FIELD OF THE INVENTION

The invention relates to a bismuth containing composition for use in prophylaxis or treatment of an infection by Pseudomonas (P.) aeruginosa. The invention further relates to the concomitant use of a bismuth containing compound and an antibiotic for use in prophylaxis or treatment of an infection by P. aeruginosa. The invention further relates to an inhalation device comprising a bismuth containing compound, and optionally an antibiotic, in particular for use in the treatment of a P. aeruginosa infection.

BACKGROUND

Pseudomonas aeruginosa

P. aeruginosa is a rod-shaped, obligate aerobic Gram-negative bacterium which is one of the most common and serious sources of nosocomial infections and can be detected in a variety of medical devices (Gellatly 2013). It can cause various infections mostly in immune- compromised patients, such as postoperative patients, burn patients, HIV patients and patients using medical devices (such as ventilators, intubation tubes, artificial tissues, etc.) and so on (Ruiz 2004). The relatively large genome (6.3M) of P. aeruginosa encodes a large number of environmental response factors and virulence factors to deal with various environmental pressures and establish acute or chronic infections. P. aeruginosa is able to quickly adjust gene expression in response to environmental stresses, such as antibiotics, oxidative stress, and other adverse conditions in the host environment through a variety of sensing and regulatory pathways, which play an important role in the pathogenesis (Balasubramanian 2013).

Infections caused by this bacterium usually lead to serious diseases such as acquired pneumonia, respiratory tract infection, urinary tract infection, keratitis, colitis, meningitis (Shupp 2010, Martin 2011). A recent survey showed that P. aeruginosa was isolated in at least 21% of patients with hospital-acquired pneumonia (Rello 2003). In the United States, the latest national nosocomial infection surveillance showed that P. aeruginosa is the second frequently isolated nosocomial pathogen after Staphylococcus aureus (NNIS System 2003). According to reports, among ventilator-associated pneumonia and burn infections, the infection caused by P. aeruginosa is the most serious, with a mortality of 30% (NNIS System 2003), while the mortality of patients with compromised immunity increased by 40% (Afessa 2000). In addition, patients suffering from cystic fibrosis (CF) disease are extremely prone to P. aeruginosa infection, with an infection rate of as high as 44%, and chronic airway inflammation with recurrent P. aeruginosa infection is the main cause of morbidity and mortality in CF patients (Kerem 1990). In the affected area, as host cells die due to infection, and tissue epithelial cells are destroyed, P. aeruginosa can penetrate the barrier and enter the blood, causing sepsis and bacteremia. In the 1997 SENTRY study conducted in the United States, Canada, and Latin America, P. aeruginosa was the most common pathogen among a total of 4,267 hospital and community-acquired bloodstream infections (Diekema 1999). The infectious ability of P. aeruginosa is due to its numerous virulence factors and defense mechanisms to ensure that it establishes acute and chronic infections and escapes from unfavorable environments (Balasubramanian 2013). The pathogenicity of P. aeruginosa depends on the diversified synergy between related genes, and thus also exhibits a complex gene regulatory network (Balasubramanian 2013).

Antibiotic resistance

One other reason why P. aeruginosa has attracted widespread attention is due to multi-drug resistance. Antibiotic resistance is increasingly threatening global health due to the abuse and misuse of antibiotics. In particular, it is a huge challenge to fight multidrug-resistant (MDR) "ESKAPE" microorganisms (Enterococcus spp., Staphylococcus aureus, Klebsiella spp., Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp. (as well as other Enterobacteriaceae)). The development of multi-drug resistance severely impairs the effectiveness of antibacterial chemotherapy, especially for Gram-negative pathogen-related infections. In the past several decades, there appeared no new chemical classes of broad- spectrum antibiotics and only a few narrow-spectrum drugs have been developed, while the existing drugs are rapidly losing their effects (Piddock 2016, Gill 2015, Fischbach 2009). Gram- negative bacteria are naturally resistant to most antibiotics due to the low permeability of the outer membrane (Hancock 1985, Hancock 1997). The outer leaflet of the outer membrane is composed of the polyanionic molecule lipopolysaccharide (LPS) stabilized by cross-bridges of divalent cations, thereby preventing the entry of antibiotics. P. aeruginosa is considered to be naturally resistant to several types of antibiotics (Mesaros 2007); this is usually due to the low membrane permeability (Angus 1982), the production of antibiotic- modifying enzymes (Lister 2009) and the constitutive expression of the multi-drug efflux systems (Li 2015, Poole 2001). In addition, chronic infection mediated by biofilms is also a difficult point in the clinical treatment of P. aeruginosa infection, especially of the CF patients, because the bacteria are embedded in biofilms, making it difficult for antibiotics to reach the site of action, thus increasing the antibiotic resistance by 1000 times (Costerton 1995, Govan 1996, Favre-Bonte 2003).

Inappropriate use of antibiotics, as well as the process of biological evolution, has prompted a growing number of P. aeruginosa strains with multi-drug resistant (MDR) mutations and extensively-drug resistant (XDR) mutations, and even Pandrug resistant (PDR) mutations. Multiple drug-resistance may also appear in the course of chronic infection, leading to repeated infections that are difficult to cure. Therefore, new treatment strategies for multi- drug-resistant P. aeruginosa also need to be developed urgently.

BRIEF SUMMARY

The present disclosure provides a composition comprising a bismuth containing compound for use in the treatment or for prophylaxis of an infection by P. aeruginosa. The bismuth containing compound can either be administered as a single compound treatment or combined with an antibiotic. In some aspects the combination is administered as one single composition. In other aspects, the combination is administered subsequently via the same administration route or via different administration routes.

In some aspects, the bismuth containing compound contains a bismuth ion or a bismuth salt. In some aspects, the bismuth salt is one of bismuth subsalicylate, bismuth subcitrate, bismuth subnitrate, bismuth gallate, or bismuth potassium citrate, or any combination thereof.

In some aspects, the bismuth containing compound, optionally combined with an antibiotic is administered for the treatment of a lung infection. In some aspects, the composition, optionally combined with an antibiotic is administered by inhalation. In some aspects, it is administered intravenously, orally or topically.

The present disclosure also provides an inhalation device comprising the bismuth containing compound. In some aspects, the inhalation device comprises both the bismuth containing compound and the antibiotic.

In some aspects, the antibiotic is one of tetracycline-related, a macrolide-related, a rifamycins-related, a (fluoro)quinolone-related, a glycopeptide-related, an aminocoumarin- related, or a bacteriocin-related antibiotic, aztreonam, chloramphenicol, or trimethoprim. In some aspects, the antibiotic is one of tetracycline-related, a macrolide-related, a rifamycins- related, a (fluoro)quinolone-related, a glycopeptide-related, an aminocoumarin-related, or a bacteriocin-related antibiotic, chloramphenicol, or trimethoprim. In some aspects, the antibiotic is one of aztreonam, chloramphenicol, trimethoprim, tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, or nisin, or any combination thereof. In some aspects, the antibiotic is one of chloramphenicol, trimethoprim, tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, or nisin, or any combination thereof.

In some aspects, the P. aeruginosa is multi-drug resistant and/or resistant to the concomitantly administered antibiotic.

In some aspects, the P. aeruginosa has formed or is able to form a biofilm. In some aspects, treatment with the composition, optionally combined with the antibiotic, leads to disruption of the biofilm or the prevention of biofilm formation.

In some aspects, the treatment leads to a reduction in bacterial load in a patient treated with the bismuth containing composition, optionally in combination with antibiotic treatment. In some aspects, the treatment or the prophylactic treatment leads to a reduced risk of attracting a P. aeruginosa infection or a reduced risk of a P. aeruginosa biofilm formation in a patient treated with the composition, optionally in combination with antibiotic treatment.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Evaluation of the antimicrobial activity against P. aeruginosa PAO1 for combinations of several metal salts and 23 antibiotics from different families. Fractional inhibitory concentration (FIC) index values below 0.5 are considered synergistic. Darkest rectangles have a FIC index value > 2.0 and are considered antagonistic.

Figure 2: Combinations of antibiotics and bismuth subsalicylate (BSS, 16μM) preferentially inhibit P. aeruginosa strains and not, or to a lesser extent other Gram-negative or Gram- positive bacteria. The used strains are listed in Table 2. FIC index values below 0.5 are considered synergistic. Darkest rectangles have a FIC index value > 2.0 and are considered antagonistic. Figure 3: Heat plots of microdilution checkerboard assay for 12 selected combinations of antibiotics and BSS. The intensity in the color is related to bacterial growth.

Figure 4: Wavelength-scanning curves for PVD (pyoverdine, Fig.4A) and PCH (pyochelin, Fig.4B) supplemented with iron, bismuth (BSS) or both. C) The MICs of i.e. eravacycline (ERC) in the presence or the absence of 16 μM BSS when supplementing with different concentrations of PVD or PCH. The PVD or PCH alone do not influence the MIC of ERC in the absence of BSS. D) The relative production and secretion of pyoverdine in the presence of BSS. All the tests were performed in triplicate and all the data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; by Student's t-test.

Figure 5: A) The relative iron uptake related genes expression under the treatment of BSS. fpvA is the PVD primary receptor. fpvQ alternative PVD receptor. pvdA and pvdF are PVD synthesis related genes. pchD and pchE PCH synthesis related genes. pvdS, is an alternative sigma factor regulated by the global iron regulator Fur. fur is the global repressor of the iron uptake system. B) The binding activity of Fur repressor to the P. aeruginosa Fur-box operator (e.g promoter of pvdS) depends on the presence of Bi or Fe. In the case of Bi, the affinity of Fur for the Fur-box is higher than in the case of iron. C) Total iron concentration inside the cells of PAO1 after the treatment of BSS. D) Free iron concentration in the cell lysate after 2 h treatment with different concentrations of BSS or heat as a positive control. E) The MICs of six selected antibiotics in the presence or the absence of 16 μM BSS when supplement with different concentrations of FeCI3. All the tests were performed in triplicate and all the data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; by Student's t-test. Figure 6: A) effect of different BSS dosages on the electron transport chain quantified by an extracellular oxygen consumption assay. B) Intracellular NADH concentration after the treatment of different BSS doses. C) Effect of BSS in the activity of NADH dehydrogenases.

The bacterial inner membrane was collected and treated with different concentrations of BSS for 2 hours, then the membrane-bound NADH-quinone oxidoreductase activity was measured. D) BSS induced PMF disruption measurement in a dosage-related way. CCCP was used as a positive control. E) Intracellular ATP levels at different BSS concentrations. CCCP was used as a positive control. F) BSS dosage related ethidium bromide efflux inhibition. CCCP was used as a positive control. G) Intracellular accumulation of ERC at different BSS dosages. The data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; by Student's t-test. Figure 7: A, B, C) Time killing curves for ofloxacin (OFL), chloramphenicol (CHL), rifampicin (RIF), tigecycline (TGC), eravacycline (ERC) omadacycline (OMC), azithromycin (AZM), doxycycline (DOC), minocycline (MIN) and BSS monotherapy and combination therapy against PAO1 during 24 h of incubation at the indicated concentration. D, E) The addition of BSS thwarts the resistance-evolution of eravacycline (D) and ofloxacin (E) resistance in the PAO1 strain. The MIC is expressed in micromolar.

Figure 8: BSS enhance the anti-biofilm ability of antibiotics. The established biofilms were treated with the antibiotics alone or combined with 32 μM of BSS for 24 hours. The biofilms were washed three times with sterile PBS, dispersed by ultrasound and the number of bacteria was determined by plating. All the tests were performed in triplicate and all the data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; by Student's t-test. Figure 9: Ex vivo bacteremia model for antibiotics, bismuth drugs, and their combined antimicrobial activity. A) Killing kinetic for the antimicrobial activity of the antibiotics, bismuth of the combinations after 4h of incubation in human blood. 10 μl of serial decimal dilutions of each one of the combinations of antibiotics with/without bismuth drugs (32 μM) tested in blood were dropped and the activity can be observed because of the reduction in the CFU/ml. B) The appearance of the treated blood after 24 h of treatment. Dark grey color indicates those combinations for which hemolysis and therefore bacterial growth was observed (the blood samples turn black if the bacteria grow). Light grey indicates the therapy controlled the infection (the blood remains red after 24h of incubation with the bacteria). BC, bismuth citrate. BSS, bismuth subsalicylate. All the tests were performed in triplicate.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention provides a composition comprising a bismuth containing compound for use in the treatment or for prophylaxis of an infection by P. aeruginosa, preferably in a patient. Treatment in this context is defined as administration of the composition with the intention to reduce P. aeruginosa numbers in said patient or with the intention to stabilize said numbers or contain the P. aeruginosa at a certain site, for instance to prevent the occurrence of sepsis. Prophylaxis in this context is defined as reducing the risk of developing a clinical manifest infection, which is defined according to the Centers for Disease Control and Prevention guidelines that were modified to accept community-acquired infections and to exclude asymptomatic bacteriuria (Garner 1988). It should be noted that treatment and prophylaxis in this context is not meant to be absolute and that it may not work for each and every patient. Treatment and prophylaxis should thus be understood to have the intention to treat or prevent. The skilled person will appreciate that many persons carry P. aeruginosa without having a clinical manifest infection. A subclinical infection, also called pre-infection or inapparent infection, is as such not of great concern but can result in a life-threatening infection and for instance sepsis when the immune system of the patient is compromised. Since subclinical infections often occur without overt signs, their existence is only identified by microbiological culture. In this context, prophylaxis of infection by P. aeruginosa includes and preferably is the same as treatment of a subclinical P. aeruginosa infection but it can also include the treatment of a specific patient population that is known to be at risk of a P. aeruginosa infection. Patients at risk of P. aeruginosa infection are, for instance, patients on mechanical ventilation or any other medical device connected, patients with a medical prosthesis, patients burned, patients immunocompromised or patients having cystic fibrosis (Restrepo 2018). In patients being treated with a bismuth containing compound for use according to the invention, it can be advantageous to co-administer an antibiotic in order to more effectively combat a (sub)cl inical infection with P. aeruginosa.

In a preferred embodiment, a composition for use according to the invention is provided, wherein the composition is administered concomitant with an antibiotic. It is preferred to combine the bismuth and antibiotic treatment in particular when a clinical manifest infection is present. In such a case, the composition is preferably for the treatment of an infection by P. aeruginosa, but it can also be for the prevention of a clinical infection when a (sub)cl inical infection is suspected or expected. In a preferred embodiment the P. aeruginosa is a carbapenem-resistant or/and multi-drug-resistant (MDR) P. aeruginosa. With MDR in this context is meant that the P. aeruginosa does not respond to antimicrobial drugs from three or more categories based on the action mechanism or target of the drug. Preferably, P. aeruginosa is defined as being MDR when the organism is resistant to beta-lactams (3 ^rd and 4 ^th generation cephalosporins, monobactams, carbapenems and/or ureidopenicillins), quinolones and aminoglycosides (Pang 2019, Pachori 2019).

The invention unexpectedly showed that several antibiotics most of them in use against Gram-positive bacteria that were, in itself, not effective against P. aeruginosa, were very effective and highly P. aeruginosa-specific when combined with low dosages of bismuth- containing compounds.

This synergistic effect, which is probably due to restoring sensitivity or susceptibility to the antibiotic, was seen with several classes of antibiotics. Without being bound by theory, it is thought that the restoration of susceptibility to antibiotics in P. aeruginosa by bismuth is because of the iron metabolism impairment after the bismuth uptake by the dedicated and specific systems for iron uptake unique of P. aeruginosa:

1) Via the production of extracellular Fe ³⁺ chelating molecules termed siderophores (e. g. pyoverdine and pyochelin) and the uptake of ferrisiderophores via TonB-dependent receptors (TBDR).

2) Via the uptake of xenosiderophores (not produced by the bacterium itself).

3) Via the uptake of the heme molecule from the host hemoproteins.

4) Via the extracellular reduction of Fe3+ to Fe2+ involving phenazine compounds and a Fe2+ dedicated iron uptake system, termed the Feo system (Cheryl 2016).

The inventors have shown that, independent of other alternative mechanisms, the major P. aeruginosa siderophores (pyoverdine and pyochelin) bind bismuth efficiently. In the case of the most important one, which is also a virulence factor (pyoverdine) the affinity for bismuth is higher than for iron. Even more so, bismuth replaces iron when it is bound to the pyoverdine. The reduction of iron (III) to iron (II) is required to be released for pyoverdine inside the cells (Ganne 2017). This suggest that bismuth cannot be released because it cannot be reduced, so the pyoverdine recycling system is impaired and the levels of pyoverdine are reduced and therefore, the virulence of the bacteria.

However, this is not the only way in which bismuth impairs siderophores production since its effect is extendable also to the regulation in the genes involved in iron uptake and siderophores synthesis which is controlled by the repressor Ferric Uptake Regulator (Fur). In the presence of iron inside the cell, it can be bind to the repressor Fur, and the Fur-Fe complex can bind to the Fur-box in several iron uptake related gene promoters. As a consequence, iron-related uptake genes (including the main siderophores) are downregulated. In absence of iron, Fur repressor cannot bind the Fur box and the iron uptake genes are upregulated (Hosni 2013). Interestingly, once inside, bismuth enhances Fur expression in an unknown way and as consequence, iron uptake related genes are even more downregulated. So, at the end bismuth is (1) impairing the iron uptake because of its ability to bind efficiently to the iron siderophores, (2) once inside, it cannot be released from pyoverdine (and probably other siderophores) so this virulence factor cannot be recycled, the levels are reduced and the virulence of the bacteria is impaired, and (3) once inside the cells, bismuth not only enhances the expression of Fur repressor but also binds Fur repressor (Fur- Bi complex) to the Fur-box better than iron, enhancing the repression of the dedicated iron uptake system genes.

Next to being a siderophore, pyoverdine is an important virulence factor required for pathogenesis. It is also involved in triggering the production of other virulence factors as proteases, exotoxins or in biofilm formation (Lamont 2002). The effect of bismuth on pyoverdine metabolism and the unique iron uptake system of P. aeruginosa has not been described prior to the present invention, thus has a devastating effect on P. aeruginosa's virulence through multiple pathways.

The inventors have surprisingly found that as a final consequence of bismuth addition, the iron levels inside the cells are not just reduced but also bismuth replaces iron impairing critical processes of the cells, like respiration, ATP synthesis or efflux pumps activity, inducing also antibiotic accumulation.

In a preferred embodiment, a composition for use according to the invention is provided, wherein the bismuth containing compound is not a bismuth-thiol compound. Bismuth-thiol containing compounds have been used in combination with antibiotics before, but they are not preferred as they failed to show synergistic effects in P. aeruginosa (Wu 2002). This is likely because the bismuth-thiol containing compounds do not bind to the siderophores and, thus act through a different, less optimal mechanism of action. The current invention surprisingly shows that several bismuth ions or bismuth salts do show a synergistic effect when combined with several antibiotics and this effect is not observed for combinations of other metals in similar conditions. It is thus preferred that composition for use according to the invention contains a bismuth ion or a bismuth salt, preferably selected from the group consisting of bismuth subsalicylate, bismuth subcitrate, bismuth subnitrate, bismuth gallate, and bismuth potassium citrate. Such bismuth salts are also already approved for treating bacterial infections by the FDA and EMA, in contrast to bismuth-thiol containing compounds. A composition for use according to the invention can be administered by any suitable route such as orally, intravenously, topically or by inhalation. The antibiotic which is administered concomitantly can also be, independently from the administration route of the bismuth composition, be administered intravenously, orally, topically, or by inhalation. The bismuth compound and the antibiotic can also be combined in one composition and be administered by any of the before mentioned routes. In a preferred embodiment, a composition for use according to the invention is provided, wherein the composition and the antibiotic are, independently from one another, administered intravenously, orally, topically, or by inhalation. The skilled person is quite well able to decide the optimal route of administration, depending on the site of infection, the in-house or out-house patient setting, and the approved route of administration of the bismuth compound and/or the antibiotic. For instance, in an acute in-hospital setting, intravenous administration may be first choice wherein this route of administration may be less preferred when the patient is out of hospital. In such setting, e.g., oral administration may be more preferred.

In one preferred embodiment, a composition for use according to the invention is provided, wherein the infection is a lung infection and, wherein preferably the composition is administered by inhalation.

In one embodiment, therefore, the invention provides an inhalation device comprising a composition comprising a bismuth compound as defined above, the composition preferably being for use according to the invention. The inhalation device can be one of a metered-dose inhaler (MDI) or a dry powder inhaler (DPI). Preferably, if the composition is administered by inhalation, e.g., through the use of an inhalation device, the antibiotic is administered by inhalation as well. However, there may be situation in which the antibiotic is not to be inhaled (e.g., as it has not been approved for inhalation) and in such case the antibiotic is preferably administered orally or intravenously.

Typically, for bismuth in helicobacter and the trivalent therapy the dosage is about 420 mg of bismuth subcitrate potassium, 4 times per day during 10 days. For a 75 kg patient this amounts to about 22.4mg/kg per day. Such dosage correlates with the used in vitro concentrations of about 16 μM. In a preferred embodiment, the composition for use is administered such that a bismuth dose of 1 - 100 mg/kg/day is achieved. Preferably, the bismuth dose is between 5 - 50 mg/kg/day, more preferably, between 10 - 40 mg/kg/day. Also preferred is a dose of 1 - 5 mg/kg/day. In one preferred embodiment, the compositions for use is administered at even lower dosage ranges, such a bismuth dose of 0.05 - 5 mg/kg/day. This is particularly preferred when the composition is administered in the lung. Preferably, the bismuth dose is between 0.1 - 2 mg/kg/day, more preferably between 0.5 and 1 mg/kg/day. These dosages in mg are, for instance when a bismuth salt like bismuth potassium citrate is used, having an MW of about 700 Dalton. The dosages may be adjusted by the skilled person if the bismuth compound used has much higher or lower molecular weight. The skilled person is perfectly able to determine the optimal dosage of the antibiotic to be administered in combination with the bismuth compound. The standard dosage of antibiotics for different indications can easily be found in literature or in databases such as, e.g., on the website drugs.com. Typically, such information is also made available to the skilled person through medical databases, such as for instance in the Netherlands "Het Farmacotherapeutisch Kompas" (https://www.farmacotherapeutischkompas.nl/). The following is information from "Het Farmacotherapeutisch Kompas" and is used to illustrate examples of dosages that can be given in combination with bismuth. However, typically, dosages of the various antibiotics could be twice, four times, or eight times lower than the advised dosages from mono treatment, because of the effect of co-treatment with bismuth. Azithromycin, for instance, is an antibiotic in the macrolide family of medications, used to treat infections of the upper and lower respiratory tract, skin, uncomplicated Chlamydia, among others. Typical dosage of azithromycin is 500 mg once per day for 3 days or 500 mg on day followed by 250 mg per day for up to 5 days.

Aztreonam is an antibiotic, primarily used to treat chronic respiratory tract infections caused by P. aeruginosa in patients older than 6 years. Typical dosage of aztreonam is 75 mg per inhalation, three times per day for 28 days.

Ciprofloxacin is an antibiotic in the fluoroquinolone family of medications, used to treat infections of the lower respiratory tract, acute exacerbations of chronic sinusitis, genitourinary tract, among others. Typical dosage of Ciprofloxacin is 500-750 mg, twice per day, for 7-14 days.

Clarithromycin is an antibiotic in the macrolide family of medications, used to treat infections of the upper and lower respiratory tract, skin, and H. pylori, among others. Typical dosage of Clarithromycin is 250 mg, twice per day, for 6-14 days.

Doxycycline is an antibiotic in the tetracyclines family of medications, used to treat community-acquired pneumonia, urinary tract infections, acne, otitis media acuta, among others. Typical dosage of doxycycline is 200 mg on day 1, thereafter 100 mg per day, for 5 - 10 days.

Eravacycline is an antibiotic in the tetracycline family of medications, used to treat intra- abdominal infections in adults, among others. Typical dosage of Eravacycline is 1 mg per kg bodyweight every 12 hours by infusion, for 4-14 days. Erythromycin is an antibiotic in the macrolide family of medications, used to treat acne, acute faryngotonsilitis, acute rhinosinusitis, SOAs, infections of the skin, community acquired pneumonia, among others. Typical dosage of Erythromycin is 2 g per day, separated over the day, for light to moderate infections and up to 4 g per day for severe infections.

Levofloxacin is an antibiotic in the fluoroquinolone family of medications, used to treat infections of the respiratory tract, urinary tract, among others. Typical dosage of Levofloxacin is 500 mg once or twice per day for 7-14 days.

Minocycline is an antibiotic in the tetracyclines family of medications, used to treat infections of the respiratory tract, urinary tract, gastrointestinal tract, among others. Typical dosage of Minocycline is 200 mg on day one, followed by 100 mg once or twice per day for up to 3 days. For Streptococcus haemolyticus, treatment duration is at least days.

Ofloxacin is an antibiotic in the fluoroquinolone family of medications, used to treat cystitis, complicated infections of the urinary tract, community-acquired pneumonia, chlamydia, among others. Typical dosage of Ofloxacin is 200-800 mg per day, max 400 mg per dose, treatment duration 7-10 days.

Rifabutin is an antibiotic in the rifamycin family of medications, used to treat Mycobacterium infections of patients suffering from AIDS, and pulmonary tract, among others. Typical dosage of Rifabutin is 300 mg once per day, for an indefinite period, to be used as prophylaxis.

Rifampicin is an antibiotic in the rifamycine family of medications, used to treat tuberculosis, pulmonary and extrapulmonary, among others. Typical dosage of Rifampicin is 10 mg/kg body weight, once per day, for up to 9 months.

Tetracycline is an antibiotic in the tetracyclines family of medications, used to treat a number of infections, including urinary tract, respiratory tract, otitis media acuta, acne, cholera, brucellosis, plague, malaria, and syphilis, among others. Typical dosage of tetracycline is 250 mg, four times per day, with a max of 2 g per day.

Tigecycline is an antibiotic in the tetracycline family of medications, used to treat intra- abdominal infections and infections of the skin, among others. Typical dosage of Tigecycline is 100 mg by infusion on day one, followed by 50 mg every 12 hours, for 5-14 days.

Trimethoprim is an antibiotic used to treat acute uncomplicated infections of the urinary tract and respiratory tract, maintenance of chronic respiratory infections, among others. Typical dosage of Trimethoprim is 300 mg once per day, of 200 mg twice per day, for 3-5 days, for upper respiratory infections at least 7 days.

Vancomycin is an antibiotic in the glycopeptide family of medications, used to treat complicated skin and soft tissues infections, bacterial pneumonia such as community- acquired-, hospital-acquired-, and ventilator-associated pneumonia, infectious endocarditis, among others. Typical dosage of Vancomycin is 15 mg twice per day, or 7.5 mg four times per day, followed by a per-person calculated dosage and treatment duration.

In one preferred embodiment, a composition for use according to the invention is provided, wherein the antibiotic is, dependent on the type of antibiotic, administered at a dose of 0.1 - 100 mg/kg, preferably at a dose of 0.5 - 50 mg/kg, more preferably at a dose of 1 - 30 mg/kg. If we take the example of tetracycline which is, in monotherapy, administered at 2 g/day maximum, a dosage to start with in combination with bismuth could be 0.2 g/day, or even lower. This is based on the 8 - 16x more effectiveness in vitro if tetracycline is combined with bismuth when compared with tetracycline alone. Optimal dosages are then found by routine experimentation in pre-clinical and clinical trials.

In one preferred embodiment, a composition for use according to the invention is provided, wherein the infection is a skin infection, e.g., after burn wounds, surgery, or linked to catheters and other medical devices and wherein preferably the composition is administered topically. The composition is also preferably applied topically in the case of ear or eye infections.

In one preferred embodiment, a composition for use according to the invention is provided, wherein the infection is a gastro-intestinal infection, and wherein preferably the composition is administered orally.

The intestinal tract is considered the most important reservoir of P. aeruginosa in intensive care units (ICUs). Gut colonization by P. aeruginosa underlies the development of invasive infections such as gut-derived sepsis. Intestinal colonization by P. aeruginosa is associated with higher ICU mortality rates. The translocation of endogenous P. aeruginosa from the colonized intestinal tract is an important pathogenic phenomenon (Okuda 2020).

The lungs are a major site of P. aeruginosa infection in ill patients; however, a considerable number of such infections occur through direct contamination of the lungs by gastrointestinal flora or through hematogenous spread from the intestine to the lungs (Zaborina 2006). In particular, the presence of highly virulent strains of P. aeruginosa within the intestinal tract alone is the main source of sepsis and death among immunocompromised patients, even in the absence of established extraintestinal infection and bacteremia (Marshall 1993, Osmon 2004, Zaborina 2006). Furthermore, the lethal effects of intestinal P. aeruginosa are dependent upon its ability to adhere to and disrupt the intestinal epithelial barrier (Alverdy 2000).

In each case, the concomitant administration of the antibiotic is preferably by the same route and more preferably the antibiotic and the bismuth compound are present in a single composition for administration. However, if it is not feasible to administer the antibiotic by the same route as the bismuth containing compound, the antibiotic could also be given through any other suitable route of administration.

In one preferred embodiment, an inhalation device containing both a composition comprising a bismuth containing compound as defined in any of the preceding claims, and an antibiotic is provided. The composition and the antibiotic contained in the inhalation device are preferably for use in the treatment or for prophylaxis of an infection, preferably a lung infection, by P. aeruginosa.

In one preferred embodiment, a composition or device according to the invention is provided, wherein the treatment is for a patient suffering from cystic fibrosis. Cystic fibrosis is a genetic disease that affects the production of secretions such as mucus in the body. In patients with cystic fibrosis, there is an overproduction of thick mucus in the lungs, which leads to inflammation and a high risk of the lungs becoming infected with bacteria. P. aeruginosa is one of the most common types of bacteria that tend to infect the lungs of patients with cystic fibrosis. P. aeruginosa lung infection in cystic fibrosis is a long-term debilitating disease and may be life threatening because it severely damages the lung tissue and does not allow the patient to breathe normally (Malhotra 2019, Waters 2019).

In one preferred embodiment, a composition or device according to the invention is provided, wherein the treatment is for a patient suffering from or at risk of suffering from a P. aeruginosa infection, wherein the patient is or has recently been on mechanical ventilation. With recently is meant less than 2 weeks, preferably less than 1 week, more preferably less than 2 days prior to the start of the treatment with a composition according to the invention. Ventilator-associated pneumonia is the most common infection in intensive care unit patients associated with high morbidity rates and elevated economic costs. P. aeruginosa is one of the most frequent bacteria linked with this entity, with high attributable mortality despite adequate treatment that is increased in the presence of MDR strains, a situation that is becoming more common in intensive care units. The invention shows that the composition for use according to the invention can be synergistically used with a variety of different antibiotics. In a preferred embodiment, a composition for use or a device according to the invention is provided, wherein the antibiotic is selected from the group consisting of tetracycline-related, a macrolide-related, a rifamycins-related, a (fluoro)quinolone-related, a glycopeptide-related, an aminocoumarin- related, or a bacteriocin-related antibiotic, aztreonam, chloramphenicol, and trimethoprim. More preferably, the antibiotic is selected from the group consisting of tetracycline-related, a macrolide-related, a rifamycin-related, a (fluoro)quinolone-related, a glycopeptide-related, an aminocoumarin-related, or a bacteriocin-related antibiotic, chloramphenicol, or trimethoprim. In one preferred embodiment, the antibiotic is selected from the group consisting of aztreonam, chloramphenicol, trimethoprim, tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, and nisin. In a more preferred embodiment, the antibiotic is selected from the group consisting of chloramphenicol, trimethoprim, tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, and nisin.

As already mentioned, the bismuth composition for use according to the invention is capable of restoring the sensitivity of a P. aeruginosa towards an antibiotic to which the P. aeruginosa is resistant. The composition is thus extremely useful in settings wherein MDR P. aeruginosa are to be treated. In one preferred embodiment, therefore, a composition for use or device according to the invention is provided, wherein the P. aeruginosa is an MDR P. aeruginosa. Preferably, the P. aeruginosa is resistant to the concomitantly administered antibiotic. In such case, the P. aeruginosa is made sensitive by the composition of the invention and can be treated accordingly. However, antibiotics to which the P. aeruginosa is sensitive already can also be used because the composition of the invention will render the P. aeruginosa even more sensitive to that antibiotic making it possible to lower the dose of the antibiotic and subsequent adverse effects. The composition for use according to the invention also lowers the risk of or even prevents a P. aeruginosa treated with an antibiotic to build-up resistance to that antibiotic in two ways. On one side, the composition aims the total eradication of the infection switching the activity of the antibiotics used from bacteriostatic to bactericidal which is difficult for antibiotic-resistance-development. On the other side, bismuth directly impairs the development of resistance to antibiotics in the compositions (e.g. ofloxacin and eravacycline). It must be added that the combinations with bismuth even overcome the newly developed resistance to tetracyclines and quinolones generated in the antibiotic- resistance-development assays.

A composition for use according to the invention, thus also solves, at least in part, the widespread problem of antibiotic resistance build-up. In one embodiment, therefore, the invention provides a composition comprising a bismuth compound, preferably a bismuth compound that does not contain bismuth-thiol, for use in preventing, impairing development, or treating antibiotic resistance in an P. aeruginosa strain in an animal, preferably a human, more preferably a patient, comprising administering to the animal a therapeutical effective amount of the bismuth compound, preferably in combination with an antibiotic. In a preferred embodiment, the antibiotic is an antibiotic to which the P. aeruginosa strain is resistant to or is at risk of developing resistance to.

The current invention also presents antimicrobial activity in complex matrices as formed biofilms or in blood. In a preferred embodiment, a composition for use or device according to the invention is provided, wherein the P. aeruginosa has formed a biofilm structure. Of course, the combination will work also in bacteria that do not form a biofilm but, in particular in biofilms, the resistance of bacteria to antibiotics could be several hundred times higher than for bacteria not contained in a biofilm. Because of the synergistic effect, a composition for use according to the invention is able to break such resistance and lower the concentration of antibiotics necessary for treating biofilm-forming P. aeruginosa. In a more preferred embodiment, a composition for use or device according to the invention is provided, wherein the treatment leads to disruption of the biofilm structure. One preferred embodiment provides a composition for use or device according to the invention, wherein the treatment leads to a reduction in bacterial load.

Further provided is a method of treating an individual suffering from or at risk of attracting a P. aeruginosa infection, the method comprising administering to the patient an effective amount of a composition comprising a bismuth compound, preferably in combination with an antibiotic, thereby reducing the risk of attracting a P. aeruginosa infection or treating the P. aeruginosa infection. In one embodiment, the composition is administered concomitant with an antibiotic. In one embodiment, the bismuth-containing compound is not a bismuth-thiol compound. In one embodiment, the bismuth-containing compound contains a bismuth ion or a bismuth salt, preferably selected from the group consisting of bismuth subsalicylate, bismuth subcitrate, bismuth subnitrate, bismuth gallate, and bismuth potassium citrate. In one embodiment, the bismuth compound does not contain a bismuth-thiol. The term "bismuth thiol" has the generally accepted meaning, i.e., a compound that results from the chelation of bismuth with a thiol compound, which is an -SH functional group bearing compound. The composition and the antibiotic may, independently from one another, be administered by any suitable route, such as intravenously, orally, topically, or by inhalation. In one embodiment, the infection is a lung infection. In one embodiment, the patient is suffering from cystic fibrosis or is (or was) on mechanical ventilation. In one embodiment, the antibiotic is selected from the group consisting of tetracycline-related, macrolide-related, rifamycin-related, quinolone-related, glycopeptide-related, aminocoumarin-related, and bacteriocin-related antibiotics, aztreonam, chloramphenicol, and trimethoprim. In one embodiment, the antibiotic is selected from the group consisting of tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin, rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, nisin, aztreonam, chloramphenicol, and trimethoprim. In one embodiment, the antibiotic is selected from the group consisting of tetracycline, doxycycline, minocycline, tigecycline, eravacycline, omadacyline, azithromycin, clarithromycin, erythromycin, telithromycin, rifabutin, rifampicin, rifapentine, rifaximin, ciprofloxacin, levofloxacin, nalidixic acid, ofloxacin, vancomycin, coumermycin Al, nisin, chloramphenicol, and trimethoprim. The P. aeruginosa may be a multidrug-resistant P. aeruginosa and/or the P. aeruginosa may be resistant to the concomitantly administered antibiotic. The P. aeruginosa may form or may have formed a biofilm structure and the treatment may lead to disruption of the biofilm structure. The treatment may lead to a reduction in bacterial load. In case of prophylaxis, the prophylaxis may lead to a reduced risk of attracting a P. aeruginosa infection or may lead to a reduced risk of a P. aeruginosa biofilm formation.

EXAMPLES

EXAMPLE 1: Synergy of different metal salts with antibiotics against P. aeruginosa PAO1 (Figure 1, Table 1).

Material and method example 1: The MIC values were determined in triplicate using standard broth microdilution in accordance with Clinical and Laboratory Standards Institute (CLSI) recommendations. Briefly, the drugs were two-fold diluted in MHB, and then mixed with an equal volume of bacterial suspension in a 96-well microliter plate, resulting in the final bacterial concentration of 5x10 ⁵ CFUs mL ^-1 . The MIC values were defined as observing no visible bacterial growth under the lowest concentration of antibiotics after the overnight incubation at 37°C. For the large antibiotics-metal screening of synergism and considering the activity of the metal alone, 16 μM of bismuth-based compounds or their sodium counterparts, 8 μM of auranofin, 2 μM of silver nitrate, and 32 μM of gallium nitrate, scandium acetate or indium acetate were used in the combination. The FIC index (FICI) was calculated according to the formula FICI = FICa + FICb = MICab/MICa + MICba/MICb where MICa is the MIC (Minimal Inhibitory Concentration) for the antibiotic alone, MICb is the MIC for the bismuth compound alone, MICab is the MIC for the antibiotic in the presence of a MICba concentration of bismuth compound. The FICI was interpreted according to EUCAST as follows: antagonistic, FICI≥2; indifferent, 1<FICI<2; additive, 0.5<FICI≤1; synergistic, FICI≤0.5. For the FICI calculations, twice the highest concentration tested was used in the cases where the MIC was not reached.

Results example 1: Bismuth based salts were highly active sensitizing PAO1 to several antibiotics (regardless of their activity) at low concentrations (considering that no activity (or scarce activity) for bismuth salts alone was observed) overcoming natural resistance to such antibiotics. This is the case of tetracyclines, rifamycins, macrolides-related antibiotics or antibiotics such trimethoprim, chloramphenicol, coumermycin A1, vancomycin or the antimicrobial peptide nisin since P. aeruginosa is highly resistant to them (Table 1). This synergism was not observed for other trivalent metals as scandium or indium (with exceptions as (e.g.) indium acetate and tetracyclines) and neither for gallium, and in clinical use methal proposed to be used as antimicrobial in P. aeruginosa infections (Figure 1, Table 1). Other antimicrobial metals used in the clinic as auranofin (Au) or silver nitrate neither synergized with antibiotics against P. aeruginosa (Table 1). Although not included in Table 1, no synergism in such conditions was observed for other antibiotics as aminoglycosides (e.g. gentamycin, amikacin, kanamycin, streptomycin or neomycin), colistins (e. g. polymyxin E and polymyxin B), bacitracin, clorobiocin, novobiocin, fosfomycin, fusaric acid, metronidazole, linezolid, pentamidine or most beta-lactam related antibiotics (e.g ampicillin, carbenicill in, cephalexin, meropenem or oxacillin). EXAMPLE 2: Bismuth-antibiotics detected synergisms are highly P. aeruginosa-specific (Figure 2, Table 2).

Material and methods example 2: Bismuth subsalicylate (BSS) at 16 μM in combination with the previously reported synergistic antibiotics was tested as described in example 1 against a set of Gram-negative and Gram-positive strains, including 4 reference strains belonging to the three main Pseudomonas genotypes, as well as 9 clinically isolated P. aeruginosa strains. As Gram-negative were tested: P. aeruginosa (strains PAO1, PAK, PA14, ATCC27853, NR-31040, NR-31041, HM-214, AUMC-Pa-1, AUMC-Pa-2, AUMC-Pa-3, AUMC-Pa- 4, AUMC-Pa-5 and AUMC-Pa-6), Escherichia coli LMG 8223, Acinetobacter baumannii LMG 01041, Klebsiella pneumoniae LMG 20218, Salmonella enterica LMG 07233 and Shigella flexneri NR-5. As Gram-positive: Staphylococcus aureus ATCC43300, Mycobacterium marinum mUSA and Streptococcus pneumoniae D39. FICI values were calculated as indicated in example 1.

Results for example 2: Overall, combinations of antibiotics and (e.g.) BSS (16μM) preferentially inhibit P. aeruginosa strains and not other Gram-negative or Gram-positive bacteria (Figure 2, Table 2) although some exceptions were observed for the combination of BSS with rifamycins against Klebsiella pneumoniae, rifamycins and macrolides against Acinetobacter baumannii and rifamycins, macrolides and tetracyclines against Escherichia coli (Figure 2, Table 2). As indicated in example 1, synergism was observed for both active antibiotics (e.g some quinolones) and non-active antibiotics (e.g. macrolides, tetracyclines or chloramphenicol) (Table 2).

EXAMPLE 3: Checkerboard assay for 12 selected combinations of antibiotics and BSS (Figure 3).

Material and methods example 3: A standard checkerboard broth micro-dilution test was carried out to test the synergistic effects of the combined antibacterial agent. Because of the MIC of BSS is higher than 1 mM, the 64 μM BSS was used as the highest concentration to test how low concentration of BSS can show the synergistic effect in combination to azithromycin, chloramphenicol, rifampicin, ofloxacin, nalidixic acid, nisin, doxycycline, tigecycline, eravacycline, tetracycline, minocycline, and omadacycline. The percentage of growth for the different combinations was calculated with respect to negative no-treated control. All the experiments were performed in triplicate. Results for example 3: Overall, strong synergism was also observed at lower concentrations of BSS (Figure 3). This effect was noticeable in the case of combinations with ofloxacin, tetracycline, minocycline and omadacycline. In these cases, using 4-fold less BSS a similar reduction in the MIC than with 16 μM was observed (Figure 3).

EXAMPLE 4: Interaction of BSS with the major siderophores of P. aeruginosa, pyoverdin (PVD) and pyochelin (PCH).

Material and Methods example 4: Considering the affinity of PCH and specially PVD for iron (III) we tested the binding ability of these siderophores for BSS. For that, the PVD was prepared in sterile water to a final concentration of ImM and PCH was prepared in sterile water with 10% methanol to a final concentration of ImM. The PVD or PCH was mixed at different molar ratios (see Figure 4A, B) with FeCI3 or BSS. After that, luL of the mixture was measured by wave scan at 250nm~550nm using a nanodrop.

To test the importance of PVD and PCH in the action mechanism of bismuth we explored the effect of the external addition of these siderophores in the combined activity of bismuth and eravacycline (e.g.). For that, the MICs for eravacycline (ERC) was evaluated in the presence or the absence of 16 μM BSS when supplementing with different concentrations of PVD or PCH (Figure 4C).

Finally, we explored the levels of PVD after bismuth treatment. For that, overnight culture of PAO1 was diluted 1:100 into fresh Casamino acid medium (0.5% Casamino acids, 0.1 mM MgSO4, 7 mM potassium phosphate buffer, pH 7.0). At given time points, the PVD from culture supernatants was measured as OD405 normalized by the cell density of bacterial cultures (OD600) as previously described (Imperi 2009).

Results example 4: As indicated in Figure 4A, PVD showed an amazing ability to bind bismuth even replacing iron from the complex Fe-PVD. In figure Figure 4A, when bismuth is bound to PVD the spectra signal switch to the left and the maximum absorbance peak is obtained at approx. 360 nm while PVD alone or the complex Fe-PVD present the maximum absorbance peak at 400 nm (Figure 4A). The addition of BSS to Fe-PVD formed complexes switch the signal to the left, which suggest the ability of bismuth to replace iron in the siderophore (Figure 4A). In the case of PCH, the addition of bismuth to PCH induces a switch in the absorbance to the right moving the maximum absorption peak from the 255nm observed for PCH alone or the Fe-PCH complex to 275nm for the Bi-PCH complex. Unlike PVD, in the case of PCH, the data suggest that the bismuth binding affinity was similar to iron since the curve obtained for the combination suggests a mixture of PCH-Bi and PCH-Fe complexes (Figure 4B). Next, we explored the role of the siderophores in the combined effect of bismuth and antibiotics using ERC as an example. The PVD or PCH alone did not influence the MIC of ERC in the absence of BSS, however, when exogenous PVD was added to the culture the synergistic effect of low-concentrations of BSS and eravacycline was antagonized when the concentration of PVD reached equal-molecular concentrations as bismuth (Figure 4C). Surprisingly, and despite the fact that the binding affinity test for bismuth and iron for PCH showed a similar binding capacity when exogenous PCH was added to the culture the synergistic effect of bismuth was enhanced (Figure 4C).

Finally, we explored the effect of bismuth in the recycling of PVD. Considering that Fe has to be reduced inside the cell to be released from PVD and that bismuth cannot be reduced, we quantify the production of PVD after BSS treatment. As indicated in Figure 4D, BSS severely reduce the levels of PVD even at low concentrations. PVD play a critical role in the virulence and the infectivity of P. aeruginosa, so the reduction in the levels of this siderophore impaired the pathogenicity of this bacteria.

EXAMPLE 5: Bismuth and iron relation: bismuth impairs the dedicated P. aeruginosa iron uptake system depriving intracellular iron levels. Iron impairs the synergistic activity of bismuth.

Material and methods example 5: Overnight cultured PAO1 cells were diluted 1: 100 into CA-MHB and incubated at 37 °C to an OD600 of 1. The cells were treated with 16 μM BSS for 30 min and no treated cells were used as a negative control. The cells were collected and resuspended in ImL TRIzol reagent (Life technologies). Total bacterial RNA was extracted by chloroform-isopropanol precipitation. The residual DNA was digested with RNase-free recombinant DNase I (Roche), the RNA was dissolved in RNase-free water, and cDNA was synthesized using random primers and reverse transcriptase (Invitrogen™). SYBR II Green supermix (BioRed) was used for RT-PCR. Selected iron uptake genes were analyzed as: fpv/\, fpvB, pvdA, pvdF, pchD, pchE, pvdS and fur. The ribosomal gene rpsL was used as an internal reference control. Primer sequences and names are listed in Table 3 (Figure 5A).

For bismuth Fur interaction, fur gene was amplified from PAO1 chromosome and primers listed and cloned fused to a poly-Histidin tag into the plasmid pET28a. The resulting plasmid was transferred into E. coli BL21(DE3). The BL21(DE3) cells harbouring PET28a-fur plasmids were cultured overnight and then diluted by 1:100 to fresh LB medium supplemented with 50 pg/mL kanamycin. Cells were grown (37 °C and 200 rpm) to OD600 of 0.4-0.6 and then induced by ImM p-D-thiogalactoside (IPTG) and the bacteria were further incubated at 18 °C for 20 h. The bacteria were harvested by centrifugation (8000g, 4 °C for 30 min) and the cell pellets were resuspended with lysis buffer (50 mM Tris, 300mM NaCI, 10% glycerol pH = 7.6) and lysed by sonication. The lysates were centrifuged (12,000g, 4 °C for 30 min). The supernatant was incubated with a Ni-NTA for two hours for 2 h at 4°C and then washed three times with the lysis buffer with 20 mM imidazole. The Fur protein was eluted with 300 mM imidazole in the same buffer and the protein concentrations were quantified by a BCA analysis. To analyze the interaction of Fur and the Fur-box in the presence of iron ( Fe( II )) and bismuth, the electrophoretic mobility shift assay (EMSA) was performed. A DNA fragments (200 ng) containing the Fur-box sequence was incubated with 1 μM purified recombinant Fur protein at 37°C for 30 min in a 20 μL reaction (10 mM Tris-HCI, pH 7.6, 4% glycerol, 100 mM NaCI, 10 mM-β-mercaptoethanol) including the indicated concentration of BSS or FeSO4 (Figure 5B). Samples were loaded onto a 6% native polyacrylamide gel in 0.5x Tris-borate- EDTA (TBE) buffer that had been pre-run for 1 hour and then run at 100 V for 2 hours on ice. The gel was stained in 0.5x TBE containing ethidium bromide for 10 min and bands were visualized with a molecular imager ChemiDoc XRS + (Bio-Rad) (Figure 5B).

For the intracellular total iron quantification (Figure 5C), overnight cultured PAO1 was diluted 1:100 into fresh MHB with the indicated concentration of BSS and incubated for 24 h at 37 °C under continuous shaking. The cells were collected by centrifugation and washed three times with 20 mM Tris-HCI pH 7.2 buffer. The bacterial cells were lysed by sonication. The lysates were treated at 90 °C for 20 min to release the protein-bound iron. The total iron in the cell lysate was detected by incubating samples for 1 h with 10 mM Ferene-S and measuring absorbance at 593 nm. The standard curve was prepared using the FeCI3, while the BSS does not affect the absorbance at 593 nm when incubated with Ferene-S. Total bacterial protein concentrations were quantified using a BCA analysis for calibration. All the tests were performed in triplicate. In the case of Iron displacement analysis (Figure 5D), overnight cultured PAO1 cells were collected by centrifugation and washed three times with 20 mM Tris-HCI pH 7.2 buffer. The bacterial cells were lysed by sonication. The lysate was treated with the indicated concentrations of BSS for 2 hours. Release of protein-bound iron in the lysate was detected by incubating samples for 1 h with 10 mM Ferene-S and measuring absorbance at 593 nm. The lysate which was treated at 90 °C for 20 min was used as a positive control. All the tests were performed in triplicate. Finally, to analyze the effect of iron on the synergistic relationship of BSS and antibiotics, a MIC test was perfomed for selected antibiotics and BSS (16 μM) in the presence of increasing concentrations of iron (FeCl ₃ ) (Figure 5E). All the tests were performed in triplicate.

Results of example 5: We initially explored the expression of genes related to iron uptake in P. aeruginosa after bismuth treatment by RT-PCR. The results (Figure 5A) showed that the P. aeruginosa siderophores pyoverdine (PVD) and pyochelin (PCH) synthesis related genes (pvdA, pvdF, pchDE) and the PVD primary receptor fpvA were significantly downregulated under the treatment of BSS(Figure 5A). It also hindered the generation of PVD, so PVD levels are reduced in two ways, inhibiting the production and impairing the recycling (Figure 5A and Figure 4D). No effect was observed in the alternative PVD receptor fpvB and neither in the alternative sigma factor pvdS (Figure 5A). However, an upregulation of the fur repressor was observed (Figure 5A). In iron-limited conditions, ferripyoverdine (Fe- PVD) is captured by cells mainly through the PVD primary receptor FpvA, resulting in the increased expression of biosynthetic operons and the receptor gene in a positive feedback- loop. This process only is interrupted by the global repressor Fur, when the intracellular iron level is sufficiently high. The binding of Fur to operators is mediated by Fe, but also including Zn, Co or Mn. The high expression of fur genes and the low expression of siderophore synthesis genes and the receptor gene fpvA suggest that bismuth could be able to bind Fur protein, forming a Fur-Bi complex able to inhibit the expression of iron uptake genes by binding Fur-box in the promoter operators. To prove it, Fur protein was cloned and purified. After that, an electrophoretic mobility shift assay was performed, showing that bismuth was able to promote the binding of Fur to its target (the Fur box containing DNA) with higher affinity than iron (II) (Figure 5B). Collectively, these results suggest that bismuth inhibits the iron uptake by binding with Fur. Fur activity is therefore enhanced, and the iron uptake related genes are repressed.

As a consequence of this iron uptake inhibition, the concentration of iron inside the cells is reduced when the cells are treated with bismuth (Figure 5C). The ability of bismuth to bind Fur repressor better than iron suggest that bismuth could be able to bind other enzymes replacing iron. As demonstrated in Figure 5D, bismuth is not only reducing the intracellular levels of iron but also replacing iron as a cofactor in enzymes.

Finally, we tested the influence of the iron concentration on the synergistic activity of BSS for six different antibiotics (e.g.): azithromycin, chloramphenicol, doxycycline, rifampicin, ofloxacin and eravacycline. As indicated in Figure 5E, supplementation with exogenous FeCl ₃ antagonized the synergistic effect of low-concentration BSS and antibiotics but unlike PVD, a concentration higher than equimolecular was usually necessary to completely impairs the BSS activity. It could be related to the higher binding capacity of bismuth to PVD (Figure 4A).

EXAMPLE 6: Iron replacement by bismuth severely affect critical metabolic processes in P. aeruginosa. Inside into the action mechanism of bismuth.

Material and methods example 6: Next, we explored critical processes in which clusters Fe-S are involved and that could be targeted by bismuth according to the previous results. For that, we analyzed the effect of iron in the electron transfer chain (ETC) since Fe-S clusters play a critical role in the enzymes involved in this process. Firstly, the bacterial oxygen consumption was measured using an extracellular oxygen consumption assay kit (Abeam). An overnight culture of PAO1 was diluted 1:100 into a fresh MHB medium and grown at 37°C to the late log phase (OD600=1). The bacterial concentration was adjusted to 1x10 ⁶ CFU/ml in a fresh MHB medium containing the indicated concentration of BSS (Figure 6A). 150 μl of the bacterial resuspension was transferred to each well of a 96-well plate containing 10 μl of O ₂ consumption reagent. Each well was sealed with 50 μl mineral oil provided by the kit to isolate the air. The fluorescence signal was monitored at excitation/emission of 380/650 nm every 10 min for 3 hours with a luminometer. After that and to confirm the activity of bismuth in the electron transfer chain, we measured the bacterial NADH levels using an amplite fluorimetric NAD/NADH assay kit (AAT Bioquest, Inc., USA). Bacteria were cultured in a fresh MHB medium at 37°C to an OD ₆₀₀ of 1. Then the bacteria were treated with the indicated concentration of BSS (Figure 6B) for 1 hour, collected, suspended in PBS and lysed by sonication. The supernatant was collected by centrifugation at 12,000g for 10 minutes at 4°C. The NADH level was measured according to the manufacturer's instructions. The standard curve was prepared using the standard NADH provided by the kit. The total protein level was measured by a BCA analysis kit. NADH-quinone oxidoreductase enzyme assay: overnight cultured PAO1 cells were treated with indicated concentrations of BSS (Figure 6C) for 2 hours and then cells were collected by centrifugation and washed three times with sterile saline. To prepare spheroplasts, the cells were resuspended in 30 mM Tris-HCI, pH 8.0, containing 20% sucrose at room temperature. EDTA iron (III) salt (pH 7.5) and lysozyme were added to achieve final concentrations of 10 mM and 1 mg/mL, respectively, and incubate at 21°C for 60 minutes. The spheroplasts were centrifuged at 16000 g for 30 min at 4°C. Resuspend the spheroplast pellet in 20 mL of 0.1 M phosphate buffer pH 7.5, which contains 20% sucrose. DNase and MgSO4 were added to achieve final concentrations of 2 mg/mL and 20 mM, respectively; and incubate the spheroplast mixture at 37°C for 60 minutes. The spheroplasts were broken by ultrasonication for 15 min, pulsation at 10 sec/10 sec on-off, on ice. The larger cell debris were removed by centrifugation at 800 g for 10 min at 4°C. The lysate was centrifuged at 75,000 g for 30 minutes at 4°C (Beckmann Optima MAX Tabletop Ultracentrifuge) to obtain a crude inner membrane. The membrane was resuspended in 50 mM phosphate buffer (pH 7.5) containing 5 mM MgSO4. The cell debris were removed by centrifugation at 800 g for 10 minutes at 4°C. The inner membranes were separated by centrifugation at 75000 g for 1 hour at 4°C and the membrane preparation was stored at -80°C. For the membrane-bound NADH-quinone test, bacterial inner membrane (1 mg/mL) were resuspended in the phosphate buffer containing magnesium sulfate. The ubiquinone-1 (0.1) and potassium cyanide (KCN) were added to achieve final concentrations of 200 μM and 5 mM, respectively; and BSS was added as desired and the reaction mixture was incubated for 2 hours at 37°C. The reaction was initiated by adding 200 μM NADH immediately before each experiment. The NADH oxidase activity was measured by following the decrease in absorbance at 340 nm using a luminometer for 2 hours. The membrane which was treated with 90 °C for 10 min was used as a negative control (Figure 6C). All the tests were performed in triplicate.

Proton motive force assay: overnight cultured PAO1 cells were collected via centrifugation and washed with 50 mM potassium phosphate buffer (pH = 6) and potassium phosphate- EDTA (5 mM) buffer, then the cells were re-suspended in 1 mL the same phosphate-EDTA buffer. BCECF-AM (2 mM, 10 μl) was added and incubated for 1 hour at room temperature. The cells were concentrated and resuspended in 120 μl phosphate-EDTA buffer and incubated on ice for 4 hours. 2 μl cell suspension was added to fresh phosphate-EDTA buffer (200 μl) with the indicated concentration of BSS or CCCP (40 μg/ml) as a positive control (Figure 6D). The fluorescence signal was monitored at excitation/emission of 500/522 nm by a luminometer. The data shown are representative of results from six technical replicates. Bacterial intracellular ATP levels were determined using a BacTiter-GloTM Microbial Cell Viability Assay (Promega) kit. The bacteria were cultured at 37°C in CA-MHB medium to an OD600 of 1. Subsequently, the bacteria were treated with the indicated concentration of BSS or CCCP (40 μg/ml) as a positive control at 37 °C for 4 hours (Figure 6E). 50 μl BacTiter-GloTM reagent was mixed with 50 μl cell culture and incubated for 5 minutes at room temperature. Luminescence was measured with a luminometer. The ATP concentration was calculated using a standard curve made with a commercial ATP solution. Relative ATP levels were calculated by the OD600. All the tests were performed in triplicate.

Ethidium bromide (EtBr) efflux assay: the bacteria were cultured in a CA-MHB medium at 37 °C to an OD ₆₀₀ of 1. The cells were washed with 10 mM HEPES buffer and adjusted to the OD ₆₀₀ =0.5. EtBr was added at a final concentration of 5 μM and cells were incubated for 30 min at 37 °C. The cells were collected by centrifugation and resuspended in a fresh CA-MHB medium containing the indicated concentration of BSS or 40 mg/l CCCP as control (Figure 6F). Then the fluorescence signal was monitored at excitation/emission of 525/600 nm every 5 min for 120 min at 37°C with a luminometer (Varioskan Flash; Thermo Scientific). Data shown are representative of results from three technical replicates.

Tetracycline accumulation assay: Tetracyclines have strong ultraviolet absorption and fluorescence properties in the near-visible region. Therefore, we used fluorescence detection to analyze the accumulation of tetracycline. PAO1 was grown in MHB to an OD ₆₀₀ of 1 and then incubated with 50 mg/L of ERC in the presence of different concentrations of BSS or not for 15 min (Figure 6G). The bacteria were collected by centrifugation and washed three times with PBS. The bacterial cells were lysed in PBS by sonication. The fluorescence of ERC was measured at excitation/emission of 400/520 nm with a luminometer. Total bacterial protein concentrations were quantified using a BCA analysis for calibration.

Results of example 6: The oxygen molecule acts as the electron acceptor in the last step of the electron transport chain. The oxygen consumption rate usually represents the rate of electron transport and respiration, so we evaluated the oxygen consumption ratio in BSS treated Pseudomonas, observing a dose-related reduction in the oxygen consumption which is related to reduce ETC activity and respiratory rate (Figure 6A). Parallelly, we observed an increment in the intracellular NADH levels ina BSS dosa-dependent manner (Figure 6B). NADH should be oxidated to NAD in the first step of the ETC by the first enzyme of the route, the NADH oxidase enzyme. Interesting this enzyme is richer of the ETC system in Fe-S clusters. The accumulation of NADH suggests that Bismuth could be impairing the activity of this enzyme so we tested it. For that, the NADH-quinone oxidoreductase enzymes were purified form PAO1 membranes and after that we checked the effect of BSS at different dosages. As indicated in Figure 6C, BSS inhibited the NADH oxidoreductase. The results suggest the inhibition of the ETC at this point by BSS. In the process of electron transfer through the ETC, protons are pumped across the membrane to generate a proton gradient for ATP synthesis. The inhibition of ETC is related to the dissipation of PMF and therefore the generation of ATP and the activity of multidrug efflux pumps which highly depend on the PMF are impaired. The destruction of PMF was detected by monitoring the fluorescence intensity of P. aeruginosa cells (treated with different doses of BSS) labelled with the BCECF-AM probe. Carbonyl cyanide meta-chlorophenyl (CCCP), a compound known as proton uncoupler, was used as a positive control due to its ability to reduce PMF (Figure 6D). Correspondingly, a bismuth- dosage related decrease in ATP levels and a decrease in efflux pump (which requires ATP for their activity) were observed upon the treatment by BSS (Figures 6D, E). As consequence of efflux pumps inhibition antibiotics cannot be removed and they are accumulated inside the cells killing them (Figure 6F, e.g. for eravacycline accumulation). The efflux pumps inhibitor PaβN was used as a positive control for eravacycline accumulation.

EXAMPLE 7: Bismuth combinations inhibit the antimicrobial resistance development in two ways: aiming the total bacterial eradication after the treatment with the combination and thwarting the antibiotic resistance evolution.

Material and methods for example 7: to analyze the bactericidal/bacteriostatic nature of the combinations a time-depending killing assay was performed. For that, an overnight culture of PAO1 was diluted 1:100 into fresh MHB and incubated for 2.5 h at 37 °C under continuous shaking to the early log phase (OD ₆₀₀ =0.4~ 0.6). Then, the cells were challenged by either antibiotic or BSS (16 μM) alone or in combination with nine selected antibiotics (ofloxacin, chloramphenicol, rifampicin, azithromycin, doxycycline, minocycline, tigecycline, eravacycline and omadacycline). At the indicated time points (Figure 7A, B and C), the samples were decimal serially diluted in PBS andlO μl of the dilutions were spotted on LB agar. Colony counts were determined after incubating overnight at 37°C. All experiments were performed with three biological replicates.

For the antibiotic resistance evolution assay in vitro a standard sequential passaging technique at increasing concentrations of the antibiotic was performed. P. aeruginosa PAO1 cells were grown in CA-MHB medium to exponential phase at 37 °C. The MICs of i.e. eravacycline (ERC) or ofloxacin (OFL) in the presence or the absence of 16 μM BSS was determined by standard broth microdilution in 96-wells plates and monitored for 25 days. The plate was incubated for 18 hours under static conditions at 37 °C. The sub-MIC (0.25 x MIC) bacterial suspension from the ERC, OFL or BSS+ERC, BSS+OFL combinations were diluted in CA-MHB to a final concentration of ≈10 ⁶ CFU mL-1 to prepare the inoculum for the next day MIC experiment. Experiments were performed with biological replicates. Results of example 7: As indicated in Figures 7A, B and C, the combination of BSS and the antibiotics acted bactericidally reducing the population of PAO1 at the exponential phase by more than 10,000-fold upon exposure to the drug combinations, while the antibiotics alone or BSS alone didn't affect the cells. The fast bactericidal activity is especially important in the case of bacteriostatic antibiotics as (e.g.) azithromycin o the tetracyclines-related drugs since a fast bactericidal killing activity impairs the development of resistances and or persistence.

Another important characteristic of a good synergistic relation is repression of the resistance evolution, which is a key factor to break the increasing antibiotic resistance tendency. To get a better understanding of BSS on the development of antibiotic resistance, we performed serial passages of PAO1 with sub-MIC (0.25 x MIC) of ERc and OFL in the absence and presence of BSS (16 μM, corresponding to 0.25 x MIC) for 25 days. As shown in Figure 7D and E, sequential passaging on ERC or OFL alone for 25 days resulted in 32 and 64-fold increases in MIC for P. aeruginosa respectively, while BSS prevented the evolution of resistance when co-administered, resulting just in 2-fold increases in MIC of the tested antibiotics. These results suggested that the combination of antibiotics and bismuth could effectively suppress the emergence of antibiotic resistance in P. aeruginosa.

Finally, we selected 3 ERC and 3 OFL resistant strains that were sequenced to identify the mutations involved in such a resistance (Table 4), and we tested the resistance to other tetracyclines and quinolones in the presence of BSS or absence. Resistances to OFL were related to mutations in the DNA gyrase enzyme involved in DNA replication while in the case of ERC it was related to mutations in efflux pumps. As expected, the MIC of the ERC/OFL- resistant strains to other tetracyclines/ quinolones also increased significantly (Table 4). However, and surprisingly, BSS overcomes these resistances (Table 4) sensitizing the cells to the antibiotics. This data suggest that bismuth could sensitize P. aeruginosa to these antibiotics independently of the resistant mechanism developed.

EXAMPLE 8: Antimicrobial combined activity of bismuth and antibiotics was extensible to biofilms growing P. aeruginosa.

Material and methods for example 8: for biofilm formation and treatment, overnight cultured PAO1 cells were diluted 1: 100 into CA-MHB and incubated at 37 °C to an OD600 of 1. The bacterial suspension was standardized to an OD ₆₀₀ =0.025 in fresh CA-MHB. 1 mL of bacterial suspension was added into a glass tube and incubated at 37 °C for 24 hours to form the biofilm. Since the biofilms have strong antibiotic resistance, we used 1x, 1/2x, 1/4x and 1/8xMIC of antibiotics 911 selected antibiotics, Figure 8) combined with 32 μM BSS to kill the established biofilm for 24h. Finally, the biofilm was washed three times with sterile PBS and treated with 2 mL fresh CA-MHB containing the indicated concentration of antibiotics or BSS or the combination to cover the biofilms at 37°C for 24h. The tube was washed 3 times with sterile PBS, the biofilm was dispersed by ultrasound, and the number of bacteria was determined by plating. The experiment was performed in triplicate.

Results of example 8: Infections caused by biofilms, including on medical devices, are an important cause of treatment failure and recurrence of infections. The strong resistance to antibiotics makes them difficult to treat, and it is a major challenge in the treatment of clinical infections since biofilms caused chronic P. aeruginosa infection reducing the therapeutical options of the patients. We analyzed the synergistic effects of bismuth with 11 antibiotics on biofilms. Since the biofilms have strong antibiotic resistance, we used 32 μM BSS combined with antibiotics to kill the established biofilm for 24 h. Even though the biofilms have strong antibiotics resistance, azithromycin exhibits good bactericidal activity against P. aeruginosa biofilms. The results showed that the sub-MIC of azithromycin can efficiently kill the biofilm associated cells, while the combination of BSS and azithromycin slightly enhanced the killing efficacy (Figure 8). For another macrolide antibiotic, the sub-MIC of clarithromycin showed a weak killing effect on the biofilms, while the combination significantly enhanced the killing efficacy (Figure 8). The combination of BSS and chloramphenicol or rifampicin significantly enhanced the killing efficacy on the biofilm- associated cells, while chloramphenicol and rifampicin alone showed very weak effects on biofilms (Figure 8). Moreover, ofloxacin itself has a certain killing effect on the biofilm and its killing effect was further enhanced when it was used in combination with BSS (Figure 8). In the case of the tetracycline-related antibiotic tested, the results showed that the combination of BSS and tetracyclines significantly enhanced the killing efficacy on the biofilm-associated cells (modestly in the case of tetracycline, Figure 8). The best biofilm inhibition activity was observed for the combination with tigecycline and doxycycline (Figure 8), but a good reduction was observed for combinations at low dosages of eravacycline and also in the case of omadacycline and minocycline (Figure 8). The combination of BSS and tetracyclines significantly enhanced the killing efficacy on the biofilm-associated cells.

EXAMPLE 9: Antimicrobial activity of the combinations in a bacteremia model. Material and methods for example 9: Human blood of healthy individuals was obtained from Sanquin (certified Dutch organization responsible for meeting the need in healthcare for blood and blood products, https://www.sanquin.nl/) and was infected with 5x10 ⁷ CFU/mL of PAO1. The blood was distributed at 0.5 mL in 2 mL Eppendorf tubes and treated with the indicated concentration of antibiotics (azithromycin, chloramphenicol, doxycycline, eravacycline, ofloxacin, rifampicin) or bismuth compounds (bismuth subcitrate (BC) and BSS) or the combination at 37 °C (Figure 9). At 4 h post-infection, the samples were decimal serially diluted in PBS and drops of 10 μl of each dilution plated on LB agar to track the number of remaining viable bacteria. At 24 h post-infection, the samples were photographed to track the development of infection. The experiment was performed in triplicate.

Results of example 9: Although we have observed that bismuth and antibiotics have significant synergistic and bactericidal effects in vitro, considering the complexity of the bacterial infection environment, it is still unclear whether this synergistic effect can play a role in organic environments infections. Then, we tested the synergistic effects in an ex vivo bacteremia model through whole blood infection with P. aeruginosa and quantifying the progress of the infection in the blood by counting the remaining viable bacteria in the blood after four hours of different treatment. The results showed that the combination of bismuth compounds and antibiotics showed very good bactericidal effects in blood, while bismuth or antibiotics alone could not control the blood infection of P. aeruginosa (Figure 9A). It is worth noting that some of these combined therapies can even control the infection for more than 24 hours, just after a single dosage (Figure 9B). In fact, even at the lower tested concentration of ERC and OFL the infection was completely controlled, and also for azithromycin combined with BSS. A concentration-related effect was observed for the other antibiotics combinations (Figure 9B).

Table 1. Antimicrobial activity of dif ^: erent metal salts in combinations to anti aiotics against P. aeruginosa. Bismuth salts present high synergistic activity.

Table 1 (continued). Antimicrobial activity of different metal salts in combinations to antibiotics against P. aeruginosa. Bismuth salts present high synergistic activity.

Table 2. Antimicrobial activity of BSS and antibiotics against a broad panel of bacteria. High specificity towards P. aeruginosa is observed.

Table 2 (continued). Antimicrobial activity of BSS and antibiotics against a broad panel of bacteria. High specificity towards P. aeruginosa is observed.

Table 2 (continued). Antimicrobial activity of BSS and antibiotics against a broad panel of bacteria. High specificity towards P. aeruginosa is observed.

Table 3: List of primers used.

Table 4: Antimicrobial resistance to quinolones and tetracyclines overcome after direct resistance evolution assays. OR strais are ofloxacine resisitan strains of PAO1. ER strains are eravacycline resistant strains of PAO1.

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