COMBINATIONS OF LYSOBACTIN AND AMINOGLYCOSIDES AGAINST DISEASES CAUSED BY GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA IN NON-HUMAN ANIMALS

Abstract

The present invention relates to the combination of lysobactin with an aminoglycoside antibiotic for use in the treatment of diseases caused by Gram-positive and Gram-negative bacteria in non-human animals. The invention proved to be particularly useful relating to the treatment of mastitis in non-human animals. Particularly bovine mastitis can be well addressed by the present invention.

Claims

1. A method for treatment of a disease caused by Gram-negative and/or Gram-positive bacteria in a non-human mammalian animal in need thereof, comprising administering a combination of lysobactin and at least one aminoglycoside antibiotic to the non-human mammalian animal.

2. The method of claim 1, wherein the non-human mammalian animal is a female animal having an udder.

3. The method of claim 2, wherein the female non-human mammalian animal having an udder is a cow and the disease is bovine mastitis.

4. The method of claim 2, wherein the combination is administered intramammarily.

5. The method of claim 3, wherein the bovine mastitis is a clinically manifest bovine mastitis.

6. The method of claim 3, wherein the bovine mastitis is a subclinical bovine mastitis.

7. The method of claim 4, wherein the lysobactin is provided at a dose of 25 to 1000 mg per udder quarter.

8. The method of claim 1, wherein the at least one aminoglycoside antibiotic is kanamycin or neomycin.

9. The method of claim 3, wherein the bovine mastitis is caused by Staphylococcus bacteria, Streptococcus bacteria, Trueperella bacteria, Corynebacterium bacteria and/or Escherichia bacteria.

10. The method of claim 9, wherein the bovine mastitis is caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalacticae, Streptococcus agalacticae and/or E. coli bacteria.

11. The method of claim 9, wherein the bovine mastitis is caused by Trueperella pyogenes and/or E. coli bacteria.

12. The method of claim 9, wherein the bovine mastitis is caused by Corynebacterium bovis and/or E. coli bacteria.

13. A pharmaceutical composition comprising lysobactin and at least one aminoglycoside antibiotic.

14. The method of claim 3, wherein the combination is administered intramammarily.

15. The method of claim 14, wherein the bovine mastitis is a clinically manifest bovine mastitis.

16. The method of claim 14, wherein the bovine mastitis is a subclinical bovine mastitis.

17. The method of claim 14, wherein the lysobactin is provided at a dose of 25 to 1000 mg per udder quarter.

18. The method of claim 8, wherein the at least one aminoglycoside antibiotic is neomycin.

19. The pharmaceutical composition of claim 13, wherein the at least one aminoglycoside antibiotic is kanamycin or neomycin.

20. The pharmaceutical composition of claim 14, wherein the amount of lysobactin is present at a dose of 25 to 1000 mg per udder quarter and the amount of the at least one aminoglycoside antibiotic is present at a dose of 25 to 1000 mg per udder quarter.

Description

EXAMPLES

[0053] The present invention will be further elucidated with reference to the following examples and figures without being limited to them.

[0054] FIG. 1 shows the kill kinetics of lysobactin against Staphylococcus aureus and Streptococcus uberis (example 2)

[0055] FIG. 2 shows the MIC changes of lysobactin against Staphylococcus aureus and Streptococcus uberis during serial passaging (example 4)

[0056] FIG. 3 shows the efficacy of lysobactin in a Staphylococcus aureus acute mouse mastitis model (example 5)

[0057] FIG. 4 shows the efficacy of lysobactin in a Streptococcus uberis challenge mouse mastitis model (example 6)

[0058] FIG. 5 shows the concentration-time profile of lysobactin in milk after intramammary (IMAM) application to lactating Holstein cows (example 7)

[0059] FIG. 6 shows the kill kinetics in milk of lysobactin and neomycin, either alone or in combination at 1:1 and 2:1 dose ratios, against Staphylococcus aureus and Streptococcus uberis (example 10)

[0060] FIG. 7 shows the kill kinetics in milk of lysobactin and neomycin, either alone or in combination at 1:5 and 1:10 dose ratios, against Staphylococcus aureus and Streptococcus uberis (example 10)

[0061] FIG. 8 shows the kill kinetics in milk of lysobactin and neomycin, either alone or in combination, against E. coli (example 10)

[0062] FIG. 9 shows the kill kinetics of combinations of lysobactin, either with neomycin or kanamycin, against Staphylococcus aureus (example 11)

[0063] FIG. 10 shows the kill kinetics of combinations of lysobactin with florfenicol for Staphylococcus aureus and Streptococcus uberis (example 12)

[0064] FIG. 11 shows the efficacy of lysobactin and neomycin, either in combination or alone, in a Staphylococcus aureus acute mouse mastitis model (example 13)

[0065] FIG. 12 shows the efficacy of lysobactin and neomycin, either in combination or alone, in a Streptococcus uberis acute mouse mastitis model (example 14)

EXAMPLE 1: In Vitro Antibacterial Activity of Lysobactin Alone Against Mastitis Pathogens

[0066] The in vitro antibacterial activity of lysobactin against common mastitis pathogens such as Staphylococcus aureus, Coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, Trueperella pyogenes, Escherichia coli, or Klebsiella pneumoniae was assessed by microbroth dilution MIC methodology as described by the Clinical and Laboratory Standards Institute (CLSI) in order to obtain the Minimal Inhibitory Concentration (MIC), which is defined as the lowest concentration of a substance that prevents visible growth of a bacterium. The results expressed as MIC.sub.90 are summarized in the following table, where the MIC.sub.90 is defined as the concentration at which the growth of at least 90% of the strains of a given species is inhibited.

TABLE-US-00002 MIC.sub.90 [μg/ml] Tested bacteria of lysobactin Staphylococcus aureus 0.5 Coagulase-negative staphylococci 0.5 Streptococcus uberis 0.125 Streptococcus dysgalactiae 0.25 Streptococcus agalactiae 0.25 Trueperella pyogenes 0.5 Escherichia coli >16 Klebsiella pneumoniae >16

EXAMPLE 2: In Vitro Kill Kinetics of Lysobactin Alone for Mastitis Pathogens

[0067] In order to assess the ability of lysobactin to kill bacteria in milk, flasks containing different concentrations of lysobactin in store-bought full-fat milk were inoculated with 1-2×10.sup.6 colony forming units/ml of a representative strain of either Staphyloccus aureus or Streptococcus uberis. The flasks were incubated for 24-48 hours in a shaking water bath at 35+/−2° C., and viable bacterial counts in each flask were determined at several time-points by diluting and plating samples on agar plates. A reduction of the number of viable bacteria in the initial inoculum by at least 99.9% is defined as bactericidal activity.

[0068] The kill kinetics of lysobactin against Staphylococcus aureus ATCC 29740 and Streptococcus uberis ATCC 27958 in milk were determined for concentrations of lysobactin of 4, 8, 16, 32 and 64 μg/mL. Results are depicted in FIG. 1 (CFU: colony forming units). A cidality at 24 h and 48 h, respectively, can be postulated for S. aureus and S. uberis.

EXAMPLE 3: In Vitro Assessment of Spontaneous Resistance Development Against Lysobactin Alone

[0069] The frequency of spontaneous resistance development was assessed by plating at least 1×10.sup.9 colony forming units of the respective bacterial strain on agar plates containing lysobactin at either 4× or 8× the MIC and incubating the plates at 35+/−2° C. After 48 h, the number of colonies that had grown on the plates at lysobactin concentration above the MIC was divided by the number of bacteria that was initially plated. The resulting number is defined as the spontaneous resistance frequency, and is an indication for the likelihood of resistant isolates to appear during an infection.

[0070] As a conclusion, lysobactin at 4- and 8-fold MIC displays a very good resistance profile: no resistant isolates were detectable. The results are summarized in the following tables:

[0071] Staphylococcus aureus ATCC 29740:

TABLE-US-00003 24 h 48 h Lysobactin plated obtained frequency of obtained frequency of concentration CFU colonies resistance colonies resistance 2 μg/ml (4 × MIC) 3.52 × 10.sup.9 0 <2.84 × 10.sup.−10 0 <2.84 × 10.sup.−10 4 μg/ml (8 × MIC) 3.52 × 10.sup.9 0 <2.84 × 10.sup.−10 0 <2.84 × 10.sup.−10

[0072] Streptococcus uberis ATCC 27958:

TABLE-US-00004 24 h 48 h Lysobactin plated obtained frequency of obtained frequency of concentration CFU colonies resistance colonies resistance 2 μg/ml (4 × MIC) 2.4 × 10.sup.10 0 <4.17 × 10.sup.−11 0 <4.17 × 10.sup.−11 4 μg/ml (8 × MIC) 2.4 × 10.sup.10 0 <4.17 × 10.sup.−11 0 <4.17 × 10.sup.−11

EXAMPLE 4: In Vitro Assessment of Resistance Development Against Lysobactin Alone During Serial Passaging

[0073] The appearance of MIC changes during constant exposure of bacteria to sub-MIC concentrations of lysobactin was assessed by serial passaging experiments. On the first day the MICs of S. aureus and S. uberis were assessed by microbroth dilution MIC methodology as described by the Clinical and Laboratory Standards Institute (CLSI). Each day for the following 33 consecutive days, bacteria from the well containing the highest concentrations allowing full growth were collected and used to inoculate new 96-well plates and to assess the MIC during constant exposure to lysobactin. The MIC obtained on each day was plotted over time. The results are shown in FIG. 2. Rifampicin, which is known to result in quick changes of the MIC, was used as positive control.

[0074] The results indicate a very good profile of lysobactin for resistance development during constant exposure, as the MICs for S. aureus and S. uberis remain constant over the 33 days period.

EXAMPLE 5: Efficacy of Lysobactin Alone in an Acute Mouse Mastitis Model with S. aureus

[0075] The efficacy of lysobactin (formulated in a hydrogel at pH 4.7) was tested in a Staphylococcus aureus acute mouse mastitis model established at the University of Sherbrooke, Canada (Brouillette et al, Vet. Microbiol. (2004) 4:253-262), that is hereby incorporated by reference. Both the abdominal mammary glands (L4 and R4) of lactating CD-1 mice were intramammarily infected with 100 CFU (colony forming units) of S. aureus. The mice were treated intramammarily with lysobactin four hours after infection. Each treatment group contained at least 3 mice (6 quarters), 14 hours later (18 hours after inoculation) mice were sacrificed, mammary glands were harvested and the CFU content evaluated by plating 10-fold serial dilutions of mammary gland homogenates. The CFU content was expressed as log.sub.10 count. The detection limit was 200 CFU/g of gland. Glands with less than 200 CFU/g were regarded as cleared. The results are shown in FIG. 3.

[0076] Intramammary instillation of 50 μg lysobactin reduces the median CFU content by ca. 4 log.sub.10, 400 μg lysobactin eliminates the infection from all infected glands.

EXAMPLE 6: Efficacy of Lysobactin Alone in an Acute Mouse Mastitis Model with S. uberis

[0077] The efficacy of lysobactin (formulated in a hydrogel at pH 4.7) was tested in a Streptococcus uberis challenge mouse mastitis model. The results are shown in FIG. 4.

[0078] Twenty lactating mice were experimentally infected on the fourth pair of mammary glands with S. uberis around 12-15 days after birth of the offspring. Four hours after inoculation, groups of four mice were treated on the same glands with lysobactin at 100, 200, 400, or 800 μg/gland formulated as hydrogel. The fifth group was treated solely with the hydrogel vehicle as negative control. Eighteen hours after infection the animals were euthanized, the glands were removed, homogenized, and bacterial colony forming units (CFUs) were determined by established microbiological methods. Subsequently CFU/mL homogenate as well as CFU/g of gland were calculated. The detection limit was approximately 100 CFU/g of gland. Antimicrobial activity of lysobactin against Strep uberis was determined by comparison of the mean CFUs/gland of the different dosing groups and the negative control group.

[0079] Glands of all animals of the control group showed an optimum infection rate (≥10.sup.7 CFU/g of gland) 18 hours after infection. With one exception in the highest dosing group all glands in the lysobactin treated groups had bacterial counts below the limit of detection (10.sup.2 CFU/g). It can be concluded that lysobactin formulated as hydrogel has outstanding antibacterial efficacy against S. uberis at intramammary doses between 100 and 800 μg/gland.

EXAMPLE 7: Lysobactin Alone In Vivo Cattle Data—Milk Pharmacokinetic Study in Lactating Holstein Cows

[0080] The study was designed as a non-pivotal study suitable to investigate the milk pharmacokinetics of the active substance lysobactin after single intracisternal application to lactating dairy cows.

[0081] The active substance was provided in a paraffin based service formulation suitable for intracisternal application containing 150 mg lysobactin in 10 g oily suspension.

[0082] The test item was administered as single intracisternal treatment at a dose rate of 150 mg lysobactin to a single hind quarter of four lactating dairy cows each.

[0083] The dairy cows on study (Holstein cows) represented the target population in age, lactation number, lactation stage, milk yield and breed. The animals were stalled in a tie-barn and were fed with standard feed for dairy cows consisting of corn and grass silage and milk performance feed. Milking was twice daily at a 12 hour interval using a bucket-type milking device.

[0084] Frequent milk sampling was performed from the treated and respective control quarters prior to (0 h) and over a period of 168 h after treatment (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 84, 96, 120, 144, and 168 h) by manual stripping of the respective udder quarters. Milk samples at routine milking times were gathered prior to milking

[0085] Concentrations of the active substance lysobactin in milk were analyzed by HPLC with detection by tandem mass spectrometry. The limit of quantitation was 0.05 mg/L.

[0086] Pharmacokinetic evaluation of milk concentration data was based on non-compartmental methods and comprised PK-parameters to adequately describe the absorption, distribution and elimination profile of the active substance in milk.

[0087] Summarized results derived from the treated udder quarters are presented in the following table. No lysobactin was detected in the control samples (untreated udder quarters).

Mean Milk Pharmacokinetic Results of Lysobactin after Single Treatment

[0088]

TABLE-US-00005 C.sub.max.sup.1 T.sub.max.sup.2 t.sub.1/2.sup.1 AUC.sub.inf.sup.1 AUC.sub.0-12 h.sup.1 AUC.sub.0-24 h.sup.1 Matrix mg/L h h mg*h/L mg*h/L mg*h/L Milk 342 4 11.6 2321 1870 2213 Dose applied to 1 quarter per cow was 150 mg lysobactin; Means are given as 1) geometric mean, 2) median

[0089] The concentration time curve of lysobactin is depicted in FIG. 5.

EXAMPLE 8: Lysobactin Alone In Vivo Cattle Data—Dairy Cow Udder Infection Model with S. aureus

[0090] Fifteen healthy lactating dairy cows were experimentally infected with the mastitis pathogen Staphylococcus aureus on all four udder quarters. As soon as an udder quarter showed clinical symptoms of mastitis, such as swelling, pain, abnormal milk consistency, it was treated with either Lysobactin paraffine based ointment at two different concentrations, or Ubrolexin® (Cephalexin+Kanamycin, Boehringer Ingelheim), or saline solution as negative control. Treatments were randomly assigned to 42 udder quarters in total, either 50 mg lysobactin per quarter (in 11 quarters), or 150 mg Lysobactin (in 11 quarters), or Ubrolexin® (in 9 quarters), or saline solution (in 11 quarters). Only udder quarters that were bacteriologically positive for the challenge organism immediately prior to treatment (n=42) were eligible for assessment of microbiological and clinical cure. The diseased quarters were treated with these intramammary formulations two times with an interval of 24 hours in between. Udders were clinically examined and milk samples were taken before treatment and several times after that until three weeks after the second administration. Milk was inspected visually for deviation in its consistency and samples were evaluated for presence of the challenge organism and for somatic cell count to confirm the diagnosis mastitis. Diseased udder quarters were considered microbiologically cured when Staphylococcus aureus found in milk samples shortly before treatment could not be isolated from any milk sample taken within the time period between the third and the twenty-first day after the second treatment. Clinical cure was achieved when the local symptoms of mastitis had completely disappeared, and recovery of somatic cell counts (SCC) as parameter of udder inflammation was attained when all counts remained below 500.000 cells per mL of milk in the period mentioned above.

[0091] The microbiological cure rate on Day 3 and Day 21 after second treatment was 73% and 27% for Lysobactin 50 mg, 73% and 45% for Lysobactin 150 mg, 78% and 44% for Ubrolexin®, and 0% and 0% for saline solution, respectively. The local clinical cure rate on Day 3 and Day 21 after second treatment was 45% and 73% for Lysobactin 50 mg, 37% and 82% for Lysobactin 150 mg, 33% and 67% for Ubrolexin®, and 55% and 73% for saline solution, respectively. The SCC recovery rate on Day 3 and Day 21 after second treatment was 36% and 45% for Lysobactin 50 mg, 36% and 91% for Lysobactin 150 mg, 56% and 67% for Ubrolexin®, and 36% and 27% for saline solution, respectively.

[0092] It can be concluded that Lysobactin 150 mg shows similar or even better efficacy in comparison to the positive control product Ubrolexin® and superiority to Lysobactin 50 mg and Saline solution in the parameters microbiological and clinical cure, and SCC recovery.

EXAMPLE 9: Lysobactin Alone In Vivo Cattle Data—Dairy Cow Udder Infection Model with S. uberis

[0093] Seventy-two healthy lactating dairy cows were experimentally infected with the mastitis pathogen Streptococcus uberis on two udder quarters per cow. As soon as an udder quarter showed clinical symptoms of mastitis (e.g. heat, swelling, redness, pain, abnormal milk consistency), it was treated with either lysobactin paraffine based ointment at two different concentrations, or Ubrolexin® (cephalexin+kanamycin, Boehringer Ingelheim).

[0094] Treatments were randomly assigned to 41 (clinical) or 37 (microbiological) udder quarters in total, either 50 mg lysobactin per quarter (in 15/13 quarters), or 150 mg lysobactin (in 15/14 quarters), or Ubrolexin® (in 11/10 quarters) as positive control. The diseased quarters were treated with these intramammary formulations two times with an interval of 24 hours in between. Udders were clinically examined and milk samples were taken before treatment and several times after that until three weeks after the second administration. Milk was inspected visually for deviation in its consistency and samples were evaluated for presence of the challenge organism to confirm the diagnosis mastitis. Diseased udder quarters were considered microbiologically cured when Streptococcus uberis found in milk samples shortly before treatment could not be isolated from any milk sample taken within the time period between the seventh and the twenty-first day after the second treatment. Clinical cure was achieved when the local symptoms of mastitis completely disappeared or were at the most of very slight nature.

[0095] The microbiological cure rate on Day 7 and Day 21 after second treatment was 84.6% and 92.31% for lysobactin 50 mg, 71.4% and 85.71% for lysobactin 150 mg, and 30% and 30% for Ubrolexin®, respectively. The local clinical cure rate on Day 7 after second treatment was 86.7% both for lysobactin 50 mg and lysobactin 150 mg, and 36.4% for Ubrolexin®, respectively.

[0096] It can be concluded that lysobactin 50 and 150 mg were clearly more efficacious in comparison to the positive control product Ubrolexin® in the pivotal parameters microbiological and clinical cure.

EXAMPLE 10: In Vitro Kill Kinetics of Combinations of Lysobactin with Neomycin for Mastitis Pathogens

[0097] In order to assess the synergistic activity of lysobactin and neomycin in store-bought full-fat milk in vitro, flasks containing different concentrations of lysobactin and neomycin, either alone or in combination, were inoculated with 1-2×10.sup.6 colony forming units/ml of a representative strain of either Staphyloccus aureus, Streptococcus uberis or E. coli.

[0098] The flasks were incubated for 24-48 hours in a shaking water bath at 35+/−2° C., and viable bacterial counts in each flask were determined at several time-points by diluting and plating samples on agar plates.

[0099] A reduction of the number of viable bacteria achieved by the combination compared to the most active single compound by at least 99% after 24 h is defined as synergistic activity (NCCLS/CLSI M26-A Vol. 19, No. 18).

[0100] The kill kinetics of lysobactin and neomycin, either alone or in combination, against Staphylococcus aureus ATCC 29740 in milk were determined for concentration ratios of lysobactin:neomycin of 1:1 (2 μg/ml each) and 2:1 (2 μg/ml of lysobactin and 1 μg/ml neomycin).

[0101] Results are depicted in FIG. 6.

[0102] After 24 h a synergistic activity of the lysobactin:neomycin combinations could be observed. The synergy is extremely visible at ratios of at least 2:1, particularly at a ratio of 1:1.

[0103] The kill kinetics of lysobactin and neomycin, either alone or in combination, against Streptococcus uberis ATCC 27958 in milk were determined for concentration ratios of lysobactin:neomycin of 1:1 (16 μg/ml each) and 2:1 (16 μg/ml of lysobactin and 8 μg/ml neomycin).

[0104] Results are also depicted in FIG. 6.

[0105] After 24 h a synergistic activity of the lysobactin:neomycin combinations could be observed at both ratios.

[0106] The kill kinetics of lysobactin and neomycin, either alone or in combination, against Staphylococcus aureus ATCC 29740 in milk were determined for concentration ratios of lysobactin:neomycin of 1:5 (1 μg/ml lysobactin plus 5 μg/ml neomycin and 1.5 μg/ml lysobactin plus 7.5 μg/ml neomycin, respectively).

[0107] Results are depicted in FIG. 7.

[0108] After 24 h a synergistic activity could be observed. The synergy is extremely visible already at lysobactin:neomycin ratios of at least 1:5 (i.e. 0.2). Said synergy is not dependent on the absolute amount of lysobactin and neomycin used, as evidenced by FIG. 7.

[0109] The kill kinetics of lysobactin and neomycin, either alone or in combination, against Streptococcus uberis ATCC 27958 in milk were determined for concentration ratios of lysobactin:neomycin of 1:10 (1 μg/ml lysobactin plus 10 μg/ml neomycin and 2 μg/ml lysobactin plus 20 μg/ml neomycin, respectively).

[0110] Results are also depicted in FIG. 7.

[0111] After 24 h a synergistic activity could be observed. At a lysobactin:neomycin ratio of 1:10 (0.1) the synergy is highly visible regardless of absolute amount of lysobactin and neomycin.

[0112] Taken together, a synergistic activity of lysobactin:neomycin combination could be shown. Said synergistic activity being very visible at ratios ranging from 2:1 to 1:5 (0.2) and 2:1 to 1:10 (0.1) for Staphylococcus aureus ATCC 29740 and Streptococcus uberis ATCC 27958, respectively.

[0113] The kill kinetics of lysobactin and neomycin, either alone or in combination, against E. coli P4:032 in milk were determined for concentration ratio of lysobactin:neomycin of 4:1 (256 μg/ml lysobactin and 32 μg/ml neomycin).

[0114] Results are depicted in FIG. 8.

[0115] Although neomycin as well as the lysobactin:neomycin combination were able to reduce the bacterial cell count below the detection limit after 8 h, the combination, showed a faster onset of killing

[0116] Furthermore the combination substantially prevented regrowth of the bacteria after 24 h which cannot be achieved by the respective sole actives.

[0117] Therefore, a synergistic activity was observed here as well with regard to the Gram-negative E. coli bacteria.

EXAMPLE 11: In Vitro Kill Kinetics of Combinations of Lysobactin Either with Neomycin or Kanamycin for Staphylococcus aureus ATCC 29740

[0118] To investigate whether the above referred to synergy does apply to more aminoglycoside antibiotics beyond neomycin combined with lysobactin, the kill kinetics of kanamycin as combination partner were tested as well.

[0119] Kill kinetics were performed as described in example 10.

[0120] The kill kinetics of lysobactin either with neomycin or kanamycin against Staphylococcus aureus ATCC 29740 in milk were determined for a concentration ratio of lysobactin:neomycin or lysobactin:kanamycin of 1:4 (0.5 μg/ml lysobactin plus 2 μg/ml neomycin or kanamycin; 1 μg/ml lysobactin plus 4 μg/ml neomycin or kanamycin and 2 μg/ml lysobactin plus 8 μg/ml neomycin or kanamycin, respectively).

[0121] Results are depicted in FIG. 9.

[0122] For all concentrations evaluated the lysobactin:kanamycin combination showed similar activity compared to the lysobactin: neomycin combination.

[0123] From that it can be concluded that the combination of lysobactin with aminoglycoside antibiotics per se conveys an unexpected synergy.

COMPARATIVE EXAMPLE 12: In Vitro Kill Kinetics of Combinations of Lysobactin with Florfenicol for Staphylococcus aureus ATCC 29740 and Streptococcus uberis ATCC 27958

[0124] Synergistic activities of two different antibiotic agents are not reliably predictable, as shown hereafter.

[0125] For example, the combination of lysobactin with florfenicol did not reveal synergistic activity for neither Staphylococcus aureus ATCC 29740 nor Streptococcus uberis ATCC 27958 in milk under test conditions depicted in FIG. 10.

EXAMPLE 13: Efficacy of a Combination of Lysobactin with Neomycin Trisulfate in an Acute Mouse Mastitis Model with S. aureus

[0126] The efficacy of lysobactin and neomycin trisulfate, either alone or in combination, was tested in a Staphylococcus aureus acute mouse mastitis model established at the University of Sherbrooke, Canada (Brouillette et al, Vet. Microbiol. (2004) 4:253-262), as described in example 5. Samples were formulated in paraffin.

[0127] The results are shown in FIG. 11.

[0128] Intramammary instillation of 50 μg lysobactin, 1600 μg neomycin trisulfate and the combined 50 ng lysobactin plus 1600 μg neomycin trisulfate reduces the median CFU content by ca. 2.7, 2.5 and 5.9 log.sub.10, respectively.

EXAMPLE 14: Efficacy of a Combination of Lysobactin with Neomycin Trisulfate in an Acute Mouse Mastitis Model with S. uberis

[0129] Twenty-three lactating CD-1 mice were experimentally infected 10-15 days after birth of the offspring with the mastitis pathogen Streptococcus uberis (ATCC 27958) on the left and right fourth mammary gland.

[0130] Four hours after infection the animals were treated intrammarily with the test items. The study comprised four treatment groups with four mice each and a control group with seven mice.

[0131] The mice of the treatment groups received paraffin formulations of lysobactin at 15 μg/gland or neomycin trisulfate at 1600 μg/gland, or two combinations of both with lysobactin at 15 μg plus neomycin trisulfate at 800 μg or 1600 μg per gland.

[0132] Eighteen hours after infection the mice were killed and the mammary glands were removed and bacterial CFU were evaluated microbiologically.

[0133] The results are shown in FIG. 12.

[0134] The control group receiving paraffin as placebo was in the expected range with CFU of around 7 log.sub.10/g of tissue. Intramammary instillation of 15 μg lysobactin, 1600 μg neomycin trisulfate and the combined 15 μg lysobactin plus 1600 μg neomycin trisulfate reduces the mean CFU content by ca. 2.6, 1.5 and 3.8 log.sub.10, respectively.

[0135] It can be concluded that the lysobactin/neomycin trisulfate combination dosed at 15/1600 μg/gland was superior in its efficacy against S. uberis ATCC27958 compared to their single components at the same dosage.

EXAMPLE 15: Lysobactin and Neomycin Trisulfate In Vivo Cattle Data—Dairy Cow Udder Infection Model with S. aureus

[0136] Fifteen healthy lactating dairy cows were experimentally infected with the mastitis pathogen Staphylococcus aureus on all four udder quarters. As soon as an udder quarter showed clinical symptoms of mastitis, such as swelling, pain, abnormal milk consistency, it was treated with either lysobactin miglyol based suspension at a dose of 50 mg, or with neomycin trisulfate miglyol based suspension at a dose of 400 mg, or with a combination of both, i.e. 50 mg lysobactin and 400 mg neomycin trisulfate.

[0137] Additionally a fourth group of udder quarters received miglyol as negative control. Treatments were randomly assigned to 60 udder quarters in total. Only udder quarters that were bacteriologically positive for the challenge organism immediately prior to treatment (n=49) were eligible for assessment of microbiological and clinical cure. The diseased quarters were treated with these intramammary formulations two times with an interval of 24 hours in between. Udders were clinically examined and milk samples were taken before treatment and several times after that until three weeks after the second administration by study personnel blinded to treatment.

[0138] Milk was inspected visually for deviation in its consistency and samples were evaluated for presence of the challenge organism and for somatic cell count to confirm the diagnosis mastitis. Diseased udder quarters were considered microbiologically cured when Staphylococcus aureus found in milk samples shortly before treatment could not be isolated from any milk sample taken within the time period between the third and the twenty-first day after the second treatment. Clinical cure was achieved when the local symptoms of mastitis had completely disappeared, and recovery of somatic cell counts (SCC) as parameter of udder inflammation was attained when all counts remained below 200.000 cells per mL of milk in the period mentioned above.

[0139] The microbiological cure rate on Day 3 and Day 21 after second treatment was 71% and 14% for lysobactin 50 mg, 100% and 42% for neomycin trisulfate 400 mg, 69% and 54% for the combination of lysobactin 50 mg plus neomycin trisulfate 400 mg, and 10% and 30% for the negative control miglyol, respectively.

[0140] The local clinical cure rate on Day 3 and Day 21 after second treatment was 71% and 71% for lysobactin 50 mg, 67% and 100% for neomycin trisulfate 400 mg, 77% and 85% for the combination of lysobactin 50 mg plus neomycin trisulfate 400 mg, and 80% and 70% for the negative control miglyol, respectively.

[0141] The SCC recovery rate on Day 3 and Day 21 after second treatment was 35% and 36% for lysobactin 50 mg, 17% and 50% for neomycin trisulfate 400 mg, 46% and 69% for the combination of lysobactin 50 mg plus neomycin trisulfate 400 mg, and 30% and 30% for the negative control miglyol, respectively.