Patent Description:
Antimicrobial agents have been used for the last <NUM> years to treat patients who have infectious diseases. Since the <NUM>, these drugs have greatly reduced illness and death from infectious diseases. However, these drugs have been used so widely, and for so long, that the infectious organisms the antimicrobial agents are designed to kill have adapted to them. As a result, the medicines become ineffective and infections persist in the body, increasing the risk of spread to others.

Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi.

Thus, antimicrobial resistance is an increasingly serious threat to global public health that requires action across all government sectors and society.

All classes of microbes develop resistance: fungi develop antifungal resistance, viruses develop antiviral resistance, protozoa develop antiprotozoal resistance, and bacteria develop antibiotic resistance.

The problem with antibiotic resistance is a common phenomenon. Fungal infections that are resistant to treatment are an emerging public health problem. Overall, antifungal resistance is still relatively uncommon, but the problem will likely continue to evolve unless more is done to prevent further resistance from developing and prevent the spread of these infections. Although most antifungal resistance occurs in Candida species, resistance in other types of fungi, such as Aspergillus, is also an emerging issue.

Since fungi are eukaryotes, just like the human hosts they infect, there are only few distinct targets that can be employed for antifungal drug development. Hence antifungals are mostly restricted to drugs targeting a few metabolic pathways.

One target for antimicrobial agents is the biofilm which is a product of a microbial developmental process. Biofilms are formed by microbial cells stuck to each other and surrounded by the self-produced extracellular polymeric matrix. Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in a hostile environment. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behaviour in which large suites of genes are differentially regulated. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae.

Biofilms are estimated to be associated with <NUM>% of microbial infections and growth of micro-organisms in biofilms may enhance their resistance to antimicrobial agents. In addition, biofilm bacteria are up to <NUM>-fold more tolerant and/or resistant to antibiotics than planktonic cells.

In most fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium. For example, the virulence of Candida albicans is mediated by a transformation from planktonic cells into hyphae. The hyphal form, i.e. filamentous cells, has the ability to invade tissue and induce inflammation, mediated by candidalysin, a cytotoxic peptide toxin that destroys the epithelial cells (<NPL>).

Antimicrobial resistance is not only problematic in primary infections, but also in secondary infections. Without effective antimicrobials for prevention and treatment of infections, medical procedures such as organ transplantation, cancer chemotherapy, diabetes management and major surgery become very high risk. Furthermore, fungal infections have become one of the major causes of morbidity and mortality in immunocompromised patients, such as those with HIV/AIDS, tuberculosis or receiving chemotherapy. Despite increased awareness and improved treatment strategies, the frequent development of resistance to the antifungal drugs used in clinical settings contributes to the increasing toll of mycoses.

<CIT> discloses that the antibacterial effect of an antifungal agent such as miconazole can be enhanced by a buffering system comprising an organic acid.

<CIT> discloses calcium sugar acid salts, such as calcium gluconate, and their ability to reduce TSST-<NUM> production of S.

<CIT> discloses the effect of zinc gluconate on salmonella in pigs.

<CIT> discloses the effect of a series of lactones on Streptococcus pneumoniae.

Thus, there is an emerging need for novel antimicrobial agents.

The present inventors have developed a method for preventing and/or reducing biofilm formation. When the biofilm formation is reduced or prevented, the individual microbial cells can no longer attach to the surface. Hence, further infection is prevented and the microbial cells, which no longer form a biofilm, are discarded. The present inventors have shown that treatment with a compound of Formula XX,
<CHM>
or a lactone thereof, such as a compound of formula XIX or XXI,
<CHM>
reduces the presence of biofilm of fungi species, and to have a cytotoxic effect on several fungi species. Furthermore, the present inventors have shown that a compound of formula I is useful as an antibacterial agent.

Thus, in one aspect, the present invention concerns a compound of Formula XX, or a lactone thereof, or a pharmaceutical composition comprising a compound of Formula XX, or a lactone thereof, for use in the treatment and/or prevention of bacterial infections.

In one embodiment, the present invention relates to glucono-δ-lactone for use in the treatment of a bacterial infection caused by Gardnerella vaginalis, Mobiluncus spp, Ureaplasma urealyticum, Mycoplasma hominis, Prevotella spp, Enterococci, Bacteroides spp, Peptostreptococcus spp, Porphyromonas gingivalis, Escherichia coli, Pseudomonas aeruginosa, Acetinobacter baumanii, Streptococcus pyogenes, Beta-Hemolytic Streptococci Group C, Beta-Hemolytic Streptococci Group G, and/or Streptococcus agalactiae.

In a further aspect, the present invention concerns a compound of Formula XX, or a lactone thereof, such as a compound of Formula XIX or XXI, for use in preventing preterm birth caused by bacterial vulvovaginitis.

The references to methods of treatment in this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for the treatment of the human (or animal) body by therapy (or for diagnosis).

In one aspect, the present invention concerns a pharmaceutical composition comprising a compound of Formula XX,
<CHM>
or a lactone thereof, such as a compound of formula XIX or XXI,
<CHM>
for use in the treatment and/or prevention of a bacterial infection selected from the group consisting of Gardnerella vaginalis, Mobiluncus spp, Ureaplasma urealyticum, Mycoplasma hominis, Prevotella spp, Enterococci, Bacteroides spp, Peptostreptococcus spp, Porphyromonas gingivalis, Escherichia coli, Pseudomonas aeruginosa, Acetinobacter baumanii, Streptococcus pyogenes, Beta-Hemolytic Streptococci Group C, Beta-Hemolytic Streptococci Group G-, and/or Streptococcus agalactiae infection.

In one embodiment, compounds XIX, XX and XXI are in equilibrium in aqueous solution.

In a preferred embodiment, the compound of Formula I is glucono-δ-lactone (GDA, Formula XIX),
<CHM>.

In one embodiment, the compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, is oligomerized to form an oligomer. In another embodiment, the compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, is polymerized to form a polymer.

In one embodiment, the oligomer or polymer comprises one compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI,, i.e. is a homo-oligomer/polymer wherein one compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, is the monomer. In another embodiment, the oligomer or polymer is a mixed oligomer/polymer, i.e. a hetero-oligomer/polymer.

In one embodiment, the oligomer or polymer further comprises lactic acid, i.e. is a lactic acid oligomer/polymer.

In one aspect, the present invention concerns a compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, or a pharmaceutical composition comprising said compound for use in the treatment and/or prevention of a bacterial infection selected from the group consisting of Gardnerella vaginalis, Mobiluncus spp, Ureaplasma urealyticum, Mycoplasma hominis, Prevotella spp, Enterococci, Bacteroides spp, Peptostreptococcus spp, Porphyromonas gingivalis, Escherichia coli, Pseudomonas aeruginosa, Acetinobacter baumanii, Streptococcus pyogenes, Beta-Hemolytic Streptococci Group C, Beta-Hemolytic Streptococci Group G-, and/or Streptococcus agalactiae infection.

In one embodiment, the bacterial infection is a urogenital infection. In one embodiment, the bacterial infection is a vaginal infection.

In one embodiment, the infection is an infection in a mammal, i.e. the subject in need of said treatment is a mammal. Preferably, the mammal is a human. In one embodiment, the human is a woman. Said woman may be a woman who is pregnant.

In one embodiment, the infection is dermatitis and/or eczema. Said dermatitis and/or eczema may be Seborrhoeic dermatitis. The infection may also be a secondary infection of said dermatitis or eczema.

The term "secondary infection" as used herein refers to a sequela or complication of a root cause. Said root cause may be a primary infection.

In one embodiment, the infection is acne of all severities, or acneiform conditions such as rosacea, perioral or periorbital dermatitis.

In one embodiment, the infection is furunculosis, carbunculosis or folliculitis.

In one embodiment, the infection is an infection of the face, scalp, torso and/or groin. The infection may be an infection of infected skin wounds in said areas. The infection may also be located in skinfolds of the body.

In one embodiment the infection is impetigo or erysipelas.

In one embodiment, the infection is an infection of the feet. Said infection of the feet may be associated with diabetic foot wounds. In one embodiment, the infection of the feet is secondary to ingrown toenails or blisters of the feet.

In one embodiment, the infection is a secondary infection arising after an animal bite. Said animal may be an insect. In one embodiment, the infection is a secondary infection arising after insect bites, mosquito bites, tic bites, erythema migrans or lymphadenosis benigna cutis.

In one embodiment, the infection is a secondary infection of herpes simplex, dermatological, oral or genital, or a secondary infection of herpes zoster or varicella zoster.

In one embodiment, the infection is a secondary infection of injury to the skin, such as burns or cuts.

In one embodiment, the bacterial infection is periodontitis.

In one embodiment, the bacterial infection is bacterial vaginosis.

In one aspect, the present invention concerns a compound of formula XX,
<CHM>
or a lactone thereof, such as a compound of formula XIX or XXI,
<CHM>
for use in the treatment of bacterial vaginosis, wherein the bacterial vaginosis is caused by Gardnerella vaginalis, Mobiluncus spp, Ureaplasma urealyticum, Mycoplasma hominis, Prevotella spp, Enterococci, Bacteroides spp, Peptostreptococcus spp, Porphyromonas gingivalis, Escherichia coli, Pseudomonas aeruginosa, Acetinobacter baumanii, Streptococcus pyogenes, Beta-Hemolytic Streptococci Group C, Beta-Hemolytic Streptococci Group G, and/or Streptococcus agalactiae.

In one embodiment, the infection is secondary to oral, nasal or anogential colonization of group A or group B streptococci or multiresistant bacteria.

In one embodiment, the infection is perianal streptococcal dermatitis.

In some embodiments, the present invention relates to a pharmaceutical composition comprising a compound of formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, for use in the treatment of a bacterial infection. In one embodiment, the pharmaceutical composition is formulated as a tablet, orally disintegrating tablet (or orally dissolving tablet (ODT)), lozenge, gum, chewing gum, cream, lotion, gel, emulsion, solution, foam, ointment, spray, suspension mouthwash, mouth rinse, oral rinse, mouth bath, nail polish, dermal patch or shampoo. In one embodiment, said solution as adapted for use in a bandage, dressing and/or compress.

In one embodiment, the pharmaceutical composition comprises at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt %, such as at least <NUM> wt % of the compound of Formula (I).

In one embodiment, the pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutical composition comprises no more than <NUM> wt %, such as no more than <NUM> wt %, such as no more than <NUM> wt %, such as no more than <NUM> wt %, such as no more than <NUM> wt %, such as no more than <NUM> wt % of the compound of Formula (I).

In one embodiment, the pharmaceutical composition comprises <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt %, such as in <NUM> to <NUM> wt % of the compound of Formula (I).

In one embodiment, the pharmaceutical composition comprises no more than <NUM> wt % water, such as no more than <NUM> wt % water.

An "antimicrobial agent", as used herein, refers to an agent that is capable of decreasing or eliminating or inhibiting the growth of microorganisms such as that term is known in the art (exemplary microorganisms include microbes such as bacteria, fungi, viruses and other pathogens). Similarly, the term "antifungal agent" refers to an agent that is capable of decreasing or eliminating or inhibiting the growth of fungi, and the term "antibacterial agent" refers to an agent that is capable of decreasing or eliminating or inhibiting the growth of bacteria.

In one embodiment, the pharmaceutical composition is formulated as a tampon, vagitorium, vaginal aerosol, vaginal cup, vaginal gel, vaginal insert, vaginal patch, vaginal ring, vaginal sponge, vaginal suppository, vaginal cream, vaginal emulsion, vaginal foam, vaginal lotion, vaginal ointment, vaginal powder, vaginal shampoo, vaginal solution, vaginal spray, vaginal suspension, vaginal tablet, vaginal rod, vaginal disc, vaginal device, and any combination thereof, or wherein the pharmaceutical composition is present on a sanitary article, such as a tampon, a sanitary napkin, an incontinence pad or diaper, or a panty liner.

In one embodiment, the pharmaceutical composition is adapted for administration at least once daily, such as at least twice daily, such as at least three times daily.

In one embodiment, the pharmaceutical composition is adapted for administration no more than every second day, such as no more than every third day, such as no more than once a week.

In one embodiment, the pharmaceutical composition is adapted for administration for no more than six days.

In one embodiment, the pharmaceutical composition is adapted for administration during at least one week, such as during at least two weeks, such as during at least three weeks, such as during at least four weeks.

In one embodiment, the pharmaceutical composition is adapted for administration for at least once daily during at least a week.

In one embodiment, the pharmaceutical composition is formulated to release the compound according to Formula I over an extended period of time, such as over at least <NUM> hours, such as over at least <NUM> hours, such as over at least <NUM> hours after administration.

The term "biofilm" as used herein refers to an aggregate of microorganisms in which microbial cells adhere to each other and/or to a surface. These adherent cells are often covered with a matrix of extracellular polymeric substance, e.g. comprising extracellular DNA, proteins, and polysaccharides, which is produced by the cells. Microbial cells growing in a biofilm are often physiologically distinct from planktonic cells of the same organism. Such biofilms may form on any living or non-living surfaces.

In one embodiment, the bacterial infection is bacterial vulvovaginitis. With cervical ripening or cervical insufficiency, the said infection may migrate to the uterus and cause chorioamnionitis and subsequently preterm birth. There is evidence supporting that excessive inflammation, such as a vaginitis or cervicitis, through prostaglandin production can cause premature contractions and preterm birth even in the absence of manifest chorioamnionitis. The preterm neonate can subsequently face invasive bacterial infection; pneumonia, meningitis or sepsis, due to neonatal immunodeficiency in the preterm infant. Especially group A and B streptococci and multiresistent bacteria can give rise to serious perinatal infection in the infant and postpartum endometritis in the newly delivered woman and be the cause of both neonatal and maternal serious morbidity and mortality.

Thus, in one aspect, the present invention concerns a compound of Formula XX, or lactone thereof, such as a compound of Formula XIX or XXI, for use in preventing preterm birth caused by bacterial vulvovaginitis. In a preferred embodiment, said compound is administered vaginally. The compound may be formulated as a tampon, vagitorium, vaginal aerosol, vaginal cup, vaginal gel, vaginal insert, vaginal patch, vaginal ring, vaginal sponge, vaginal suppository, vaginal cream, vaginal emulsion, vaginal foam, vaginal lotion, vaginal ointment, vaginal powder, vaginal shampoo, vaginal solution, vaginal spray, vaginal suspension, vaginal tablet, vaginal rod, vaginal disc, vaginal device, and any combination thereof, or wherein the compound is present on a sanitary article, such as a tampon, a sanitary napkin, an incontinence pad or diaper, or a panty liner.

Yeast strains (Table <NUM>) were grown at <NUM> in complete medium YPD (<NUM>% yeast extract, <NUM>% peptone, <NUM>% glucose) or minimal medium consisting of YNB (yeast nitrogen base without amino acids and ammonium sulphate, FORMEDIUM™, CYN0505) supplemented with <NUM>% ammonium sulphate, <NUM>% glucose and <NUM> L-proline. If needed <NUM>% agar was used to solidify media. The liquid minimal medium (YNB (yeast nitrogen base without amino acids and ammonium sulphate, FORMEDIUM™, CYN0505) supplemented with <NUM>% ammonium sulphate, <NUM>% glucose and <NUM> L-proline) was used for biofilm assay (biofilm medium).

In the experiments on the impact of pH on biofilm the pH values (from <NUM> to <NUM>) were obtained using either different potassium phosphate buffers at the final concentration <NUM>, or by the addition of citric acid, lactic acid, and gluconic acid to the biofilm medium.

Biofilm was measured in liquid culture as described [<NPL>. <NPL>] with some modifications. Prior the biofilm assay, yeast cultures were grown in liquid YPD medium for <NUM> hours until stationary phase (OD<NUM> <NUM>-<NUM>)<NUM>, cells were then pelleted by centrifugation (<NUM>), washed with sterile water and cells were further inoculated into test biofilm medium (YNB (yeast nitrogen base without amino acids and ammonium sulphate) supplemented with <NUM>% ammonium sulphate, <NUM>% glucose and <NUM> L-proline pH7. <NUM>) at final concentration <NUM> OD<NUM>/ml and incubated in <NUM>-well flat-bottom polystyrene microtiter plates (Sigma Aldrich, Corning® Costar® culture plates, CLS3596-50EA) for <NUM> hours at <NUM> thermostat. At defined time points crystal violet (HT901-8FOZ; Sigma Aldrich) was added to the media at the final concentration <NUM>%, in addition total biomass was measured. After <NUM> hours of cells staining, plate wells were washed four times with <NUM>µl of water to remove planktonic cells, biofilms were then dried and dissolved in <NUM>µl of <NUM>% ethanol. Total biomass and crystal violet biofilm staining measurements were performed at OD<NUM> with FLUOstar OPTIMA plate reader, BMG LABTECH. Crystal violet biofilm measurements were normalized to the total biomass (OD<NUM>Biofilm/OD<NUM> total biomass).

To compare the effects of different hydroxylated carboxylic acids at low concentrations, the biofilm formation was measured <NUM> after addition of <NUM> wt% of glyceric acid, xylonic acid, citric acid, gluconic acid, and lactic acid, under unbuffered conditions. The data are shown in Table <NUM> and in <FIG>.

As can be seen from Table <NUM>, gluconic acid is the most efficient compound, followed by lactic acid and citric acid. Replacing lactic acid by gluconic acid resulted in ><NUM>% less biofilm formation (<NUM>% vs <NUM>% of control). Given that the biofilm formation is very sensitive to low pH and that gluconic acid only induces a moderate lowering of the pH in comparison with lactic acid and citric acid, the result is striking.

In order to further evaluate the effect of gluconic acid in preventing biofilm formation, the biofilm formation at different pH were determined (as described in Example <NUM>) for gluconic acid, lactic acid and citric acid.

As can be seen from Table <NUM>, Gluconic acid shows strong effects on the biofilm formation of Candida albicans, while the effects from lactic acid and citric acid are much less pronounced. Furthermore, gluconic acid shows strong effect also at pH values up to around at least <NUM>, whereas the effect seen with lactic acid and citric acid starts diminishing already at pH <NUM>. In addition, gluconic acid results in a complete loss of biofilm formation at pH <NUM>. The data are summarized in Table <NUM>.

Candida glabrata is much more complicated to treat, compared to Candida albicans. However, a clear effect is obtained by longer treatment, i.e. <NUM> with gluconic acid (Table <NUM>).

Based on these results it was concluded that gluconic acid has superior effect in targeting biofilm formation by candida compared to lactic acid and citric acid. In contrast to the effect observed for lactic acid and citric acid, the effect observed for gluconic acid is not merely a pH-related effect. The effect is present even at pH <NUM>.

It is thus concluded that gluconic acid is useful as antifungal compound, as indicated by reduction of biofilm formation. The compound is physiologically and pharmaceutically acceptable. Gluconic acid is thus useful for providing pharmaceutical formulations for use in treating vulvovaginal candidosis.

Lactonization/oligomerization of gluconic acid:
Gluconic acid (GA) (<NUM> wt% in H<NUM>O, <NUM>) was poured in an open vial and heated to <NUM>. After <NUM>, the mixture was cooled down to room temperature, whereas it solidified.

In order to analyze the composition of aqueous solution of gluconic acid (GA), GA (<NUM> wt% in H<NUM>O) was dissolved in DMSO-d<NUM> and analyzed by <NUM>H- and <NUM>C-NMR.

Lactonized/oligomerized gluconic acid (cf. above) was dissolved in DMSO-d<NUM> and analyzed by <NUM>H- and <NUM>C-NMR, see Table <NUM>.

It was concluded that the GA form a complex mixture of different lactones as well as oligomerized material upon prolonged dehydration.

In water solution glucono-δ-lactone (GDA) is in equilibrium with gluconic acid (GA, <NPL>). GDA (<NUM>) was added to distilled H<NUM>O (<NUM>), pH <NUM> buffer, pH <NUM> buffer, or pH <NUM> buffer at <NUM>. The optical rotation and pH were measured over time. Optical rotation, measured at <NUM>, sodium D line, C=<NUM>/mL, path length=<NUM>. The optical rotation of GDA is approximately <NUM> °. The optical rotation of gluconic acid is approximately <NUM> ° [<NPL>].

This experiment shows that GDA is slowly hydrolyzed to a mixture of GDA and GA (<FIG>). The equilibrium is pH-dependent and relevant concentrations of GDA are present at all buffered conditions.

Pellet of lactonized/oligomerized gluconic acid (<NUM>, duplicate samples) were added to buffer solution of pH <NUM> (<NUM> KH<NUM>PO/ortho phosphoric acid, <NUM>) at <NUM>. Samples (<NUM>) were taken every hour (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) and new buffer solution (<NUM>) was added. The samples were diluted <NUM> times with biofilm medium (vide supra) and the amount of biofilm formation was measured after <NUM> as described above. As seen from <FIG>, the released GA significantly reduces the amount of biofilm formation in Candida albicans. Further, the hydrolysis of the pellet is seemingly slow enough to provide a preventive effect for at least up to <NUM> hours, likely far more. The effect is less pronounced with Candida glabrata (<FIG>). The data are summarized in Table <NUM>.

Pellets of glucono-δ-lactone (GDA) (<NUM>, duplicate samples) were added to buffer solution of pH <NUM> (<NUM> KH<NUM>PO<NUM>/ortho phosphoric acid, <NUM>) at <NUM>. Samples (<NUM>) were taken at fixed time points (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) and new buffer solution (<NUM>) was added. The samples were diluted <NUM> times with biofilm medium (vide supra) and the amount of biofilm formation was measured after <NUM> as described above. As seen from <FIG>, the released GDA significantly reduces the amount of biofilm formation in C. Further, the hydrolysis of the pellet is seemingly slow enough to provide a preventive effect for at least up to <NUM> hours, likely far more. The effect is less pronounced with C. glabrata (<FIG>).

The results show that biofilm formation of both C. albicans and C. glabrata was reduced in the presence of GDA. In addition to diminished biofilm formation, GDA may affect the viability of mature biofilm of C. albicans and C.

Viability of biofilms of C. albicans and C. glabrata after treatment with glucono-δ-lactone (GDA) at different concentration and different time periods was evaluated by staining the cells with XTT. XXT is a colorimetric assay for quantification of cellular viability, and cytotoxicity. The assay is based on the cleavage of the tetrazolium salt XTT, a conversion that only occurs in viable cells. The mature biofilm was exposed to GDA for <NUM>. Then the cells were washed <NUM> times with PBS, after which the XTT reaction mixture was added. After <NUM> the optical density measured at <NUM>.

The XTT assay showed a strong decrease in viability for C. glabrata already after <NUM> of incubation (<FIG>). The effect was less pronounced for C. albicans but clearly seen after <NUM> (<FIG>).

Furthermore, mature biofilm (grown for <NUM> in YNB, <NUM>% glucose, <NUM> proline) of C. albicans and C. glabrata was incubated with GDA of different concentrations (<NUM> - <NUM>/ml) at <NUM>. For this purpose biofilm medium (YNB, <NUM>% glucose, <NUM> proline) was removed and GDA was added, which was dissolved either in water at concentration <NUM>, <NUM>, <NUM> and <NUM>/ml. After incubation with GDA for <NUM> or <NUM>, 5µl of cells were plated at serial dilution (<NUM>:<NUM> to <NUM>:<NUM>) on the agar medium YPD to estimate cell survival. The plated cells were incubated for <NUM> at <NUM> and visually analyzed. The cells from the mature biofilm treated with water were used as a control. It was found that the GDA decreases cell viability of C. albicans and C. glabrata, particularly at high concentrations. At the concentrations of <NUM> and <NUM>/ml after <NUM> of incubation the cell viability was decreased by about <NUM> times for both C. albicans and C. After <NUM> of incubation with <NUM>/ml of GDA the cell viability of C. albicans was decreased by about <NUM> times (data not shown). glabrata proved to be more sensitive to GDA (<FIG>).

To monitor the C. albicans cell morphology we studied the biofilm development also using microscopy and microfluidics. After the yeast cells were inoculated, hyphae started to form within first hour of incubation in the biofilm medium (YNB supplemented with <NUM> proline and <NUM>% glucose, pH7. <FIG> shows untreated cells after <NUM>. A pellet of glucono-δ-lactone (<NUM>) was added to buffer solution of pH <NUM> (<NUM> KH<NUM>PO<NUM>/ortho phosphoric acid, <NUM>) at <NUM>. A sample was taken after <NUM> and diluted <NUM> times with biofilm medium and added to C. After <NUM> most treated cells were planctonic (<FIG>).

Other Candida sp. studied were also sensitive (i.e. cell viability measure using the XTT assay, cf Example <NUM>) to glucono-δ-lactone (GDA). However, they displayed different levels of sensitivity. Candida albicans SC5314 displayed the lowest susceptibility and Candida krusei silicone isolate A4-<NUM> displayed the highest susceptibility. The GDA-toxicity is mediated through cell wall damage as the cells exposed to GDA had lower viability on the medium with calcofluor white compared to that supplemented with osmotic stabilizer (<NUM> sucrose) and compared to the untreated cells on these media. Table <NUM> summarizes qualitative effects shown by GDA.

To conclude, (i) GDA can break mature biofilm formed by C. albicans and C. glabrata, (ii) upon exposure to GDA, C. albicans transforms into yeast form, while the viability of C. glabrata decreases, (iii) the effect is clear even on other strains, i.e. C. tropicalis and C.

DL-Lactic acid (<NUM>, <NUM> mmol) and D-gluconic acid (<NUM>% in water, <NUM>, <NUM> mmol) was mixed in a test tube and heated to <NUM>. After <NUM>, the temperature was increased to <NUM>. After a total of <NUM>, the reaction mixture was allowed to reach rt. The reaction mixture solidified upon cooling.

DL-Lactic acid (<NUM>, <NUM> mmol) and D-gluconic acid (<NUM>% in water, <NUM>, <NUM> mmol) were mixed in a test tube and heated to <NUM> under vaccum. After a total of <NUM>, the reaction mixture was allowed to reach rt. The reaction mixture became almost totally solid upon cooling.

DL-Lactic acid (<NUM>, <NUM> mmol) was pre-heated at <NUM> under vacuum. After for <NUM>, D-gluconic acid (<NUM>% in water, <NUM>, <NUM> mmol) was added. After another <NUM> under vacuum at <NUM>, the reaction mixture was allowed to reach rt. The reaction mixture solidified upon cooling.

D-gluconic acid (<NUM>% in water, <NUM>) and citric acid monohydrate (<NUM>, <NUM> wt. %) were mixed in a test tube and heated to <NUM>. After a total of <NUM>, the reaction mixture was allowed to reach rt. The reaction mixture did not solidify upon cooling.

From these results we show that gluconic acid can be oligomerized with lactic acid to form a solid, in contrary to pure gluconic acid or lactic acid that both are liquids.

The bacterial strain Escherichia coli K12 was used for the biofilm study. This strain was maintained on LB medium at <NUM>. The biofilm was studied in synthetic medium M9 (x1 M9 minimal salts (Sigma M6030), <NUM> MgSO<NUM>, <NUM> CaCl<NUM>, and <NUM>% glucose), which contained either phosphate buffer, citric acid, lactic acid, gluconic acid or glucono-δ-lactone to obtain media with different pH (<NUM>-<NUM>).

The overnight culture of E. coli K12 (OD<NUM>~<NUM>) was washed with sterile water and inoculated to the final concentration <NUM> OD/ml of M9 medium with different pH of different compounds. The biofilm development was studied in <NUM>-well flat-bottom polystyrene microtiter plates (Sigma Aldrich, Corning® Costar® culture plates, CLS3596-50EA). The biofilm was stained with crystal violet.

During the biofilm experiment (<NUM>-<NUM> hours) the bacterial strain had increased its biomass (<NUM>-<NUM>-fold) only in the M9 medium supplemented with phosphate buffer at pH <NUM> and pH <NUM> (<FIG>). The biomass was lower on other media and we observed the decrease in bacterial biomass with lowering of the pH of the medium. Acids (citric acid, lactic acid, gluconic acid), and glucono-δ-lactone had inhibiting effect on the growth. In addition to the growth inhibition, the lowering of the pH of the medium resulted in decreased biofilm formation. The lowest amount of biofilm was observed in the medium supplemented with gluconic acid. The second most effective in biofilm inhibition was lactic acid followed by glucono-δ-lactone. Comparing to the phosphate medium, the biofilm on gluconic acid medium was <NUM>-fold lower at pH <NUM> and <NUM>-fold lower at pH <NUM>. At pH <NUM> the gluconic and lactic lowered the biofilm ~<NUM>-fold. The glucono-δ-lactone at pH <NUM> decreased the biofilm formation <NUM> times at <NUM> and <NUM> times at <NUM> correspondingly.

The lowering of pH of the media had clear effect on the E. coli biofilm development, which is likely associated with bactericidal effect (the lower biofilm was accompanied with lower biomass).

Gardnerella vaginalis, Lactobacillus iners, and Lactobacillus crispatus were recovered by CCUG (Culture Collection of University of Gothenburg). Subculture plates were made for G. vaginalis on Chocolate-GL plates and Lactobacillus spp. agar plates at <NUM>% CO<NUM>, <NUM>. The inoculums were prepared from the subculture plates. Colonies were inoculated to culture tubes with <NUM> test medium and vortexed <NUM> with about <NUM> glass beads à <NUM> in diameter. Colonies were taken until the turbidity of the solution was OD<NUM> of <NUM>-<NUM>. The bacterial solutions were checked in light microscopy at x40 with phase contrast to make sure that the cells were dispersed. Each bacterial suspension was diluted in its test medium, <NUM>:<NUM>, to equal <NUM>-<NUM> x <NUM><NUM> CFU/ml. The microbial solutions were stored at <NUM> during the preparation of the inoculums.

The test substance solution was prepared aseptically at <NUM>/ml in sterile H<NUM>O. The first row of wells was filled with <NUM>µl substance and then <NUM> fold dilutions were done vertically in <NUM> steps. Controls were included: i) Growth controls for each strain (+ctrl) = respective microorganism in test medium without antimicrobial agent (AM), ii) no growth control (-ctrl) = test medium and substance at highest concentration tested, this is to make sure that the substance alone does not generate a colour change of test media, iii) gentamicin control for each strain.

Next, <NUM>µl microorganisms were added and the plates were gently shaken at <NUM> rpm for <NUM> seconds before incubation at <NUM> and <NUM>% RH in the CO<NUM> incubator set at <NUM>% CO<NUM>. After <NUM> and <NUM> hours of incubation, the OD was measured and ocular assessments were done. The pH was also measured for all dilutions, n=<NUM>, pH = <NUM> - <NUM>.

MIC microdilution assays were run in triplicate to assess the MIC values for GDA against G. vaginalis (CAMHB) and L. crispatus (IsoS-M. ) after <NUM> hours incubation at <NUM>% CO<NUM> and L. iners cultured under anaerobic conditions after <NUM> hours (Table <NUM>).

The minimum inhibitory concentration (MIC) value of GDA against Gardnerella vaginalis is much lower than the MIC value of GDA against Lactobacillus iners and Lactobacillus crispatus. Thus, GDA is more efficient against the Gardnerella vaginalis than against the benign Lactobacillus iners and Lactobacillus crispatus.

All strains were recovered from microbanks and streak and subculture plates were made. The aerobes on TSA plates, the fastidious aerobes on horse blood plates and the anaerobe on FAA plates. Throughout the study, the aerobes were cultured under aerobic conditions, the fastidious at <NUM> % CO<NUM> and the anaerobe under strict anaerobic conditions at <NUM>. At the time of testing, loopfuls of colonies selected from <NUM>- to <NUM>-hour agar plates for the aerobes and fastidious aerobes were suspended in <NUM> saline in <NUM> tubes containing <NUM> glass beads à <NUM> in diameter.

The anaerobes were suspended in Concept <NUM> and the diluent was reduced Brucella broth supplemented with hemin (<NUM>µg/ml), vitamin K1 (<NUM>µg/ml), and lysed horse blood (<NUM>%). The cell suspensions were then vortexed vigorously for <NUM> minute to obtain a turbid suspension. Each suspension of the aerobes and fastidious aerobes was adjusted to equal OD <NUM> at <NUM> using a spectrophotometer, which correlates approximately to <NUM>-<NUM> x <NUM> CFU/ml with most species. For the anaerobe, the OD measurement turned out to be difficult due to the use of blood in the culture medium. Hence, it was suspended to equal <NUM> Mc Farland standard and the cell density of the inoculum checked by plate count. The aerobes and fastidious aerobes were further diluted in saline <NUM> times so that the inoculum concentration equaled to <NUM> -<NUM> X <NUM> CFU/ml. The anaerobe was not diluted but used directly as is. The different aerobic and fastidious aerobic bacterial suspensions were transferred to respective wells of a <NUM> well plate. baumannii, E. coli, and P. aeruginosa were placed in one row, the Staphylococci spp. were placed in a second row, and the fastidious aerobes were placed in a third row of the plate. The anaerobe was transferred from a <NUM> multichannel pipette reservoir.

The Microdilution Preparations and the MIC tests were performed as in example <NUM>. MIC microdilution assays were run in triplicate to assess the MIC values for glucono-δ-lactone (GDA) (Table <NUM>).

Claim 1:
A compound of Formula XX,
<CHM>
or a lactone thereof, such as a compound of formula XIX or XXI,
<CHM>
for use in the treatment and/or prevention of a bacterial infection selected from the group consisting of Gardnerella vaginalis, Mobiluncus spp, Ureaplasma urealyticum, Mycoplasma hominis, Prevotella spp, Enterococci, Bacteroides spp, Peptostreptococcus spp, Porphyromonas gingivalis, Escherichia coli, Pseudomonas aeruginosa, Acetinobacter baumanii, Streptococcus pyogenes, Beta-Hemolytic Streptococci Group C, Beta-Hemolytic Streptococci Group G-, and/or Streptococcus agalactiae infection.