiotic effects on cold and influenza-like symptom incidence and duration in children"、ago. 2009、Pediatrics 124(2):e172-e179; Peiris、JSM.、Chu、CM.、Cheng、VCC. et al.、"Clinical progression and viral load in a community outbreak or coronavirus-associated SARS pneumonia: a prospective study"、2003、Lancet 361:1767-1772; Pritzl、CJ.、Seo、Y-J.、Xia、C.、Vijayan、M.、Stokes ZD.、Hahm、B.、"A ceramide analogue stimulates dendritic cells to promote T cell responses upon virus infections"、2015、J. Immunol. 194:4339-4349; Prompetchara、E.、Ketloy、C.、Palaga、T.、"Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic"、27 feb. 2020、Asian Pac. J. Allergy Immunol. 38(1):1-9; Schloer S、Brunotte L、Goretzko J、Mecate-Zambrano A、Korthals N、Gerke V、Ludwig S、Rescher U. "Targeting the endo-lysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the anti-depressant fluoxetine"、Emerg. Microbes Infect. 2020 Dec;9(1):2245-2255. Tian、S.、Hu、W.、Niu、L.、Liu、H.、Xu、H.、Xiao、S-Y.、"Pulmonary pathology of early phase 2019 novel coronavirus (COVID-19) pneumonia in two patients with lung cancer"、2020、Journal of Thoracic Oncology; Tian、Y.、Jennings、J.、Gong、Y.、Sang、Y.、"Viral infections and interferons in the development of obesity"、2019、Biomolecules 9(11):726; The GLIMMIX procedure. SAS / STAT User's Guide. 2009、Version9.2,(https: / / support.sas.com / documentation / onlinedoc / stat / 1-31 / glimmix.pdf) Xu、Z.、Shi、L.、Wang、Y.、Zhang、J.、Huang、L.、Zhang、C. et al.、"Pathological findings of COVID-19 associated with acute respiratory distress syndrome"、2020、The Lancet Respiratory Medicine 8(4):420-422; Ye、Z.、Zhang、Y.、Wang、Y. et al. "Chest CT manifestations of new coronavirus disease 2019 (COVID-19): a pictorial review"、2020、Eur. Radiol.

Claims

1. A bacterial composition for treating and / or preventing respiratory tract diseases caused by coronaviruses, including SARS-CoV, MERS-CoV and SARS-CoV-2 viruses, comprising a bacterial composition selected from the group consisting of Streptococcus thermophilus DSM 32245, Bifidobacterium animalis subsp. lactis DSM 32246, Bifidobacterium animalis subsp. lactis DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus helveticus DSM 32244, Lactobacillus paracasei DSM 32245, Lactobacillus helveticus DSM 32246, Lactobacillus paracasei DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus paracasei DSM 32244, Lactobacillus paracasei DSM 32245, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus paracasei DSM 3224 ... 32243, Lactobacillus plantarum DSM 32244, and Lactobacillus brevis DSM 27961.

2. 2. The bacterial composition of claim 1, wherein the bacterial composition comprises 99.99% to 0.01% of Streptococcus thermophilus DSM 32245, Bifidobacterium animalis subsp. lactis DSM 32246, Bifidobacterium animalis subsp. lactis DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, and Lactobacillus brevis DSM 27961, and at least one pharmaceutically acceptable excipient.

3. 3. The bacterial composition of claim 2, wherein the bacterial composition comprises 1 to 70% by weight of Lactobacillus brevis DSM 27961, 1 to 30% by weight of Lactobacillus plantarum DSM 32244, 1 to 20% by weight of Lactobacillus paracasei DSM 32243, 1 to 20% by weight of Lactobacillus acidophilus DSM 32241, 1 to 20% by weight of Lactobacillus helveticus DSM 32242, 1 to 70% by weight of Streptococcus thermophilus DSM 32245, and 1 to 40% by weight of Bifidobacterium animalis subsp. lactis DSM 32246 and DSM 32247, and at least one pharmaceutically acceptable excipient.

4. The bacterial composition according to any one of claims 1 to 3, wherein the bacterial composition is orally administered in the form of a powder, capsule, granules or lipogel.

5. 5. The bacterial composition of claim 4, wherein the oral bacterial composition contains between 50 billion and 800 billion bacteria per gram.

6. The bacterial composition according to any one of claims 1 to 3, wherein the bacterial composition is inserted into and used in combination with a facial mask or a non-invasive ventilation (NIV) device.

7. The bacterial composition of any one of claims 1 to 3, wherein the bacterial composition is administered via the respiratory tract in a form selected from the group consisting of inhalation, insufflation, powder, solution, suspension or dispersion spray or aerosol fluid, and nasal drops.

8. 8. The bacterial composition of claim 7, wherein the administration is in the form of a fluid or powder by aerosol or insufflation comprising at least 0.001% by weight of Streptococcus thermophilus DSM 32245, Bifidobacterium animalis subsp. lactis DSM 32246, Bifidobacterium animalis subsp. lactis DSM 32247, Lactobacillus acidophilus DSM 32241, Lactobacillus helveticus DSM 32242, Lactobacillus paracasei DSM 32243, Lactobacillus plantarum DSM 32244, and Lactobacillus brevis DSM 27961 and saline or purified water.

9. 8. The bacterial composition of claim 7, wherein the composition is in the form of a fluid or powder by aerosol or insufflation, comprising 1 to 70% by weight of Lactobacillus brevis DSM 27961, 1 to 30% by weight of Lactobacillus plantarum DSM 32244, 1 to 20% by weight of Lactobacillus paracasei DSM 32243, 1 to 20% by weight of Lactobacillus acidophilus DSM 32241, 1 to 20% by weight of Lactobacillus helveticus DSM 32242, 1 to 70% by weight of Streptococcus thermophilus DSM 32245, and 1 to 40% by weight of Bifidobacterium animalis subsp. lactis DSM 32246 and DSM 32247, and saline or purified water.

10. A bacterial composition according to any one of claims 7 to 9, wherein the aerosol or insufflation fluid or powder composition contains in the range of 100,000 to 50 billion bacteria per gram.

11. 8. The bacterial composition of claim 7 in the form of a nasal spray, wherein the bacterial composition contains between 1 billion and 800 billion bacteria per gram.

12. A bacterial composition according to any one of claims 1 to 3 in the form of an oral, buccal or rectal lipogel containing between 1 and 800 billion bacteria per gram.

13. The bacterial composition of any one of claims 1 to 12, wherein the bacterial composition is used in combination with oxygen therapy or ozone therapy.

14. A bacterial composition according to any one of claims 1 to 13, wherein the bacteria used are viable, non-viable, sonicated, Tyndallized or freeze-dried.

Images