Alginate oligomers for use in overcoming multidrug resistance in bacteria

Abstract

The invention provides a method of overcoming resistance to at least one antibiotic in a multidrug resistant bacterium, said method comprising contacting said bacterium with an alginate oligomer together with the antibiotic. The multidrug resistant bacterium may be on an animate or inanimate surface and both medical and non-medical uses and methods are provided. In one aspect the invention provides an alginate oligomer for use together with at least one antibiotic in treating a subject infected, suspected to be infected, or at risk of infection, with a multidrug resistant bacterium to overcome resistance to the antibiotic in said multidrug resistant bacterium. In another aspect the method can be used to combat contamination of a site with multidrug resistant bacteria, e.g. for disinfection and cleaning purposes.

Claims

1. An antibacterial method, said method comprising contacting a bacterium with a macrolide antibiotic and an alginate oligomer having an average molecular weight of less than 30,000 Daltons, wherein the bacterium is not in a biofilm and wherein an antibacterial effect of the macrolide antibiotic to inhibit growth and/or viability of said bacterium that is not in a biofilm is increased relative to an antibacterial effect of the macrolide antibiotic to inhibit growth and/or viability of said bacterium that is not in a biofilm in the absence of said alginate oligomer.

2. The method of claim 1, said method comprising administering said macrolide antibiotic and said alginate oligomer to a subject infected with, suspected to be infected with, or at risk of infection with a bacterium that is not in a biofilm.

3. The method of claim 2 wherein said alginate oligomer and said macrolide antibiotic are administered to said subject together or separately, wherein said separate administration is simultaneous or sequential.

4. The method of claim 1, wherein said macrolide antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, troleandromycin and tylosin.

5. The method of claim 1 wherein said macrolide antibiotic is selected from the group consisting of azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

6. The method of claim 1, wherein the bacterium is a clinical strain or a clinical isolate.

7. The method of claim 1, wherein the bacterium is a Gram negative bacterium.

8. The method of claim 7, wherein the bacterium is from the family Enterobacteriacee or is a non-fermenting Gram negative bacterium.

9. The method of claim 8, wherein the bacterium is selected from the genera consisting of Pseudomonas, Acinetobacter, Stenotrophomonas, Burkholderia, Escherichia, Providencia and Klebsiella.

10. The method of claim 9, wherein the bacterium is one of Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia spp, E. coli, Providencia stuartii and Klebsiella pneumoniae.

11. The method of claim 1, wherein the bacterium is resistant to three or more classes of antibiotics selected from the group consisting of the macrolides, the β-lactams, the tetracyclines, the polypeptide antibiotics and the quinolones.

12. The method of claim 1 wherein the bacterium is resistant to one or more antibiotics selected from the group consisting of ceftazidime, imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, oxytetracycline and ciprofloxacin.

13. The method of claim 1, wherein the bacterium is resistant to one or more antibiotics that is a conventional treatment for that bacterium.

14. The method of claim 1, wherein the bacterium is an MDR strain of Pseudomonas aeruginosa, Klebsiella pneumoniae, Burkholderia cepacia, Providencia stuartii or Acinetobacter baumannii that is resistant to one or more antibiotics selected from the group consisting of ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, oxytetracycline and imipenem/cilastatin.

15. The method of claim 1, wherein the alginate oligomer has a number average degree of polymerisation of 2 to 100.

16. The method of claim 14, wherein the alginate oligomer has a number average degree of polymerisation of 2 to 35.

17. The method of claim 1, wherein the alginate oligomer has up to 100 monomer residues.

18. The method of claim 17, wherein the alginate oligomer is a 2- to 35-mer.

19. The method of claim 1, wherein the alginate oligomer has at least 70% G residues.

20. The method of claim 19, wherein the alginate oligomer has at least 80% G residues.

21. The method of claim 20, wherein the alginate oligomer has at least 85% G residues.

22. The method of claim 21, wherein the alginate oligomer has at least 90% G residues.

23. The method of claim 19, wherein at least 80% of the G residues are arranged in G-blocks.

24. The method of claim 1, wherein the alginate oligomer has at least 70% M residues.

25. The method of claim 24, wherein the alginate oligomer has at least 80% M residues.

26. The method of claim 24, wherein at least 80% of the M residues are arranged in M blocks.

27. The method of claim 2, wherein the infection with a bacteria that is not in a biofilm is of an internal or external body surface selected from the group consisting of a surface in the oral cavity, the reproductive tract, the urinary tract, the respiratory tract, the gastrointestinal tract, the peritoneum, the middle ear, the prostate, vascular intima, the eye, including the conjunctiva or corneal tissue, lung tissue, heart valves, skin, scalp, nails, the interior of wounds or the surface of adrenal, hepatic, renal, pancreatic, pituitary, thyroid, immune, ovarian, testicular, prostate, endometrial, ocular, mammary, adipose, epithelial, endothelial, neural, muscle, pulmonary, epidermis or osseous tissue; or in a body fluid selected from blood, plasma, serum, cerebrospinal fluid, GI tract contents, sputum, pulmonary secretions and semen; or in or on body tissue selected from adrenal, hepatic, renal, pancreatic, pituitary, thyroid, immune, ovarian, testicular, prostate, endometrial, ocular, mammary, adipose, epithelial, endothelial, neural, muscle, pulmonary, epidermis and osseous tissue.

28. The method of claim 2, wherein, the subject is selected from the group consisting of a subject with a pre-established infection, an immunocompromised subject, a subject undergoing intensive or critical care, a subject suffering from trauma, a subject with a burn, a subject with an acute and/or chronic wound, a neonatal subject, an elderly subject, a subject with cancer, a subject suffering from an auto-immune condition, a subject with reduced or abrogated epithelial or endothelial secretion and/or secretion clearance and a subject fitted with a medical device.

29. The method of claim 2, wherein the subject is selected from the group consisting of a subject with a condition selected from HIV, sepsis, septic shock, AIDS, a cancer of the immune system, rheumatoid arthritis, diabetes mellitus type I, Crohn's disease, COPD, COAD, COAP, bronchitis, cystic fibrosis, emphysema, lung cancer, asthma, pneumonia and sinusitis, a subject preparing for, undergoing, or recovering from chemotherapy and/or radiotherapy, an organ transplant subject, a subject resident in a healthcare institution and a smoker.

30. The method of claim 2, wherein the subject is a subject with a respiratory condition or disease.

31. The method of claim 1, wherein the bacterium that is not in a biofilm is on a surface selected from the group consisting of surfaces of food or drink processing, preparation, storage or dispensing machinery or equipment, surfaces of air conditioning apparatus, surfaces of industrial machinery, surfaces of storage tanks, surfaces of medical or surgical equipment, surfaces of aquatic/marine equipment and the surfaces of buildings and other structures.

32. The method of claim 31, wherein the surface is selected from the group consisting of food processing, storage, dispensing or preparation equipment or surfaces, tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, air conditioning conduits, cooling apparatus, food or drink dispensing lines, heat exchangers, boat hulls, dental waterlines, oil drilling conduits, contact lenses, contact lens storage cases, catheters, prosthetic devices and implantable medical devices.

33. The method of claim 1, wherein the bacterium that is not in a biofilm is in a material selected from the group consisting of clinical/scientific waste, animal or human food stuffs, personal hygiene products, cosmetics, drinking water supplies, waste water supplies, agricultural feedstuffs and water supplies, insecticide formulations, pesticide formulations, herbicide formulations, industrial lubricants, cell and tissue culture media, and cell and tissue cultures.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(1) Results presented in the Examples below show particularly that alginate oligomers may be used together with various antibiotics against MDR strains of Pseudomonas and particularly MDR strains of P. aeruginosa. The results also show that alginate oligomers may effectively be used with various antibiotics against Acinetobacter species, and particularly A. baumannii and A. lwoffii; against Burkholderia species, and particularly B. cepacia; against Providencia species, and particularly P. stuartii; against Klebsiella species, and particularly Klebsiella pneumonia; against Streptococcus, and particularly Streptococcus oralis; against Staphylococcus, and in particular MRSA; against Escherichia, and particularly Escherichia coli, and that resistance to antibiotics in these genera/species may be overcome.

(2) In this regard, the data more generally show that alginate oligomers may be particularly effective in potentiating (or improving/increasing the efficacy of) the effects of antibiotics against Acinetobacter species, and particularly A. baumannii and Burkholderia species, and particularly B. cepacia. This leads to the proposal that in one aspect the invention can be seen more generally to relate to use of alginate oligomers in conjunction (or combination) with an antibiotic to combat (or to inhibit the growth and/or viability of) Acinetobacter and/or Burkolderia (i.e. Acinetobacter and/or Burkolderia species in general), for example to treat or combat infection and/or contamination (i.e. colonisation) with these bacteria.

(3) In certain aspects, the bacterium targeted by the invention may alternatively beviewed as a clinically relevant bacterium, e.g. a bacterium that is known to be associated with disease and/or infection in subjects; especially diseases and infections that are unresponsive to at least 3 structurally and/or functionally different antibiotics, or at least 3 antibiotic classes, more particularly at least 4, 5, 6, 7 8, 9 or 10 structurally and/or functionally different antibiotics or antibiotic classes conventionally used in the treatment of that disease and/or infection. More particularly, the bacterium targeted by the invention may be from a clinically relevant MDR strain of bacteria. The bacterium may cause or result in clinically significant or clinically important infections, in other words infections which are the cause of significant clinical problems. For instance, the bacterium could be a bacterium associated with nosocomial infections, infections in the respiratory tract of patients, e.g. patients suffering from cystic fibrosis, chronic obstructive pulmonary disease, congestive obstructive airway disease/congestive obstructive airway pneumonia (COAD/COAP), pneumonia, emphysema, bronchitis and sinusitis; infections in chronic wounds (including burns), device related infections associated with implantable or prosthetic medical devices e.g. prosthetic valve endocarditis or infection of lines or catheters or artificial joints or tissue replacements or endotracheal or tracheotomy tubes. Examples of these types of bacteria include Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia spp (e.g. B. cepacia), E. coli, Klebsiella pneumoniae, Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Clostridium difficile, Mycobacterium tuberculosis, Enterococcus and Vancomycin-Resistant Enterococcus and Providencia stuartii.

(4) The bacterium targeted by the method of the invention may be the same as a bacterium that has previously been isolated from a subject. Thus, the bacterium is preferably a clinical strain or a clinical isolate. The bacterium targeted by the method of the invention may be present in or on a subject. The bacterium may be known or found to be MDR, or the bacterium may have developed MDR during the subject's treatment. In view of the requirement for MDR (or MDR status), which may or may not be or include acquired resistance, the bacterium to be treated according to the present invention will generally not be a conventional laboratory or reference strain, e.g. a strain such as Pseudomonas aeruginosa PA01 (ATCC 15692) or Staphylococcus aureus ATCC 6538. In another embodiment the bacterium will not be MRSA (methicillin resistant Staphylococcus aureus), e.g. strain 1103.

(5) In representative embodiments the bacterium may be an MDR strain of Pseudomonas aeruginosa that is resistant to one or more antibiotics selected from the penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g. polymyxins), more particularly cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, or macrolides, e.g. amikacin, ciprofloxacin, gentamicin, tobramycin, piperacillin, ticarcillin, colistin, oxytetracycline, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin; particularly ciprofloxacin, colistin, oxytetracycline, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, and especially ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

(6) In other embodiments the bacterium may be an MDR strain of Klebsiella pneumoniae that is resistant to one or more antibiotics selected from the penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g. polymyxins) e.g. cefotaxime, ceftriaxone, amikacin, gentamicin, ciprofloxacin, tobramycin, ampicillin, piperacillin, ticarcillin, colistin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, imipenem/cilastatin; cefepime, levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ertapenem, particularly ciprofloxacin, colistin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, and especially ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

(7) In other embodiments the bacterium may be an MDR strain of Acinetobacter baumannii that is resistant to one or more antibiotics selected from the penicillins, cephalosporins, carbapenems, monobactams, glycylcyclines, aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g. polymyxins). e.g. imipenem/cilastatin, ampicillin, cefepime, colistin, rifampin, tigecycline, amikacin, ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin; particularly colistin, ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, and especially ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

(8) In other embodiments the bacterium may be an MDR strain of Providencia stuartii that is resistant to one or more antibiotics selected from the penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g. polymyxins) e.g. cefotaxime, ceftriaxone, amikacin, gentamicin, ciprofloxacin, tobramycin, ampicillin, piperacillin, ticarcillin, colistin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, imipenem/cilastatin; cefepime, levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ertapenem, particularly ciprofloxacin, colistin, ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, and especially ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

(9) In other embodiments the bacterium may be an MDR strain of Burkholderia cepacia that is resistant to one or more antibiotics selected from the penicillins, cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, macrolides or polypeptides (e.g. polymyxins) e.g. cefotaxime, ceftriaxone, amikacin, gentamicin, ciprofloxacin, tobramycin, ampicillin, piperacillin, ticarcillin, colistin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, imipenem/cilastatin; cefepime, levofloxacin, norfloxacin, gatifloxacin, moxifloxacin, and ertapenem, particularly ciprofloxacin, colistin, ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, and especially ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin and imipenem/cilastatin, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin and spiramycin.

(10) The data of the Examples surprisingly shows that the alginate oligomers of the invention are particularly effective in enhancing the effects (increasing the effectiveness (or efficacy)) of antibiotics against bacteria of the genus Burkholderia. As discussed above, Burkholderia represent an important genus of bacteria since they can cause disease in humans and animals and they display intrinsic resistance to multiple classes of antibiotics (e.g. the aminoglycosides, the β lactams and/or the macrolides). Burkholderia organisms, especially Burkholderia cepacia, Burkholderia pseudomallei and Burkholderia mallei are therefore considered to be MDR bacteria naturally on account of the intrinsic resistance exhibited as their natural phenotype. Of course, strains of Burkholderia species can acquire additional resistance phenotypes. Accordingly, treatments for Burkholderia species that enhance the effects of antibiotics against such species are in high demand.

(11) Thus, it is a preferred embodiment of the invention that the target bacterium is a Burkholderia organism, e.g. selected from Burkholderia ambifaria, Burkholderia andropogonis, Burkholderia anthina, Burkholderia brasilensis, Burkholderia caledonica, Burkholderia caribensis, Burkholderia caryophylli, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia dolosa, Burkholderia fungorum, Burkholderia gladioli, Burkholderia glathei, Burkholderia glumae, Burkholderia graminis, Burkholderia hospita, Burkholderia kururiensis, Burkholderia mallei, Burkholderia multivorans, Burkholderia phenazinium, Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderia phytofirmans, Burkholderia plantarii, Burkholderia pseudomallei, Burkholderia pyrrocinia, Burkholderia sacchari, Burkholderia singaporensis, Burkholderia sordidicola, Burkholderia stabilis, Burkholderia terricola, Burkholderia thailandensis, Burkholderia tropica, Burkholderia tuberum, Burkholderia ubonensis, Burkholderia unamae, Burkholderia vietnamiensis, and Burkholderia xenovorans, in particular Burkholderia cepacia, Burkholderia mallei and Burkholderia pseudomallei and especially Burkholderia cepacia.

(12) More generally, the use of alginate oligomers to combat Burkolderia (e.g to treat or combat Burkholderia infection and/or contamination (i.e. colonisation)), or to increase (or improve) the efficacy of an antibiotic against Burkholderia represents a particular preferred and separate aspect of this invention.

(13) Accordingly, in another aspect the invention provides a method to improve the efficacy of an antibiotic, and in particular the effectiveness (or efficacy) of an antibiotic to inhibit the growth and/or viability of a Burkholderia organism (which includes inhibition of the growth of a Burkholderia population, as well as growth of a Burkholderia organism), said method comprising using said antibiotic together with (in conjunction or combination with) an alginate oligomer (which may be any alginate oligomer as defined herein and especially those indicated already as being preferred; e.g. the “high G”, “high M”, “G-block” and “M-block” oligomers). The oligomers of use in this aspect may especially be those with the sizes, size ranges and molecular weight distributions stated as preferred above. The discussion of preferred alginate oligomers of the invention applies mutatis mutandis to this aspect of the invention.

(14) More particularly, the using step may comprise contacting the Burkholderia organism with an alginate oligomer at the same or substantially the same time or prior to contacting the Burkholderia organism with the antibiotic. In particular, and in accordance with the disclosures made above (and specifically the definitions provided herein), which can be read as applying to all aspects of the present invention, the step of contacting the bacterium with the alginate oligomer may include administering the alginate oligomer and the antibiotic to a subject.

(15) The antibiotic may be selected from any antibiotic disclosed above. Preferred antibiotics may be selected from the β-lactams (e.g. the carbecephems (e.g. loracarbef); the 1st generation cephalosporins (e.g. cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g. cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime); 3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g. cefepime); the monobactams (e.g. aztreonam); the penicillins (e.g. amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin) the carbapenems (e.g. imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601)); the macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin): the quinolones (e.g. ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); and the tetracyclines (e.g. demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline).

(16) More preferably the antibiotic is selected from azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, troleandromycin, tylosin, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, aztreonam, imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin demeclocycline, doxycycline, minocycline, oxytetracycline and tetracycline, e.g. aztreonam, ciprofloxacin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin, oxytetracycline, ceftazidime and imipenem. In particularly preferred embodiments the antibiotic is selected from aztreonam, ceftazidime, azithromycin, clarithromycin and erythromycin

(17) In other embodiments the antibiotic used is not tobramycin, amikacin and/or colistin. In other embodiments the antibiotic used is not an aminoglycoside or a polypeptide antibiotic. In other embodiments the antibiotic used is not an antibiotic that has a positive charge under the conditions in which it will be used with the alginate oligomer, e.g. antibiotics with at least 3, e.g. at least 4, 5, 6 or 7 amino (—NH.sub.2) groups.

(18) The Burkholderia organism can be from any Burkholderia species, e.g. any of those disclosed herein, in particular the Burkholderia organism will be Burkholderia cepacia, Burkholderia mallei or Burkholderia pseudomallei, especially Burkholderia cepacia. In certain embodiments the Burkholderia organism is resistant to the antibiotic.

(19) In one embodiment the Burkholderia organism or population thereof will not be in a biofilm or in the process of forming a biofilm. In another embodiment the Burkholderia organism or population thereof will be in a biofilm.

(20) The method may be an in vitro or an in vivo method and may have non-medical and medical applications. In the latter instance the method can be viewed as a method for the treatment of a Burkholderia infection (e.g. a Burkholderia cepacia, Burkholderia mallei or Burkholderia pseudomallei infection) in a subject (e.g. those subjects described and preferred herein), said method comprising administering to a subject a pharmaceutically effective amount of an alginate oligomer together with a pharmaceutically effective amount of an antibiotic. This embodiment extends to any and all of the medical uses, diseases and locations of infection described herein when involving Burkholderia organisms.

(21) Thus the invention provides an alginate oligomer for use together with (or in combination or conjunction with) an antibiotic for the treatment or prevention of a Burkholderia infection in a subject. The subject may be infected, suspected to be infected, or at risk of infection with Burkholderia. The Burkholderia may be of any species mentioned herein. The term “use together” should be interpreted as discussed above.

(22) Alternatively put, the invention provides the use of an alginate oligomer for the manufacture of a medicament for use together with an antibiotic in the treatment of a Burkholderia infection in a subject. The medicament may further comprise the antibiotic.

(23) The medicament may be in the form of a single composition or formulation comprising the alginate oligomer and antibiotic(s) or separate compositions or formulations may be prepared and used, each containing the alginate oligomer or the antibiotic(s), respectively.

(24) Thus in a more particular aspect the present invention provides the use of an alginate oligomer and at least one antibiotic for the manufacture of a medicament for use in the treatment of a Burkholderia infection in a subject.

(25) Thus a further aspect of the present invention provides a product containing an alginate oligomer and an antibiotic (or antibiotics) as a combined preparation for separate, simultaneous or sequential use in the treatment of a Burkholderia infection in a subject.

(26) In a further embodiment of this aspect of the invention there is provided a method for combating contamination of a site with a Burkholderia organism, said method comprising contacting the site and/or the Burkholderia organism with (an effective amount of) an alginate oligomer together with (an effective amount of) at least one antibiotic. Such a method may particularly be an in vitro method, and the site may be any surface or location discussed herein.

(27) As noted above, in these aspects of the invention the alginate oligomer may improve the efficacy of the antibiotic, and in particular the efficacy (or effectiveness) of the antibiotic in inhibiting the growth of the Burkholderia organism.

(28) The references to “improving the effectiveness of an antibiotic to inhibit the growth and/or viability of a Burkholderia organism” should be construed in accordance with preceding discussion of what is meant by “improving the effectiveness of a macrolide antibiotic to inhibit the growth and/or viability of bacteria”.

(29) This aspect also allows the concentration of the antibiotic administered to a subject or applied to a location in order to combat a Burkholderia organism to be reduced whilst maintaining the same effectiveness. This can be beneficial if the antibiotic is expensive or associated with side effects. Minimising the use of antibiotics is also desirable to minimise development of resistance. In accordance with the invention the use of an alginate oligomer together with an antibiotic permits the antibiotic to be used at a concentration that is less than 50%, less than 25%, less than 10% or less than 5% of the amount normally administered/applied to achieve a particular level of inhibition of the growth of a Burkholderia organism in the absence of the alginate oligomer.

(30) In this aspect the alginate oligomers may be any of those discussed and in particular those stated as preferred above and the alginate oligomers will be applied to the Burkholderia organism and/or their location at a local concentration of at least 2%, at least 4%, at least 6%, at least 8% or at least 10% weight by volume.

(31) Although in certain aspects of the invention as discussed above, the bacterium may be Acinetobacter, in certain particular embodiments the MDR bacterium targeted by the methods of the invention is not Acinetobacter baumannii, or any Acinetobacter species. In another embodiment the antibiotic used against the Acinetobacter baumannii, or any Acinetobacter species, is not azithromycin, or any macrolide.

(32) By “resistant to an antibiotic” it is meant that the bacterium displays a substantially greater tolerance (reduced susceptibility) to an antibiotic as compared to a reference bacterium sensitive to the antibiotic or a typical, or a wild type, version of the bacterium. Such a substantially greater tolerance may be a statistically significant decrease in susceptibility to the antibiotic, as measured for example in standard assays, such as MIC assays. In some cases, a bacterium can be completely unaffected by exposure to an antibiotic. In this instance the bacterium can be considered fully resistant to that antibiotic.

(33) A suitable reference bacterium is Oxford Staphylococcus aureus (NCTC 6571) although many others are known in the art and are readily available. Typical, or wild type, versions of a bacterium can be obtained easily from laboratories and culture collections throughout the world.

(34) Susceptibility (and conversely resistance and tolerance) to antibiotic can be measured in any convenient way, e.g. with dilution susceptibility tests and/or disk diffusion tests. The skilled man would appreciate that the extent of the difference in tolerance/susceptibility sufficient to constitute resistance will vary depending on the antibiotic and organism under test and the test used However, a resistant bacterium will preferably be at least twice, e.g. at least 3, 4, 5, 6, 10, 20, or 50 times as tolerant to the antibiotic as the reference bacterium sensitive to the antibiotic or a typical or a wild type version of the bacterium. Preferably resistance of a particular bacteria to an antibiotic is determined using bacteria which are not in a biofilm or which do not have a biofilm phenotype.

(35) The minimum inhibitory concentration (MIC) assay (Jorgensen et al., Manual of Clinical Microbiology, 7th ed. Washington, D.C.: American Society for Microbiology, 1999; 1526-43) is a convenient dilution susceptibility test to use. This assay measures the relevant tolerance of a bacterium to antibiotics by determining the lowest concentration of antibiotic that causes complete inhibition of growth. A bacterium resistant to an antibiotic will have a substantially greater MIC value for the antibiotic than that of the reference bacterium sensitive to the antibiotic or a typical, or a wild type, version of the bacterium, e.g. the resistant bacteria will have a MIC value for the antibiotic that is at least twice or at least four times, at least eight times, at least sixteen times, at least thirty two times or at least sixty four times higher. Put in a different way, the MIC value of the resistant bacterium for the antibiotic may be at least double, at least quadruple, at least octuple, at least sexdecuple or at least duotrigenuple the MIC value of the reference bacterium sensitive to the antibiotic or a typical or a wild type version of the bacterium.

(36) Viewed alternatively, and in the context of an in vivo bacterium and/or the treatment of a bacterial infection that is resistant to multiple antibiotic classes, a bacterium may be considered resistant to an antibiotic if the bacterium has a MIC value for the antibiotic that is greater than then maximum safe circulating concentration of the antibiotic in the subject (which may be determined easily by the skilled man). More functionally, a bacterium is resistant to an antibiotic if an infection associated with that bacterium is unresponsive (i.e. there is no change in the clinical indicia of the infection) to the maximum safe dose of the antibiotic.

(37) “Overcoming resistance” should be construed accordingly as a measurable reduction in the above-described indicators of the resistance (or measurable increase in susceptibility or measurable decrease in tolerance) to the antibiotic displayed by the bacterium. Therefore “overcoming resistance” can alternatively be expressed as “reducing resistance”. It is a reference to the observed phenotype of the target bacterium and should not necessarily be considered to equate to a reversal, to any extent, at the mechanistic level of any particular resistance mechanism. As can be seen from the Examples, alginate oligomers and antibiotics have a combinatorial, e.g. synergistic, effect that makes bacteria with a phenotype that is resistant to an antibiotic more susceptible to that antibiotic. In one embodiment the alginate oligomer will measurably reduce the MIC value of the resistant bacterium to the antibiotic, e.g. the MIC value will be at least 50%, 25%, 20%, 15%, 10%, 5%, 2% or 1% of the MIC value of the bacteria for the antibiotic before treatment in accordance with the invention.

(38) Thus use of alginate oligomers according to the present invention may potentiate the effect of an antibiotic (or increase or improve its efficacy). It may render usable (or effective) an antibiotic previously thought not to be usable/effective against a particular organism, or an antibiotic which is not normally effective against a given organism (e.g. bacterium or bacterial species in question). It may also enable an antibiotic to be used at a reduced dose.

(39) The effects of alginate oligomers in overcoming resistance to antibiotics or in potentiating (etc.) the effects of antibiotics may be seen irrespective of the mechanism of resistance to the antibiotic in question. Nevertheless, particularly good results have been observed with ciprofloxacin. Resistance to this antibiotic may involve accumulation of mutations, in particular in the genes encoding DNA gyrase or topoisomerase IV. Without wishing to be bound by theory, the alginate oligomers of the invention may therefore affect this accumulation process, e.g. by preventing, slowing or halting it. However, it is not to assumed from or implied by this, that alginate oligomers may have any effect on any mechanism of resistance.

(40) In a preferred embodiment of the method of the invention the alginate oligomer overcomes resistance to at least two, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10 or all of the structurally and/or functionally different antibiotics or antibiotic classes to which the bacterium is resistant. However, as noted above, it is not required, or implied, that all of the resistance of any given MDR strain is overcome. The invention may for example be effective in overcoming resistance to certain classes of antibiotic in a given MDR strain (e.g to macrolides and/or quinolones and/or β-lactams) and this may be clinically useful, even though resistance to other antibiotics may remain. This embodiment will preferably entail the use of a plurality of antibiotics corresponding in number and identity to some or all of the antibiotic resistances overcome.

(41) In other embodiments the method of the invention overcomes resistance in an MDR bacterium (e.g. a bacterium from an MDR strain of bacteria) to at least one antibiotic that is a conventional treatment of that bacterium. Put differently, the method of the invention may overcome resistance in an MDR bacterium (e.g. bacterium from an MDR strain of bacteria) to an antibiotic to which that bacterium has acquired or developed resistance. In these embodiments the method of the invention overcomes at least one acquired resistance in an MDR bacterium (e.g. bacterium from an MDR strain of bacteria) that has acquired resistance to at least one, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 structurally and/or functionally different antibiotics or antibiotic classes. Preferably all of the acquired antibiotic resistance of the bacterium is overcome. It will be clear to the skilled reader that the invention therefore makes possible the treatment of an MDR bacterium (e.g. bacterium from an MDR strain of bacteria) with an antibiotic that had become ineffective in the treatment of that bacterium. However, as noted above, not all resistance in an MDR phenotype may be acquired and the invention is not limited to this. Thus the invention may be used in the treatment of bacteria that are innately MDR.

(42) The method of the invention may entail contacting the bacterium with more than one antibiotic. The additional antibiotic(s) can be any antibiotic, e.g. those listed above. The additional antibiotic(s) may be an antibiotic to which the bacterium is susceptible. The additional antibiotic(s) may be an antibiotic to which the bacterium is resistant. The additional antibiotic(s) may be used together with (in conjunction or combination with) the first or other antibiotics and/or the alginate oligomer. More particularly, the step of using may comprise contacting the bacterium with an alginate oligomer at the same or substantially the same time or prior to contacting the bacterium with some or all of the antibiotics in an amount effective to overcome the resistance of the bacteria to the antibiotic(s).

(43) As noted above the antibiotic(s) may conveniently be applied or administered simultaneously with the alginate oligomer, or immediately or almost immediately before or after the alginate oligomer. However the antibiotic(s) may be applied or administered at a different time point e.g. least 1 hour, at least 3 hours, at least 6 hours after the alginate oligomer. It is within the skill of the medical practitioner to develop dosage regimes which optimise the effect of the alginate oligomer and antibiotic. In these embodiments the antibiotic(s) can be applied or administered with or without a further application of an alginate oligomer. The alginate oligomer can be applied or administered in a plurality of applications prior to or with the antibiotic(s). In other embodiments the antibiotic(s) may conveniently be applied or administered before the alginate oligomer, e.g. at least 1 hour, at least 3 hours, at least 6 hours before the alginate oligomer. In these embodiments the alginate oligomer can be applied or administered with or without a further application of the antibiotic(s). The antibiotic(s) can be applied or administered in a plurality of applications prior to or with the alginate oligomer. The skilled man can easily determine what would be an appropriate dosing regime for the alginate oligomer and antibiotic(s) he intends to use.

(44) Preferred antibiotic combinations can be two or more from colistin, ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin, amikacin, gentamicin, oxytetracycline, tobramycin and vancomycin. More particularly, these may be selected from ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin imipenem/cilastatin or oxytetracycline, and still more particularly from ciprofloxacin, meropenem, ceftazidime, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and imipenem/cilastatin. In preferred embodiments the bacteria is an MDR Acinetobacter, Klebsiella, or Pseudomonas (e.g. Acinetobacter baumannii, Klebsiella pneumoniae, or Pseudomonas aeruginosa) resistant to ceftazidime, ciprofloxacin and azithromycin and the antibiotics used are ceftazidime or ciprofloxacin together with azithromycin or all of ceftazidime, ciprofloxacin and azithromycin.

(45) The location of the bacterium which may targeted in any aspect of the present invention is not restricted, and thus as indicated above, not only are medical uses covered, but also non-medical uses where the bacterium is not present on or within a clinical subject, but may for example be present at an abiotic location i.e. the invention may be carried out in vitro. The bacterium may be present on a surface. The surface is not limited and includes any surface on which a bacterium may occur. The surface may be biotic or abiotic, and inanimate (or abiotic) surfaces include any such surface which may be exposed to microbial contact or contamination. Thus particularly included are surfaces on medical equipment, or machinery, e.g. industrial machinery, or any surface exposed to an aquatic environment (e.g. marine equipment, or ships or boats or their parts or components), or any surface exposed to any part of the environment, e.g. pipes or on buildings. Such inanimate surfaces exposed to microbial contact or contamination include in particular any part of: food or drink processing, preparation, storage or dispensing machinery or equipment, air conditioning apparatus, industrial machinery, e.g. in chemical or biotechnological processing plants, storage tanks, medical or surgical equipment and cell and tissue culture equipment. Any apparatus or equipment for carrying or transporting or delivering materials is susceptible to microbial contamination. Such surfaces will include particularly pipes (which term is used broadly herein to include any conduit or line). Representative inanimate or abiotic surfaces include, but are not limited to food processing, storage, dispensing or preparation equipment or surfaces, tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, air conditioning conduits, cooling apparatus, food or drink dispensing lines, heat exchangers, boat hulls or any part of a boat's structure that is exposed to water, dental waterlines, oil drilling conduits, contact lenses and storage cases.

(46) As noted above, medical or surgical equipment or devices represent a particular class of surface on which bacterial contamination may form. This may include any kind of line, including catheters (e.g. central venous and urinary catheters), prosthetic devices e.g., heart valves, artificial joints, false teeth, dental crowns, dental caps and soft tissue implants (e.g. breast, buttock and lip implants). Any kind of implantable (or “in-dwelling”) medical device is included (e.g. stents, intrauterine devices, pacemakers, intubation tubes (e.g. endotracheal or tracheostomy tubes), prostheses or prosthetic devices, lines or catheters). An “in-dwelling” medical device may include a device in which any part of it is contained within the body, i.e. the device may be wholly or partly in-dwelling.

(47) The surface can be made of any material. For example it may be metal, e.g. aluminium, steel, stainless steel, chrome, titanium, iron, alloys thereof, and the like. The surface can also be plastic, for example, polyolefin (e.g., polyethylene, (Ultra-High Molecular Weight) polyethylene, polypropylene, polystyrene, poly(meth)acrylate, acrylonitrile, butadiene, ABS, acrylonitrile butadiene, etc.), polyester (e.g., polyethylene terephthalate, etc.), and polyamide (e.g., nylon), combinations thereof, and the like. Other examples include acetal copolymer, polyphenylsulfone, polysulfone, polythermide, polycarbonate, polyetheretherketone, polyvinylidene fluoride, poly(methyl methacrylate) and poly(tetrafluoroethylene). The surface can also be brick, tile, ceramic, porcelain, wood, vinyl, linoleum, or carpet, combinations thereof, and the like. The surfaces can also be food, for example, beef, poultry, pork, vegetables, fruits, fish, shellfish, combinations thereof, and the like. The “treatment” of any such surface (i.e. the application to any such surface of an alginate oligomer together with an antibiotic) to combat infection by an MDR bacterium is encompassed by the present invention

(48) In an infection by an MDR bacterium, which may be treated according to the present invention, the bacterium may occur in or on a surface in a subject. Furthermore, outside the context of medical treatment, bacteria may also occur on biotic surfaces. Thus the invention includes the treatment of biotic surfaces. A biotic or animate surface may include any surface or interface in or on an animal, plant or fungal body. It may accordingly be viewed as a “physiological” or “biological” surface. It may be any internal or external body surface, including of any tissue or organ, which, in the case of an animal body, may include haematological or haematopoietic tissue (e.g. blood). Dead or dying (e.g. necrotic) or damaged (e.g. inflamed or disrupted or broken) tissue is particularly susceptible to bacterial contamination, and such tissue is encompassed by the term “animate” or “biotic”. The surface may be a mucosal or non-mucosal surface.

(49) Representative biotic surfaces include, but are not limited to, any surface in the oral cavity (e.g. teeth, gingiva, gingival crevice, periodontal pocket) the reproductive tract (e.g. cervix, uterus, fallopian tubes), the peritoneum, middle ear, prostate, urinary tract, vascular intima, the eye i.e. ocular tissue (e.g. the conjunctiva, corneal tissue, lachrymal duct, lachrymal gland, eyelid) the respiratory tract, lung tissue (e.g. bronchial and alveolial), heart valves, gastrointestinal tract, skin, scalp, nails and the interior of wounds, particularly chronic wounds and surgical wounds, which may be topical or internal wounds. Other surfaces include the exterior of organs, particularly those undergoing transplantation, for example, heart, lungs, kidney, liver, heart valve, pancreas, intestine, corneal tissue, arterial and venous grafts and skin.

(50) In one aspect the surface will not be mucosal, or more particularly will not have a hyperviscous mucus coating. The skilled person will be able to determine when the mucus at a given surface is hyperviscous. In one embodiment the surface will not be the surface of a mucus-secreting tissue. More particularly in such an embodiment the surface will not be the surface of a mucus-coated tissue. The skilled person will know from his common general knowledge the tissues that secrete mucus and those that are mucus-coated.

(51) The location may also be a location that is not a surface. In other words the bacterium can be found within an material as well as on its surface. The material can be chemically heterogeneous as well as chemically homogenous. The material can also be constructed or formed from or comprise different parts or components. The material can be a part of a larger material or entity. The material may be or comprise the materials from which the above mentioned surfaces are formed. In some instances the material can be considered to be an object, which terms covers volumes of liquids wherever found. The material may comprise any of the above described surfaces. The material may be abiotic or biotic (inanimate or animate) as is discussed above in relation to surfaces. For instance, the material might be, completely or in part, a solid, a liquid, a semi solid, a gel or a gel-sol. Thus, for example, the bacterium might be present in body fluids (e.g. blood, plasma, serum, cerebrospinal fluid, GI tract contents, semen, sputum and other pulmonary secretions); tissues (e.g. adrenal, hepatic, renal, pancreatic, pituitary, thyroid, immune, ovarian, testicular, prostate, endometrial, ocular, mammary, adipose, epithelial, endothelial, neural, muscle, pulmonary, epidermis, osseous); cell and tissue culture media; cell and tissue cultures; clinical/scientific waste materials (which can comprise any of the preceding materials); pharmaceuticals (e.g. tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, sprays, compositions for use in nebulisers, ointments, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packaged powders); animal or human food stuffs (e.g. meat, fish, shellfish, vegetables, cereals, diary products, fruit juices, vegetable juices, sauces, stocks, soups, confectionary, alcoholic beverages, condiments); personal hygiene products (e.g. toothpaste, mouthwash, shampoo, soap, deodorant, shower gel); cosmetics (e.g. lip gloss, eye shadow, foundation); drinking water supplies; waste water supplies; agricultural feedstuffs and water supplies; insecticide, pesticide and herbicide formulations; industrial lubricants and so on. Liquids, semi solids, gels or gel-sols are of note. The body fluids and tissues may be treated in vitro/ex vivo as well as it being possible to treat the same in vivo.

(52) In certain embodiments the bacterium will be in a biofilm. In other embodiments the bacterium will not be in a biofilm. (e.g. will be growing planktonically). Put differently, the bacterium will be, or will not be, in a biofilm mode of growth; or will be, or will not be, in a non-biofilm mode of growth.

(53) By “biofilm” it is meant a community of microorganisms characterized by a predominance of sessile cells that are attached to a substratum or interface or to each other (some motile cells may also be present) and that are embedded in a matrix of extracellular polymers (more specifically extracellular polymers that they have produced) characterised in that the microorganisms of this colony exhibit an altered phenotype with respect to growth rate and gene transcription (for example as compared to their “non-biofilm” or free-floating or planktonic counterparts). By “in a biofilm” it is meant that the bacterium targeted by the method of the invention is within (completely or in part), on or associated with the polymer matrix of a biofilm. Viewed differently, bacteria that are “not in a biofilm” are organisms that are either in isolation, e.g. planktonic, or if in an aggregation of a plurality of organisms, that aggregation is unorganised and/or is devoid of the matrix characteristic of a biofilm. In each case, the individual bacteria do not exhibit an altered phenotype that is observed in their biofilm dwelling counterparts.

(54) It is well appreciated that Acinetobacter organisms can form a capsule from extracellular polymers (e.g. polysaccharides) that they have produced and Acinetobacter organisms are typically found with such a capsule. It is also well appreciated that the simple presence of a polymer capsule of an Acinetobacter organism is not functionally equivalent to a biofilm mode of growth and the presence of such a capsule is therefore not in itself indicative of a biofilm phenotype. Thus, it will also be appreciated that Acinetobacter organisms that are “not in a biofilm” may still be in contact a matrix of extracellular polymers that they have produced (i.e. the capsule), but such organisms will not exhibit an altered phenotype that is observed in their biofilm dwelling counterparts. Thus, in the particular case of Acinetobacter, by “in a biofilm” it is meant that the Acinetobacter organism is within (completely or in part), on or associated with the polymer matrix of a biofilm and has an phenotype characteristic of Acinetobacter organisms in a biofilm (i.e. a phenotype that is altered with respect to growth rate and gene transcription, for example as compared to “non-biofilm” or free-floating or planktonic Acinetobacter organisms. Acinetobacter organisms that are “not in a biofilm” are organisms that are either in isolation, e.g. planktonic, or if in an aggregation of a plurality of organisms, that aggregation is unorganised. In each case, the individual Acinetobacter organisms do not exhibit an altered phenotype that is observed in their biofilm dwelling counterparts.

(55) From the forgoing it is clear that the methods of the invention, i.e. those described above, have medical and non-medical applications. In particular, the invention provides a method for combating contamination of a site with bacteria that are MDR, in particular the treatment in a subject of a bacterial infection that is MDR, and also a method of combating a population of MDR bacteria. Thus, the method may be an in vitro or an in vivo method. As explained in more detail below, “combating” includes both the treatment of an existing contamination or infection, and treatment to prevent a contamination or infection from occurring, i.e. both “therapeutic”/reactionary and prophylactic treatments.

(56) Accordingly, in one aspect of the invention there is provided a method for the treatment or prevention of an infection of a subject by an MDR bacterium, said method comprising administering to said subject a pharmaceutically effective amount of an alginate oligomer together with a pharmaceutically effective amount of at least one antibiotic to which the bacterium is resistant.

(57) Thus the invention provides an alginate oligomer for use together with (or in combination or conjunction with) at least one antibiotic in the treatment or prevention of an infection of a subject by an MDR bacterium, wherein the bacterium is resistant to the antibiotic.

(58) The term “use together” should be construed as discussed above, although it is particularly meant that a pharmaceutically effective amount of the alginate oligomer is administered at the same or substantially the same time as or prior to, or after, administering a pharmaceutically effective amount of the antibiotic.

(59) Alternatively put, the invention provides the use of an alginate oligomer for the manufacture of a medicament for use together with an antibiotic in the treatment or prevention of an infection of a subject by an MDR bacterium, wherein the bacterium is resistant to the antibiotic.

(60) As noted above, the medicament may further comprise the antibiotic, and single or separate compositions or formulations may be provided and used, as discussed above.

(61) This aspect of the invention also provides the use of an alginate oligomer together with an antibiotic in the manufacture of a medicament for use in the treatment of an infection of a subject by an MDR bacterium, wherein the bacterium is resistant to the antibiotic.

(62) Also provided according to this aspect of the invention is a product containing an alginate oligomer and an antibiotic as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of an infection of a subject by an MDR multidrug resistant bacterium, wherein the bacterium is resistant to the antibiotic.

(63) The MDR bacterium can be any species of bacteria, e.g. those discussed above and mentioned as preferred, e.g. a Burkholderia organism, e.g. Burkholderia cepacia. The antibiotic can be any antibiotic e.g. those discussed above and mentioned as preferred, e.g. a macrolide, e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin or spiramycin.

(64) The subject may be any human or non-human animal subject, but more particularly may be a vertebrate, e.g. an animal selected from mammals, birds, amphibians, fish and reptiles. The animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park. Representative animals therefore include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats, cows, chickens, turkeys, guinea fowl, ducks, geese, parrots, budgerigars, pigeons, salmon, trout, cod, haddock, sea bass and carp. Veterinary uses of the invention are thus covered. The subject may be viewed as a patient. Preferably the subject is a human.

(65) The term “in a subject” is used broadly herein to include sites or locations inside a subject or on a subject, e.g. an external body surface, and may include in particular infection of a medical device e.g. an implanted or “in-dwelling” medical device. The term “in a patient” should be interpreted consistently with this.

(66) The location of the infection is not restricted and may be any of the sites or locations in a subject described above. Administering the alginate oligomer and the antibiotic to the subject preferably results in the infected location being contacted with an alginate oligomer and antibiotic in amounts sufficient to treat the infection.

(67) The infection may be acute, or alternatively chronic, e.g. an infection that has persisted for at least 5 or at least 10 days, particularly at least 20 days, more particularly at least 30 days, most particularly at least 40 days.

(68) In this aspect of the invention the infection may occur on a surface in or on the subject (i.e. a biotic surface as discussed above) and/or a surface of a medical device, particularly an implantable or “in-dwelling” medical device, representative examples of which are discussed above.

(69) In one embodiment the methods or uses of the invention may comprise a step in which the subject is identified (e.g. diagnosed) as having or suspected to have an MDR bacterial infection or being a candidate that is at risk of or susceptible to an MDR bacterial infection.

(70) In particular embodiments the invention may provide for the treatment of respiratory infections, e.g. cystic fibrosis, pneumonia, COPD, COAD, COAP, bacteraemia, septicaemia, septic shock, sepsis, meningitis, or poisoning by bacterially derived toxins.

(71) An MDR bacterial infection can occur in any subject but some subjects will be more susceptible to infection that others. Subjects who are susceptible to MDR bacterial infection include, but are not limited to, subjects whose epithelial and/or endothelial barrier is weakened or compromised, subjects whose secretion-based defences to microbial infection have been abrogated, disrupted, weakened or undermined, and subjects who are immunocompromised, immunodeficient or immunosuppressed (i.e. a subject in whom any part of the immune system is not working normally, or is working sub-normally, in other words in whom any part of the immune response, or an immune activity is reduced or impaired, whether due to disease or clinical intervention or other treatment, or in any way).

(72) Representative examples of subjects who are susceptible to MDR bacterial infection include, but are not limited to, subjects with a pre-established infection (e.g. with bacteria, viruses, fungi or parasites such as protozoa), especially subjects with HIV, subjects with bacteraemia, sepsis and subjects with septic shock; subjects with immunodeficiency, e.g. subjects preparing for, undergoing or recovering from chemotherapy and/or radiotherapy, organ (e.g. bone marrow, liver, lung, heart, heart valve, kidney, etc.) transplant subjects (including autograft, allograft and xenograft patients); subjects with AIDS; subjects resident in a healthcare institution, e.g. hospital, especially subjects in intensive care or critical care (i.e. those units concerned with the provision of life support or organ support systems to patients); subjects on respiratory ventilators; subjects suffering from trauma; subjects with burns, subjects with acute and/or chronic wounds; neonatal subjects; elderly subjects; subjects with cancer (defined broadly herein to include any neoplastic condition; malignant or non-malignant), especially those with cancers of the immune system (e.g. leukaemias, lymphomas and other haematological cancers); subjects suffering from auto-immune conditions such as rheumatoid arthritis, diabetes mellitus type I, Crohn's disease, especially those undergoing immunosuppression treatment for those diseases; subjects with reduced or abrogated epithelial or endothelial secretion (e.g. mucous, tears, saliva) and/or secretion clearance (e.g. subjects with poorly functioning cilia on mucosal tissue and/or patients with hyperviscous mucous (e.g. smokers and subjects with COPD, COAD, COAP, bronchitis, cystic fibrosis, emphysema, lung cancer, asthma, pneumonia or sinusitis)) and subjects fitted with a medical device.

(73) MDR bacteria are commonly encountered in healthcare institutions due in part to the close proximity of subjects with bacterial infections and the widespread use of antibiotics. MDR bacteria, e.g. from the genera Pseudomonas, Klebsiella, Burkholderia, Providencia and Acinetobacter, are therefore often involved in nosocomial infections and accordingly the invention can be seen as providing treatments for MDR nosocomial infections.

(74) Thus, subjects in whom MDR infections may particularly be combated according to the present invention include patients who are impaired, whether due to poor perfusion, repetitive trauma, poor nutrition, poor oxygenation or white cell dysfunction.

(75) Of particular note are subjects that have undergone physical trauma. The trauma itself might cause a weakening in or compromisation of an epithelial and/or endothelial barrier of the subject or the subject may become immunocompromised in response to the trauma (a shock response). The term “trauma” refers broadly to cellular attack by foreign bodies and/or physical injury of cells. Included among foreign bodies are microorganisms, particulate matter, chemical agents, and the like. Included among physical injuries are mechanical injuries; thermal injuries, such as those resulting from excessive heat or cold; electrical injuries, such as those caused by contact with sources of electrical potential; and radiation damage caused, for example, by prolonged, extensive exposure to infrared, ultraviolet or ionizing radiations.

(76) Also of particular note are subjects that have a burn. Any burn, in particular a severe burn, has a significant impact on the integrity of the epithelial and/or endothelial barrier of the subject and the subject will often become immunocompromised in response to the burn (a shock response).

(77) Typical burn-causing agents are extremes of temperature (e.g. fire and liquids and gases at extreme temperature), electricity, corrosive chemicals, friction and radiation. The extent and duration of exposure, together with the intensity/strength of the agent, result in burns of varying severity. Scalding (i.e. trauma associated with high temperature liquids and/or gases) is considered to be a burn.

(78) Epidermal burn severity is commonly classified in two ways. Most common is the classification by degree. First-degree burns are usually limited to erythema (redness) in the general area of the injury and a white plaque at the site of injury. The cellular trauma of these burns extends only as deep as the epidermis. Second-degree burns also display erythema in the general area of the injury but with superficial blistering of the epidermis. The cellular trauma of second-degree burns involves the superficial (papillary) dermis and may also involve the deep (reticular) dermis layer. Third-degree burns are those in which the epidermis is lost with damage to the hypodermis. Damage is typically extreme including charring. Sometimes eschar, (dry, black necrotic tissue) will be present. Third-degree burns may require grafting. In fourth-degree burns catastrophic damage of the hypodermis occurs, e.g. the hypodermis is completed lost, with damage extending to the underlying muscle, tendon, and ligament tissue. Charring and eschar are observed. Grafting is required if the burn does not prove to be fatal.

(79) Another common classification system is the classification by thickness. “Superficial thickness” burns correspond to first degree burns. The spectrum of second degree burns is covered by two classes of “partial thickness” burns. “Partial thickness-superficial” are burns that affect the epidermis only as far as the papillary dermis. “Partial thickness-deep” are burns that affect the dermis as far as the reticular dermis. “Full thickness” burns correspond to third and fourth degree burns.

(80) Some physical injuries, e.g. some burns, and cellular attacks by foreign bodies result in the formation of a wound. More specifically a wound may be considered to be a breach in, or denudement of, a tissue. Wounds may also be caused by a spontaneously forming lesion such as a skin ulcer (e.g. a venous, diabetic or pressure ulcer), an anal fissure or a mouth ulcer.

(81) Wounds are typically defined as either acute or chronic. Acute wounds are wounds that proceed orderly through the three recognised stages of the healing process (i.e. the inflammatory stage, the proliferative stage and the remodelling phase) without a protracted timecourse. Chronic wounds, however, are those wounds that do not complete the ordered sequence of biochemical events of the healing process because the wound has stalled in one of the healing stages. Commonly, chronic wounds are stalled in the inflammatory phase. In accordance with a particular aspect of the present invention, a chronic wound is a wound that has not healed within at least 40 days, particularly at least 50 days, more particularly at least 60 days, most particularly at least 70 days.

(82) As discussed above, wounds are an ideal environment for an MDR bacterial infection, particularly chronic infection, due to their lack of an epithelial barrier and the availability of substrate and surface for microbial attachment and colonisation. Problematically, infection of a wound often delays healing further and thus renders that wound more susceptible to established infection. The methods of the invention are therefore effective in the treatment and prevention of MDR bacterial infection of wounds and the use of the methods of the invention in the treatment of wounds, especially chronic wounds, represents one preferred aspect of the present invention.

(83) Therefore, in an embodiment of the invention there is provided an alginate oligomer for use together with (or in combination or conjunction with) an antibiotic in the treatment or prevention of the infection of a subject by an MDR bacterium, wherein the bacterium is resistant to the antibiotic, particularly chronic infection by an MDR bacterium in the above-mentioned subjects, in particular in subjects with respiratory diseases or disorders e.g. cystic fibrosis, COPD, COAD, COAP, pneumonia, wounds, burns and/or traumas.

(84) Through the ability to treat and prevent infection of wounds by an MDR bacterium the alginate oligomers and antibiotics of the invention as defined herein can remove one of the obstacles to wound healing and therefore the alginate oligomers and antibiotics defined above are also effective in the promotion of healing of acute and chronic wounds infected with or at risk of infection with an MDR bacterium which is resistant to any of said antibiotics

(85) By promotion of healing it is meant that the treatment accelerates the healing process of the wound in question (i.e. the progression of the wound through the three recognised stages of the healing process). The acceleration of the healing process may manifest as an increase in the rate of progression through one, two or all of the healing stages (i.e. the inflammatory stage, the proliferative stage and/or the remodelling phase). If the wound is a chronic wound that is stalled in one of the healing stages the acceleration might manifest as the restarting of the linear, sequential healing process after the stall. In other words, the treatment shifts the wound from a non-healing state to a state where the wound begins to progress through the healing stages. That progression after the restart may be at a normal rate or even a slower rate compared with the rate a normal acute wound would heal.

(86) The alginate oligomers and antibiotics of the invention may be used together (or in combination or conjunction) to treat or prevent MDR bacterial infections wherever they may occur in or on the body. Thus, in another embodiment, the infection may be an infection of a medical device by an MDR bacterium, particularly an in-dwelling medical device, e.g. endotracheal and tracheostomy tubes.

(87) The alginate oligomers and antibiotics of the invention may be used together (or in combination or conjunction) as oral healthcare agents, for example in the control of dental plaque, e.g. to reduce it or to prevent, reduce or delay its development by inhibiting growth of MDR plaque bacteria on teeth or dental/oral prostheses. The alginate oligomers and antibiotics of the invention may also be used together (or in combination or conjunction) in the treatment and prevention of MDR infections or MDR infectious disease which may occur in the oral cavity, for example gingivitis and periodontitis

(88) Conveniently, the alginate oligomers and/or antibiotics can be applied by any oral health/oral hygiene delivery system. This may be through the use of toothpastes, dental gels, dental foams and mouthwashes. Removable dentures and other removable dental prostheses may be treated outside of the oral cavity with the same compositions or other suitable pharmaceutically acceptable compositions. The alginate oligomers and/or antibiotics can also be incorporated into compositions that are applied to the oral cavity (or applied to removable dentures and other removable dental prostheses outside of the oral cavity) to form a coating that persists on surfaces over time, or that releases the alginate oligomers and/or antibiotics from the coated surfaces over time, and which inhibit the growth of MDR bacteria in the oral cavity and on the surfaces of removable dentures and other removable dental prostheses.

(89) Whilst the treatment of MDR bacterial infections of the lungs and respiratory tract and all areas of the body is generally covered by the present invention, in one embodiment, the medical uses of the invention are not directed to the treatment of (i) infections in the respiratory tract, e.g. in patients suffering from COPD's (chronic obstructive pulmonary diseases), in particular the sinuses and the lungs, in particular in the treatment of cystic fibrosis, chronic obstructive pulmonary disease, emphysema, bronchitis and sinusitis; (ii) in the middle ear of patients suffering from glue ear; or (iii) in the reproductive tract of female patients with impaired fertility; or (iv) in the digestive tract of patients with digestive tract malfunction (e.g. constipation).

(90) In specific embodiments of the invention the alginate oligomers and antibiotics of the invention may be used together (or in combination or conjunction) in the treatment or prevention of native valve endocarditis, acute otitis media, chronic bacterial prostatitis, pneumonia (in particular ventilator associated pneumonia) associated with MDR bacteria; respiratory diseases associated with MDR bacteria (which may include COPD, COAD, COAP, pneumonia, cystic fibrosis and asthma); and device related MDR bacterial infections associated with implantable or prosthetic medical devices (e.g. prosthetic valve endocarditis or the infection of lines or catheters or artificial joints or tissue replacements or endotracheal or tracheotomy tubes).

(91) In further embodiments the alginate oligomers and antibiotics of the invention are used together to control MDR infections in the eye, e.g. to reduce them, or prevent, reduce or delay their development. In particular, the alginate and antibiotics of the invention are used together to treat or prevent MDR bacterial conjunctivitis and the resultant keratoconjunctivitis sicca (also known as dry eye) that can result through the blockage of the lachrymal gland.

(92) As mentioned previously, in certain embodiments, the above MDR bacterial infections and associated conditions are, or involve, biofilm, in other words they are biofilm infections. In other embodiments the above MDR bacterial infections and associated conditions are not, or do not involve biofilm.

(93) In a further aspect the invention provides a method for combating contamination of a site with MDR bacteria, said method comprising contacting the site and/or the MDR bacteria with (an effective amount of) an alginate oligomer together with (an effective amount of) at least one antibiotic to which the bacteria are resistant. Such a method may particularly be an in vitro method, and the site may be any surface or location discussed above.

(94) “Combating contamination” includes both preventative and reactionary measures or treatments and therefore covers the prevention as well as the reduction, limitation, or elimination of contamination.

(95) By “contamination” it is meant the unwanted presence of a bacterium (e.g an MDR bacterium) at a particular site or location. Contamination can be considered to cover colonisation of a location by a bacterium (e.g an MDR bacterium), i.e. the establishment of a bacterium (e.g an MDR bacterium) at a location and the expansion of the numbers of that organism by replication or the recruitment of additional bacteria, which may be of the same or of a different type. In one embodiment the colonisation process will not involve the formation of a biofilm.

(96) The site or location of the contamination or potential contamination is not restricted and can be any of the various sites or locations described or mentioned above, e.g. it can be in vitro or in vivo, but particularly in this aspect of the invention it will be an “in vitro” or “ex vivo” site or location (e.g. an inanimate or abiotic site or location). However, the site or location may be in a subject and in which case a pharmaceutically effective amounts of the alginate oligomer and the antibiotic are administered to the subject.

(97) In one particular embodiment the various aspects of the invention can be applied to the decontamination of clinical, scientific and industrial waste materials. In another particular embodiment the various aspects of the invention can be used to decontaminate transplant tissue (e.g. heart, lungs, kidney, liver, heart valve, pancreas, intestine, corneal tissue, arterial and venous grafts and skin) and medical devices (e.g. endotracheal and tracheostomy tubes) prior to implantation. In another embodiment the various aspects of the invention can be considered to cover the use of alginate oligomers together with antibiotics as anti-MDR bacterial preservative agents in materials, especially solutions and liquids.

(98) In another embodiment, the methods of the invention may further comprise a step in which the bacteria being targeted will be determined as being, or alternatively not being in, or involving, a biofilm.

(99) In other embodiments of the methods of the invention the methods may comprise a step in which it is determined (e.g. ascertained or identified) that the bacterium is resistant to a particular antibiotic(s). In a step in place of, or in addition to, the previously described step, there may be a step in which it is determined that the bacterium is an MDR bacterium. Any convenient test can be used here, for instance those described above, or any technique for identifying known and characterised bacteria (e.g. bacteria already identified as being antibiotic and/or multidrug resistant). In a further step it may be ascertained whether or not a particular resistance is acquired or intrinsic, e.g. by comparison to typical or wild type bacteria of the same species.

(100) In any of the aspects, uses or methods of the invention the MDR bacteria and the antibiotic can be any of the bacteria and antibiotics defined above and especially any, or combinations thereof, stated as preferred. For example, the MDR bacteria may be a bacteria from an MDR strain of bacteria. Also for example, the MDR bacteria may be a Burkholderia organism, e.g. Burkholderia cepacia. Also for example the antibiotic may be a macrolide, e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin or spiramycin.

(101) The term “contacting” encompasses any means of delivering the alginate oligomer and the antibiotic to the MDR bacterium, whether directly or indirectly, and thus any means of applying the alginate oligomer and the antibiotic to the MDR bacterium or exposing the MDR bacterium to the alginate oligomer and the antibiotic e.g. applying the alginate oligomer and the antibiotic directly to the MDR bacterium or administering the alginate oligomer and the antibiotic to a subject within which or on which the MDR bacterium is present, e.g. a subject infected with an MDR bacterium.

(102) More particularly the MDR bacterium will be contacted with an effective amount of the alginate oligomer and the antibiotic, more particularly an amount of the alginate oligomer and an amount of the antibiotic that together (or in combination or conjunction) overcome the resistance of the MDR bacterium to the antibiotic and therefore inhibit the viability and/or growth of the MDR bacterium and therefore treat or prevent the infection/contamination.

(103) An “effective amount” of the alginate oligomer and the antibiotic is that amount of alginate oligomer and that amount of the antibiotic that together (or in combination or conjunction) provide measurable reduction in the resistance (or measurable increase in susceptibility or measurable decrease in tolerance) to the antibiotic displayed by the bacterium (e.g. using the above-described indicators of resistance). In certain embodiments the “effective amount” of the alginate oligomer and the antibiotic is that amount of alginate oligomer and that amount of the antibiotic that together (or in combination or conjunction) provide measurable inhibition of the growth of an MDR bacterium, or population thereof, that is being targeted, e.g. which is resistant to the antibiotic.

(104) A “pharmaceutically effective” amount of the alginate oligomer and the antibiotic is that amount of alginate oligomer and that amount of the antibiotic that together (or in combination or conjunction) provide a measurable reduction in the resistance (or measurable increase in susceptibility or measurable decrease in tolerance) to the antibiotic displayed by the MDR bacterium (e.g. using the above-described indicators of resistance) in a subject and/or a measurable treatment or prevention of the infection by an MDR bacterium that is being targeted.

(105) The skilled man would easily be able to determine what an effective/pharmaceutically effective amount of alginate oligomer and antibiotic would be on the basis of routine dose response protocols and, conveniently, the routine techniques for assessing microbial growth inhibition etc., as discussed below. The skilled man would, without undue burden, also be able to optimise these amounts to maximise the combinatorial effects of the alginate oligomer and antibiotic in his target system.

(106) By “growth of an MDR bacterium” it is meant both an increase in the size of an MDR bacterium or in the amount and/or volume of the constituents of an MDR bacterium (e.g. the amount of nucleic acid, the amount of protein, the number of nuclei, the numbers or size of organelles, the volume of cytoplasm) and an increase in the numbers of the MDR bacterium, i.e. an increase in the replication of the MDR bacterium.

(107) Typically growth of an MDR bacterium is accompanied by the enlargement of the organism. The growth of MDR bacteria can be measured with routine techniques. For instance, microscopic examination of microorganism morphology over time, or assays to measure changes in the quantities of protein or nucleic acid (e.g. DNA) in general, or the changes in the quantities of specific proteins or nucleic acids, can be used. The skilled man would easily be able to select suitable markers to follow. Conveniently, so called housekeeping genes (e.g. β-actin, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein), AHSP (alphahaemoglobin stabilising protein), and 132M (beta-2-microglobulin)), 16S RNA and virus genes, and their expression products can be monitored.

(108) By “replication of an MDR bacterium” or “replication of a bacterium” it is meant the act by which the (MDR) bacterium reproduces. Typically this is by binary fission where a microorganism divides into two. To support the division of the microorganism into two, binary fission is normally preceded by enlargement of the dividing microorganism and an increase in the amount and/or volume of cellular constituents. Replication results in an increase in the number of cells and so may be followed by any method of assessing microorganism numbers in a population. Another option is to follow the process in real time by visual examination with a microscope. The time it takes for microorganism to replicate (i.e. produce another version of itself) is the generation time, Generation time will depend on the conditions in which the (MDR) bacterium is found. The rate of replication can be expressed in terms of the generation time.

(109) By “inhibiting the growth of an MDR bacterium” or inhibiting the growth of a bacterium” it is meant that measurable growth (e.g. replication) of an (MDR) bacterium, or the rate thereof, is reduced. Preferably measurable growth (e.g. replication) of an (MDR) bacterium, or the rate thereof, is reduced by at least 50%, more preferably at least 60%, 70%, 80% or 90%, e.g. at least 95%. Preferably, measurable growth (e.g. replication) is ceased. Growth in terms of microbial size increase or expansion etc. may be inhibited independently of replication and vice versa.

(110) Suitable doses of alginate oligomer and antibiotic will vary from subject to subject and can be determined by the physician or veterinary practitioner in accordance with the weight, age and sex of the subject, the severity of the condition, the mode of administration and also the particular alginate oligomer or antibiotic selected. Typically the alginate oligomers of the invention will be applied to the location undergoing treatment at a local concentration of at least 0.5%, preferably at least 2% or at least 4%, more preferably at least 6% and most preferably at least 10% weight by volume. Typically the antibiotic of the invention will be applied to the location undergoing treatment at a local concentration of at least 1 μg/ml, preferably at least 4, at least 8, at least 16, at least 32, at least 64, at least 128, at least 256 or at least 512, 1024, 2048 or 4096 μg/ml.

(111) “Treatment” when used in relation to the treatment of a medical condition/infection in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect on the condition or in relation to the infection. Thus, not only included is eradication or elimination of the infection, or cure of the subject or infection, but also an improvement in the infection or condition of the subject. Thus included for example, is an improvement in any symptom or sign of the infection or condition, or in any clinically accepted indicator of the infection/condition (for example a decrease in wound size or an acceleration of healing time). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed infection/condition, i.e. a reactionary treatment.

(112) “Prevention” as used herein refers to any prophylactic or preventative effect. It thus includes delaying, limiting, reducing or preventing the condition (which reference includes infection and contamination, as applicable, in the different aspects of the invention) or the onset of the condition, or one or more symptoms or indications thereof, for example relative to the condition or symptom or indication prior to the prophylactic treatment. Prophylaxis thus explicitly includes both absolute prevention of occurrence or development of the condition, or symptom or indication thereof, and any delay in the onset or development of the condition or symptom or indication, or reduction or limitation on the development or progression of the condition or symptom or indication.

(113) Specifically, the alginate oligomers and antibiotics of the invention can be taken together (or in combination or conjunction) as a prophylactic treatment, for example to prevent, or at least minimise the risk, of infection or contamination by an MDR bacterium resistant to the antibiotic.

(114) The aspect of the invention concerning the combating (treatment or prevention) of infection by an MDR bacterium is of particular utility in the care of hospitalised patients as the risk of contracting an nosocomial infection (commonly known as hospital related/acquired infection or healthcare-associated infection) by an MDR bacterium can be minimised with a prophylactic regime of the alginate oligomers and antibiotics defined herein. This aspect of the invention is also of particular utility in the care of subjects suffering from trauma, subjects with a burn and subjects with wounds, all of which, as discussed above, are more susceptible to infection by MDR bacteria than a subject that is not affected similarly.

(115) Generally, subjects in need of treatment or prophylaxis according to the invention will be diagnosed as suffering or at risk from infection by an MDR bacterium, e.g. identified as having or at risk of developing an infection by an MDR bacterium.

(116) Specifically, the alginate oligomers and antibiotics of the invention can be taken together (or in combination or conjunction) as a prophylactic treatment to prevent, or at least minimise the risk, of developing an infection by an MDR bacterium resistant to the chosen antibiotic(s), including for example the infection of wounds by an MDR bacterium; native valve endocarditis, acute otitis media, chronic bacterial prostatitis, associated with an MDR bacterium; infections of the respiratory tract and lungs by an MDR bacterium (e.g. cystic fibrosis, COPD, COAD, COAP, pneumonia, or other respiratory diseases) or infection of a medical (e.g. in-dwelling) medical device by an MDR bacterium.

(117) The invention encompasses the use of a single alginate oligomer or a mixture (multiplicity/plurality) of different alginate oligomers. Thus, for example, a combination of different alginate oligomers (e.g. two or more) may be used.

(118) The invention encompasses the use of a single antibiotic or a mixture (multiplicity/plurality) of different antibiotics. Thus, for example, a combination of different antibiotics (e.g. two or more) may be used. The MDR bacterium may be sensitive to the further antibiotic(s) used or may be resistant to the further antibiotic(s) used.

(119) In one advantageous embodiment of the invention the alginate oligomers and antibiotic may be used in the methods of the invention in conjunction or combination with a further anti-microbial agent (hereinafter “further anti-microbial agent”)

(120) In the context of a medical use, such an anti-microbial agent may be any clinically-useful anti-microbial agent and particularly an antibiotic or an antiviral or antifungal agent. In the context of non-clinical uses, the anti-microbial agent may again be any anti-microbial agent used for such purposes, e.g. any disinfectant or antiseptic or cleaning or sterilising agent. The agents may be used separately, or together in the same composition, simultaneously or sequentially or separately, e.g. at any desired time interval.

(121) Thus, by way of representative example, the further anti-microbial agent may be used after the alginate oligomer and/or the antibiotic, but a preceding or simultaneous or intervening use may be beneficial in some circumstances.

(122) The choice of anti-microbial agent will of course need to be appropriate for the location undergoing treatment, but for instance anti-microbial agents, e.g. antibiotics, antifungals, antivirals, antiseptics may be used and/or sterilising conditions such as irradiation (e.g. UV, X-ray, gamma) extremes of temperature, and extremes of pH.

(123) Representative antibiotics include those listed above, especially those stated as preferred.

(124) Representative antiseptics include, but are not limited to chlorine bleach (sodium hypochlorite), quaternary ammonium compounds (e.g. benzalkonium chloride, cetyl trimethylammonium bromide, cetylpyridinium chloride), hydrogen peroxide, phenol compounds (e.g. TCP), alcohols (e.g. ethanol), Virkon™, iodine compounds (e.g. povidone-iodine), silver compounds (e.g. elemental silver nano/microparticles).

(125) Antimicrobial surfactants are another class of antiseptics. These are compounds that disrupt microbial cell membranes and other structural components and therefore inhibit growth and/or viability of microorganisms. Antimicrobial surfactants and their use in antimicrobial compositions is well known in the art should further guidance be needed the discussion of antimicrobial surfactants in “Preservative-free and self-preserving cosmetics and drugs—Principles and practice”, Ed. Kabara and Orth, Marcel Dekker, NY, NY, 1997, is explicitly incorporated by reference in its entirety. Antimicrobial surfactants may be anionic, cationic, non-ionic or amphoteric. Examples of antimicrobial anionic surfactants include, but are not limited to, sodium dodecyl sulfate (sodium lauryl sulfate), sodium dodecyl aminopropionic acid, sodium ricinoleate, bile acids, alkylaryl sulfonates, Grillosan DS7911, disodium undecylenic acid monoethanol amidosulfosuccinate. Examples of antimicrobial cationic surfactants include, but are not limited to, the quaternary ammonium compounds, the aminimides and chlorhexidine compounds. Examples of antimicrobial non-ionic surfactants include, but are not limited to, the monoesters of fatty acids, polyethyleneglycomonoesters of alkyldihydroxybenzoic acids, glucosamine derivatives and diethanolamides of N-lauroyl dipeptides. Examples of antimicrobial amphoteric surfactants include, but are not limited to, the alkyl betaines, the alkylamidopropylbetaines, the alkyl aminopropionates, the alkyliminodipropionates and the alkylimidazolines.

(126) Representative antifungals include, but are not limited to the polyenes (e.g. natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin; the imidazoles (e.g. miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole); the triazoles (e.g. fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole); the allylamines (e.g. terbinafine, amorolfine, naftifine, butenafine); and the echinocandins (e.g. anidulafungin, caspofungin, micafungin).

(127) Representative antivirals include, but are not limited to abacavir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type, II interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

(128) The further anti-microbial agent may conveniently be applied before, simultaneously with, following or between the alginate oligomer and/or the antibiotic. Conveniently the further anti-microbial agent is applied at substantially the same time as the alginate oligomer and/or the antibiotic or afterwards. For example, the further anti-microbial agent is applied at least 1 hour, preferably at least 3 hours, more preferably at least 5 and most preferably at least 6 hours after the alginate oligomer and/or the antibiotic is administered. In other embodiments the further antimicrobial may conveniently be applied or administered before the alginate oligomer and/or the antibiotic r, e.g. at least 1 hour, at least 3 hours, at least 6 hours before the alginate oligomer and/or the antibiotic. In these embodiments the alginate oligomer and/or the antibiotic can be applied or administered with or without a further application of the further antimicrobial. To optimise the anti-microbial effect of the further anti-microbial agent it can be given (e.g. administered or delivered) repeatedly at time points appropriate for the agent used. The skilled person is able to devise a suitable dosage or usage regimen. In long term treatments the alginate oligomer and/or the antibiotic can also be used repeatedly. The alginate oligomer can be applied as frequently as the antibiotic and/or the further anti-microbial agent, but will typically be less frequently. The frequency required will depend on the location of the MDR bacterium, colony composition and the anti-microbial used and the skilled person is able to optimise the dosage or usage patterns to optimise results.

(129) In an advantageous embodiment the alginate oligomer and/or the antibiotic may be used or applied after physical removal or reduction (e.g. debridement) of the colony/population comprising the MDR bacterium causing the infection at the location undergoing treatment.

(130) Following removal of, or an attempt to remove, the colony/population comprising the MDR bacterium, the location may be contacted with the alginate oligomer for between 0 and 24 hours, particularly 2 and 12 hours, more particularly 4 and 8 hours, most particularly 5 and 7 hours, e.g. 6 hours. Following this, the antibiotic, and if desired the further anti-microbial agent, may be applied. Such a scenario may be desirable or particularly applicable in a clinical setting. In the case of wounds infected by an MDR bacterium, the duration of incubation can be conveniently be designed to correspond to scheduled changes of the wound dressing.

(131) Physical removal of the colony/population comprising the MDR bacterium can be carried out with any suitable surgical, mechanical or chemical means. Conveniently this can be the use of a liquid, gel, gel-sol, semi-solid compositions or gas applied at pressure to the colony/population, sonication, laser, or by abrasive implement. A composition used in the removal itself or as a wash solution before, during or afterwards may conveniently contain the alginate oligomer and/or the antibiotic.

(132) Accordingly, in one specific embodiment there is provided a debridement or wash composition e.g. solution for wounds containing an alginate oligomer, particularly any alginate oligomer as herein defined, and/or an antibiotic, particularly any antibiotic as herein defined (e.g. a macrolide, preferably selected from azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin or spiramycin), for use in the treatments and methods of the invention. Such a debridement composition will typically be a sterile solution, particularly an aqueous sterile solution or an oil-based sterile solution, and may additionally contain proteolysis enzymes (e.g. collagenase, trypsin, pepsin, elastase), an abrasive solid phase (e.g. colloidal silica, ground pumice, ground plant or animal shell).

(133) Use of the alginate oligomers and the antibiotic in combination or conjunction with immunostimulatory agents may also be beneficial in the application of the methods of the invention in a clinical situation. These immunostimulatory agents may conveniently be used at timepoints corresponding to those described above in relation to anti-microbial agents and may optionally be used in combination with an alginate oligomer and/or the antibiotic and/or a further anti-microbial agent Suitable immunostimulatory agents include, but are not limited to cytokines e.g. TNF, IL-1, IL-6, IL-8 and immunostimulatory alginates, such as high M-content alginates as described for example in U.S. Pat. No. 5,169,840, WO91/11205 and WO03/045402 which are explicitly incorporated by reference herein in their entirety, but including any alginate with immunostimulatory properties.

(134) Use of the alginate oligomers and the antibiotic in combination or conjunction with growth factors, e.g. PDGF, FGF, EGF, TGF, hGF and enzymes may also be beneficial in the medical uses of the invention. Representative examples of suitable enzymes include but are not limited to proteases, e.g. serine proteases, metalloproteases and cysteine proteases (examples of these types of proteases are listed in EP0590746, the entire contents of which are incorporated herein by reference); nucleases, e.g. DNase I and II, RNase A, H, I, II, III, P, PhyM, R; lipases and enzymes capable of degrading polysaccharides.

(135) Use of the alginate oligomers and the antibiotic in combination or conjunction with a physiologically tolerable mucosal viscosity reducing agent could also be beneficial, e.g. a nucleic acid cleaving enzyme (e.g. a DNase such as DNase I), gelsolin, a thiol reducing agent, an acetylcysteine, sodium chloride, an uncharged low molecular weight polysaccharide (e.g. dextran), arginine (or other nitric oxide precursors or synthesis stimulators), or an anionic polyamino acid (e.g. poly ASP or poly GLU). Ambroxol, romhexine, carbocisteine, domiodol, eprazinone, erdosteine, letosteine, mesna, neltenexine, sobrerol, stepronin, tiopronin are specific mucolytics of note.

(136) Use of the alginate oligomers and the antibiotic in combination or conjunction with alpha blockers may also be beneficial in the medical uses of the invention, in the treatment of chronic bacterial prostatitis especially. Representative examples of suitable alpha blockers include but are not limited to the selective alpha-1 blockers (e.g. doxazosin, dilodosin, prazosin, tamsulosin, alfuzosin, terazosin), and the non-selective adrenergic blockers (e.g. phenoxybenzamine, phentolamine).

(137) Use of the alginate oligomers and the antibiotic in combination or conjunction with bronchodilators may also be beneficial in the medical uses of the invention, in the treatment of respiratory diseases associated with MDR bacteria especially (which may include COPD, COAD, COAP, pneumonia, cystic fibrosis, emphysema and asthma). Representative examples of suitable bronchodilators include but are not limited to the β2 agonists (e.g. pirbuterol, epinephrine, salbutamol, salmeterol, levosalbutamol, clenbuterol), the anticholinergics (e.g. ipratropium, oxitropium, tiotropium) and theophylline.

(138) Use of the alginate oligomers and the antibiotic in combination or conjunction with corticosteroids may also be beneficial in the medical uses of the invention, in the treatment of respiratory diseases associated with MDR bacteria especially (which may include COPD, COAD, COAP, pneumonia, cystic fibrosis, emphysema and asthma). Representative examples of suitable corticosteroids include but are not limited to prednisone, flunisolide, triamcinolone, fluticasone, budesonide, mometasone, beclomethasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone, halcinonide. hydrocortisone, cortisone, tixocortol, prednisolone, methylprednisolone, prednisone, betamethasone, dexamethasone, fluocortolone, aclometasone, prednicarbate, clobetasone, clobetasol, and fluprednidene.

(139) The alginate oligomers and the antibiotic can be used optionally with any other therapeutically active agent it may be desired to use, e.g. an anti-microbial agent, an anti-inflammatory agent (e.g. an anti-inflammatory steroid), an immunostimulatory agent, a mucosal viscosity reducing agent, a growth inhibitor or an enzyme or an alpha blocker, a bronchodilator or a corticosteroid. The combined use of an alginate oligomer and an antibiotic with a further therapeutically active agent (e.g. an anti-microbial or anti-inflammatory agent, an immunostimulatory agent, a mucosal viscosity reducing agent, a growth inhibitor or an enzyme or an alpha blocker, a bronchodilator or a corticosteroid) may improve the clinical effects of the active agent and this may advantageously allow the dose (e.g. the usual or normal dose) of the further therapeutically active agent to be reduced e.g. it may be used at its normal or usual dose or at a lower dose, for example at up to 50% (or at 50%) of its normal dose.

(140) In the case of medical use, the alginate oligomers and antibiotics of the invention may be administered to the subject in any convenient form or by any convenient means, e.g. by topical, oral, parenteral, enteral, parenteral routes or by inhalation. Preferably the alginate and antibiotics will be administered by topical, oral or parenteral routes or by inhalation. The alginate oligomers and antibiotics need not be in the same composition and need not be administered via the same route.

(141) The skilled man will be able to formulate the alginate oligomers and the antibiotics of the invention into pharmaceutical compositions that are adapted for these routes of administration according to any of the conventional methods known in the art and widely described in the literature.

(142) The present invention therefore also provides a pharmaceutical composition for use in any of the above-mentioned methods or uses comprising an alginate oligomer as defined herein together with at least one pharmaceutically acceptable carrier, diluent or excipient. This composition may also comprise an antibiotic as defined herein.

(143) The present invention therefore also provides a pharmaceutical composition for use in any of the above-mentioned methods or uses comprising an antibiotic as defined herein together with at least one pharmaceutically acceptable carrier, diluent or excipient. This composition may also comprise an alginate oligomer as defined herein.

(144) The invention also provides products (e.g. a pharmaceutical kit or a combined (“combination”) product) or compositions (e.g. a pharmaceutical composition) wherein the product or composition comprises an alginate oligomer as herein defined and an antibiotic, e.g. selected from the group azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin, aztreonam, imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin. Preferably the antibiotic is selected from the group ceftazidime, imipenem/cilastatin, meropenem, aztreonam, oxytetracycline, colistin, azithromycin and ciprofloxacin, preferably it is azithromycin. For example, the antibiotic may be selected from amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, tylosin, troleandomycin, aztreonam, imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin. In particular, antibiotic may selected from ceftazidime, imipenem/cilastatin, meropenem, aztreonam, oxytetracycline, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin, and it is particularly preferred that the antibiotic is selected from ceftazidime, imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin. More preferably the antibiotic is selected from aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin. In other embodiments the antibiotic used is not tobramycin, amikacin and/or colistin. In other embodiments the antibiotic used is not an aminoglycoside or a polypeptide antibiotic. In other embodiments the antibiotic used is not an antibiotic that has a positive charge under the conditions in which it will be used with the alginate oligomer, e.g. antibiotics with at least 3, e.g. at least 4, 5, 6 or 7 amino (—NH.sub.2) groups. These products and compositions are specifically contemplated as for use in the methods of the invention. The products and compositions can be pharmaceutical or non-pharmaceutical. Therefore the products and compositions of this aspect of the invention can be used in any of the methods of the invention.

(145) As discussed above, the alginate oligomers and the antibiotics proposed for use according to the invention may be used in combination with each other, for example to be administered together, in a single pharmaceutical formulation or composition, or separately (i.e. for separate, sequential or simultaneous administration). Thus, the alginate oligomers and the antibiotics of the invention may be combined, e.g. in a pharmaceutical kit or as a combined (“combination”) product.

(146) Thus as noted above, further aspects of the present invention provide products containing an alginate oligomer and an antibiotic as a combined preparation for the uses defined herein. Such products may optionally further contain a further active agent.

(147) The use of alginate oligomers as herein defined to manufacture such pharmaceutical products and pharmaceutical compositions for use in the medical methods of the invention is also contemplated.

(148) Further active agents may also be incorporated. The above and following discussion of additional active agents and excipients and the like is directly applicable in its entirety to this aspect of the invention.

(149) The active ingredient may be incorporated, optionally together with other active agents, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders (e.g. inhalable powders), lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), sprays (e.g. nasal sprays), compositions for use in nebulisers ointments, soft and hard gelatine capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like. Sterile inhalable compositions are of particular note for use in the treatment of respiratory diseases associated with MDR bacteria (which may include COPD, COAD, COAP, pneumonia, cystic fibrosis, emphysema and asthma).

(150) Examples of suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, inert alginates, tragacanth, gelatine, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol, water/glycol, water/polyethylene, hypertonic salt water, glycol, propylene glycol, methyl cellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof. Excipients and diluents of note are mannitol and hypertonic salt water (saline).

(151) The compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like. Additional therapeutically active agents may be included in the pharmaceutical compositions, as discussed above in relation to combination therapies above.

(152) In some instances it may be beneficial to administer the alginate oligomers and/or the antibiotics as defined herein to animals, e.g. to promote weight gain/growth. Administration can be achieved in the form of the pharmaceutical compositions described above, but conveniently the alginate oligomers and/or the antibiotics as defined herein may be used as a conventional feed additive, i.e. a compound that is added to animal feed in small, nutritionally inconsequential amounts. The use of feed additives in animal feeds is well established and it would be entirely routine for a skilled man to determine and use appropriate amounts of the alginates of the invention to achieve the desired effects, e.g. weight gain/growth.

(153) The relative content of the alginate oligomer and the antibiotic can vary depending on the dosage required and the dosage regime being followed and this will depend on the subject to be treated and the location and identity of the MDR bacterium, and/or the constituents of the contamination or population comprising the MDR bacterium. Preferably, the composition will comprise an amount of alginate oligomer and an amount of antibiotic that will provide a measurable reduction in the resistance (or measurable increase in susceptibility or measurable decrease in tolerance) to the antibiotic displayed by the bacterium e.g. an amount of alginate oligomer that will at least double, at least quadruple, at least octuple, at least sexdecuple or at least duotrigecuple the susceptibility of the MDR bacterium, to the antibiotic. Put in a different way, the composition will comprise an amount of alginate oligomer and an amount of antibiotic that will provide a measurable treatment of the infection being targeted. Preferably the composition or product will comprise sufficient alginate oligomer that upon administration to a subject or application to a location, the local concentration of the oligomer will be at least 2%, preferably at least 4%, 6% or 8% and most preferably at least 10% (weight by volume). The antibiotic preferably will be present in an amount that is sufficient to provide a local concentration of at least 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 64, 128, 256, 512, 1024, 2048 or 4096 μg/ml. The skilled man would know that the amounts of alginate oligomer and/or antibiotic can be reduced if a multiple dosing regime is followed or increased to minimise the number of administrations or applications.

(154) The compositions and products of this aspect will typically comprise between 1% and 99%, 5% and 95%, 10% and 90% or 25% and 75% alginate oligomer and 1% and 99%, 5 and 95%, 10% and 90% or 25% and 75% antibiotic, allowance being made for other ingredients.

(155) Parenterally administrable forms, e.g., intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should have low osmolarity to minimize irritation or other adverse effects upon administration and thus solutions should preferably be isotonic or slightly hypertonic, e.g. hypertonic salt water (saline). Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975). The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the biopolymers and which will not interfere with the manufacture, storage or use of products.

(156) For topical administration the alginate oligomer and/or the antibiotic can be incorporated into creams, ointments, gels, transdermal patches and the like. The alginate oligomers and/or the antibiotic can also be incorporated into medical dressings, for example wound dressings e.g. woven (e.g. fabric) dressings or non-woven dressings (e.g. gels or dressings with a gel component). The use of alginate polymers in dressings is known, and such dressings, or indeed any dressings, may further incorporate the alginate oligomers of the invention.

(157) Accordingly, in a further specific embodiment, the invention further provides a wound dressing comprising an alginate oligomer (which may be any alginate oligomer as herein defined) and/or an antibiotic (which may be any antibiotic as herein defined) for use, where appropriate, in the treatments and methods of the invention.

(158) Further topical systems that are envisaged to be suitable are in situ drug delivery systems, for example gels where solid, semi-solid, amorphous or liquid crystalline gel matrices are formed in situ and which may comprise the alginate oligomer and/or the antibiotic. Such matrices can conveniently be designed to control the release of the alginate oligomer and/or the antibiotic from the matrix, e.g. release can be delayed and/or sustained over a chosen period of time. Such systems may form gels only upon contact with biological tissues or fluids. Typically the gels are bioadhesive. Delivery to any body site that can retain or be adapted to retain the pre-gel composition can be targeted by such a delivery technique. Such systems are described in WO 2005/023176.

(159) For application to oral, buccal and dental surfaces, toothpastes, dental gels, dental foams and mouthwashes are mentioned specifically. Thus, in one particular aspect is included an oral health care, or oral hygiene, composition, comprising an alginate oligomer and an antibiotic (which may be any alginate oligomer or antibiotic as defined herein), particularly a mouthwash, toothpaste, dental gel or dental foam for use, where appropriate, in the treatments and methods of the invention.

(160) Inhalable compositions are also of note. The formulation of compositions suitable for inhalation is routine for the skilled man and has long been standard practice in the treatment of respiratory diseases. Inhalable compositions may, for instance, take the form of inhalable powders, solutions or suspensions. The skilled man would be able to select the most appropriate type of delivery system for his needs and be able to prepare a suitable formulation of the alginates and/or antibiotics of the invention for use in that system. Propellant-free nebulisable solutions and inhalable powder formulations are particularly preferred.

(161) As noted above, a preferred composition of the invention is a debridement composition that is used in a debridement process to remove a colony or population comprising an MDR bacterium, for example from a tissue. Typically such a composition will be liquid, but gels, gel-sols, or semi-solid compositions might be used. The composition might be used to debride the colony/population (e.g. by application to the tissue under pressure) and/or may be used to bathe the tissue before, during and/or after debridement by other means such as by surgical, mechanical or chemical processes. The skilled person is readily able to formulate debridement compositions in accordance with the invention.

(162) In the case of an MDR bacterium on an inanimate surface on in an inanimate material, the alginate oligomer and/or antibiotic may be applied to the surface or material to be treated in any convenient composition or formulation, or by any convenient means. Thus the alginate oligomer and/or antibiotic may be in liquid, gel, gel-sol, semi-solid or solid form (e.g. solutions, suspensions, homogenates, emulsions, pastes, powders, aerosols, vapours). Typically the compositions for treating such inanimate surfaces or materials will be a non-pharmaceutically acceptable composition. The choice of composition form will be dictated by the identity of the MDR bacterium on the surface or in the material and location of the surface or material. For instance, if the location is a fluid line it might be convenient to apply a fluid composition. It might also be preferred to use a composition that persists on the surface or in the part of the fluid line to be treated but that will not leach into the fluid of normal use, e.g. an adhesive gel. The skilled person is readily able to prepare suitable compositions from his common general knowledge. For instance, the alginate oligomer and/or antibiotic may be added to a paint formulation and applied to the surface to be treated, e.g. a boat hull or other part of a boat's structure that is exposed to water, or to a building or any part thereof, a tank (e.g. a storage or processing tank) or indeed to any part of any industrial machinery. Such compositions may conveniently also comprise a further anti-microbial agent, as described above, e.g. an antibiotic, chlorine bleach, TCP, ethanol, Virkon™, povidone-iodine, silver compounds, antimicrobial surfactants, etc. As the compositions need not be pharmaceutically acceptable, harsher antimicrobials can be used subject to considerations of surface damage, environmental contamination, user safety and contamination of the treated surface and interaction with the other components of the composition.

(163) The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the subject/surface by employing procedures well known in the art. Adhesive compositions are also preferred. Adhesive, sustained and/or delayed release formulations may be particularly convenient.

(164) In a further aspect the invention provides products susceptible to contamination/colonisation by MDR bacteria whose susceptible surfaces have been pretreated with an alginate oligomer and an antibiotic as defined herein.

(165) By “pretreated” it is meant that the susceptible surface is exposed to an alginate oligomer and/or an antibiotic prior to an exposure to an MDR bacterium and that the alginate oligomer and/or antibiotic persists on the surface for a duration sufficient to prevent contamination/colonisation by an MDR bacterium for an appreciable duration of time. Preferably the alginate oligomer and/or the antibiotic will persist for substantially the useful life of the surface, e.g. the pretreatment results in a substantially permanent coating of an alginate oligomer and/or an antibiotic. Thus a pre-treated surface/product is one to which the alginate olgimer and/or antibiotic is applied and on which it remains. Such a product/surface may be a coated product/surface.

(166) Non-limiting examples of products and surfaces susceptible to contamination/colonisation by MDR bacteria are described above. Particular mention may be made of medical devices (e.g. endotracheal or tracheostomy tubes) and food or drink processing, storage or dispensing equipment. Pretreatment can be achieved by any convenient means, for example any form of applying the alginate oligomer and/or antibiotic to the surface, notably coating the surface, e.g. spray drying, polymer coating with a polymer incorporating the alginate oligomer and/or antibiotic, and painting, varnishing or lacquering with paint, varnish or lacquer formulations containing the alginate oligomer and/or antibiotic. Such a “coating” composition (e.g. a paint, varnish or lacquer) containing an alginate oligomer and/or antibiotic represents a further aspect of the present invention. Alternatively, the alginate oligomer and/or antibiotic can be incorporated into the material from which the object or its susceptible parts are manufactured. This approach is suited to objects, or constituent parts thereof, manufactured from polymers such as plastics and silicones, e.g. the medical and surgical devices described above. Products comprising an inanimate surface comprising an alginate oligomer and/or antibiotic coating or coating composition, or incorporating an alginate oligomer and/or antibiotic are therefore contemplated. Non-limiting examples of such products and surfaces are described above. Of particular note are medical and surgical devices. This may include any kind of line, including catheters (e.g. central venous and urinary catheters), prosthetic devices e.g., heart valves, artificial joints, false teeth, dental crowns, dental caps and soft tissue implants (e.g. breast, buttock and lip implants). Any kind of implantable (or “in-dwelling”) medical device is included (e.g. stents, intrauterine devices, pacemakers, intubation tubes (e.g. endotracheal or tracheostomy tubes), prostheses or prosthetic devices, lines or catheters). Further products include food processing, storage, dispensing or preparation equipment or surfaces, tanks, conveyors, floors, drains, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, air conditioning conduits, cooling apparatus, food or drink dispensing lines, heat exchangers, boat hulls or any part of a boat's structure that is exposed to water, dental waterlines, oil drilling conduits, contact lenses and storage cases.

(167) The invention will be further described with reference to the following non-limiting Examples.

EXAMPLES

Example 1

Effect of G-Block Alginate Oligomers on the Minimum Inhibitory Concentrations of Various Antibiotics for Various Bacterial Strains

(168) Materials and Methods

(169) Bacterial Strains Used:

(170) PA01 Pseudomonas aeruginosa ATCC 15692 Pseudomonas aeruginosa ATCC 39324, mucoid type strain (R79)* Pseudomonas aeruginosa CFA 24-1, clinical mucoid strain (R80)* Pseudomonas aeruginosa MDR R22 from China (V1)* Pseudomonas aeruginosa MDR 301 from Poland (V2)* Klebsiella pneumoniae KP05 506 from India (V3)* Acinetobacter baumannii MDR ACB from Libya (V4)*

(171) *Non-official labels assigned for internal identification purposes only.

(172) Abbreviations used: Pseudomonas aeruginosa, (PA); Klebsiella pneumoniae (KP); Acinetobacter baumannii (ACB)

(173) Media and Bacterial Strains Used:

(174) Following retrieval from −80° C. storage, bacterial colonies were grown on blood agar with 5% sheep blood and were used to inoculate tryptone soya broth (TSB) for overnight growth. Antibiotics were diluted in cation-adjusted Mueller-Hinton broth (CAMHB) or CAMHB with G-fragments (Oligo CF-5/20 90-95% G residues) at 2%, 6% or 10%. Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich. OligoG CF-5/20 G-fragments were provided by Algipharma AS, Norway.

(175) Minimum Inhibitory Concentration assay (Jorgensen et al., Manual of Clinical Microbiology 7th ed. Washington, D.C.: American Society for Microbiology, 1999; 1526-43):

(176) Overnight bacterial cultures as described above were diluted in sterile water until the OD625 was between 0.08 and 0.10 to confirm that the cell density was equivalent to 0.5 McFarland standard.

(177) In experiments with single antibiotics, two-fold antibiotic serial dilutions were prepared in CAMHB or CAMHB supplemented G-fragments (Oligo CF-5/20 90-95% G residues) at 0%, 2%, 6% or 10% and were placed in duplicate wells of flat-bottom 96-well microtiter plates (100 μl in each well).

(178) In experiments with two antibiotics (ceftazidime and azithromycin or ciprofloxacin and azithromycin), two-fold antibiotic serial dilutions were prepared in CAMHB or CAMHB supplemented with azithromycin at either 1, 2, 4, or 8 μg/ml and G-fragments at either 0%, 2%, 6% or 10% and were placed in duplicate wells of flat-bottom 96-well microtiter plates (100 μl in each well).

(179) Bacterial cultures at 0.5 McFarland standard were diluted ten-fold in CAMHB and 5 μl added to the microtiter plates containing the antibiotic serial dilutions. Plates were wrapped in parafilm and incubated at 37° C. for 16-20 hours. MIC values for each antibiotic/antibiotic combination were determined as the lowest concentration at which there was no visible growth. Results are shown in Tables 1, 2 and 3.

(180) TABLE-US-00001 TABLE 1 Minimum inhibitory concentration (MICs) of different antibiotics for different Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii strains in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1). R79 R80 V1* V2* V3* V4* PA01 Mucoid Mucoid MDR R22 PA MDR 301 PA KP05 506 ACB Antibiotic PA PA PA (China) (Poland) (India) (Libya) Oxytetracycline 0G 8.sup.† 8.sup.† 8.sup.† +2% G 4.sup.† 4.sup.† 8 ND ND ND ND +6% G 4.sup.† 4.sup.† 4.sup.† +10% G  4.sup.† 2.sup.† 4.sup.† Azithromycin 0G 128.sup.† 128.sup.† 256.sup.† 64.sup.† 64.sup.† 32.sup.† 8.sup.† +2% G 64.sup.† 64.sup.† 128.sup.† 64 64 16.sup.† 2.sup.† +6% G 16.sup.† 16.sup.† 64.sup.† 64 32.sup.† 16.sup.† <0.25.sup.† +10% G  4.sup.† 4.sup.† 8.sup.† 32.sup.† 16.sup.† 8.sup.† <0.25.sup.† Ciprofloxacin 0G 0.125.sup.† 0.125.sup.† 1.sup.† 16.sup.† 16.sup.† 128 64.sup.† +2% G 0.0625.sup.† 0.0625.sup.† 0.125.sup.† 16 16 128 32.sup.† +6% G 0.0625.sup.† 0.03125.sup.† 0.125.sup.† 8.sup.† 8.sup.† 128 16.sup.† +10% G  0.03125.sup.† 0.03125.sup.† 0.125.sup.† 4.sup.† 8.sup.† 128 16.sup.† Primaxin 0G <1 <1 <1 128.sup.† 512.sup.† 32.sup.‡ <1 (Imipenem/ +2% G <1 <1 <1 128 256.sup.† 64.sup.‡ <1 cilastatin) +6% G <1 <1 <1 64.sup.† 256.sup.† 64.sup.‡ <1 +10% G  <1 <1 <1 32.sup.† 128.sup.† 64.sup.‡ <1 Meropenem 0G 2.sup.† <1 <1 32.sup.† 64.sup.‡ 64.sup.‡ <4 +2% G 2.sup.† <1 <1 32 64.sup.‡ 64.sup.‡ <4 +6% G <1.sup.† <1 <1 16.sup.† 128.sup.‡ 128.sup.‡ <4 +10% G  <1.sup.† <1 <1 4 128.sup.‡ 128.sup.‡ <4 Ceftazidime 0G <1 <1 <1 128.sup.† 32.sup.† >1024 512.sup.† +2% G <1 <1 <1 64.sup.† 16.sup.† >1024 512 +6% G <1 <1 <1 32.sup.† 8.sup.† >1024 512 +10% G  <1 <1 <1 8 4.sup.† >1024 256.sup.† Aztreonam 0G 8 2.sup.† <1 32.sup.† 64.sup.† 2048.sup.† 1024.sup.† +2% G 16.sup.‡ 2 <1 16.sup.† 16.sup.† 2048 512.sup.† +6% G 4.sup.† <1.sup.† <1 <4.sup.† 8.sup.† 512.sup.† 256.sup.† +10% G  2.sup.† <1.sup.† <1 <4.sup.† 8.sup.† 256.sup.† 128† .sup.†Indicates increasing MIC values with increase in G-fragment concentration .sup.‡Indicates decreasing MIC values with increase in G-fragment concentration

(181) TABLE-US-00002 TABLE 2 Minimum inhibitory concentration (MICs) of azithromycin for an MDR Acinetobacter baumannii and various strains of Pseudomonas aeruginosa and Klebsiella pneumoniae in the presence of varying concentrations of OligoCF-5/20 (0-10%). R79 R80 V1* V2* V3* V4* PA01 Mucoid Mucoid MDR R22 MDR 301 PA KP05 506 ACB Antibiotic PA PA PA PA (China) (Poland) (India) (Libya) Antibiotic 0G 128.sup.† 128.sup.† 256.sup.† 64.sup.† 64.sup.† 32.sup.† 8.sup.† +2% G 64.sup.† 64.sup.† 128.sup.† 64 64 16.sup.† 2.sup.† +6% G 16.sup.† 16.sup.† 64.sup.† 64 32.sup.† 16.sup.† <0.25.sup.† +10% G  4.sup.† 4.sup.† 8.sup.† 32.sup.† 16.sup.† 8.sup.† <0.25† .sup.†Indicates increasing MIC values with increase in G-fragment concentration .sup.‡Indicates decreasing MIC values with increase in G-fragment concentration

(182) TABLE-US-00003 TABLE 3 Minimum inhibitory concentrations (MICs) of two antibiotics in combination with each other (azithromycin with either ceftazidime or ciprofloxacin) for multi drug resistant (MDR) strains of Pseudomonas aeruginosa and Acinetobacter baumannii in the presence of varying concentrations of OligoCF-5/20 (0-10%) (MIC values are expressed in μg ml.sup.−1). V1* V4* MDR R22 PA ACB Antibiotic (China) (Libya) Ceftazidime with 0G 256.sup.† 512.sup.† azithromycin at 8 μg/ml +2% G 128.sup.† <8 μg/ml Az.sup.† +6% G 32v <8 μg/ml Az.sup.† +10% G  16.sup.† <8 μg/ml Az.sup.† Ceftazidime with 0G 128.sup.† 1024.sup.† azithromycin at 4 μg/ml +2% G 128 <4 μg/ml Az.sup.† +6% G 64.sup.† <4 μg/ml Az' +10% G  8.sup.† <4 μg/ml Az.sup.† Ceftazidime with 0G 128.sup.† 1024.sup.† azithromycin at 2 μg/ml +2% G 64.sup.† 256.sup.† +6% G 32.sup.† 2.sup.† +10% G  16.sup.† <1 Cf.sup.† Ceftazidime with 0G 128.sup.† 1024.sup.† azithromycin at 1 μg/ml +2% G 64.sup.† 512.sup.† +6% G 16.sup.† 128.sup.† +10% G  16.sup.† <1 Cf.sup.† Ciprofloxacin with 0G 16' 128 azithromycin at 8 μg/ml +2% G 16 <8 μg/ml Az.sup.† +6% G 16 <8 μg/ml Az.sup.† +10% G  <8 μg/ml Az.sup.† <8 μg/ml Az.sup.† Ciprofloxacin with 0G 16.sup.† 128.sup.† azithromycin at 4 μg/ml +2% G 16  64 Cpr, 4 Az.sup.† +6% G 8.sup.† <4 μg/ml Az.sup.† +10% G  4.sup.† <4 μg/ml Az.sup.† Ciprofloxacin with 0G 32.sup.† 128.sup.† azithromycin at 2 μg/ml +2% G 16.sup.† <0.25 Cpr 2Az.sup.† +6% G 16.sup.† <0.25 Cpr.sup.† +10% G  8.sup.† <0.25 Cpr.sup.† Ciprofloxacin with 0G 16.sup.† 64.sup.† azithromycin at 1 μg/ml +2% G 16 32.sup.† +6% G 8.sup.† <0.25 Cpr.sup.† +10% G  8.sup.† <0.25 Cpr† .sup.†Indicates increasing MIC values with increase in G-fragment concentration .sup.‡Indicates decreasing MIC values with increase in G-fragment concentration
Results and Discussion

(183) In general, treatment of planktonically growing MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii strains with increasing concentrations of OligoG CF-5/20 lowered the MIC values of the antibiotics used (Tables 1, and 2). Oxytetracycline, azithromycin and ciprofloxacin were all shown to have decreasing MICs with increasing amounts of OligoG CF-5/20 used. Thus, in the case of these antibiotics, the data appear to show that alginate oligomers may potentiate their effects. The antibiotics tested include antibiotics common in the treatment of cystic fibrosis.

(184) The magnitude of the effect was most pronounced for the MDR strain Pseudomonas aeruginosa strain R22, although all strains studied responded to treatment with the alginate oligomers and azithromycin. The results also show alginate oligomers potentiate the antibiotic azithromycin with all strains tested. Such an effect may be seen with azithromycin alone or in combination with other antibiotics.

(185) More specifically, for the MDR Pseudomonas strains, primaxin (a combination of imipenem and cilastatin), azithromycin, ceftazidime, ciprofloxacin and aztreonam were all more effective when used in combination with the alginate oligomers. Two antibiotics in conjunction with alginate oligomers were more effective against KP05 506, namely, azithromycin and aztreonam, but the data from experiments using primaxin and meropenem is inconclusive. In combination with alginate oligomers, azithromycin, ceftazidime, ciprofloxacin and aztreonam showed a more positive effect on the Acinetobacter baumannii isolate.

(186) The effects of azithromycin in conjunction with either ceftazidime or ciprofloxacin in the presence of alginate oligomers on the MDR R22 PA strain and the MDR Acinetobacter baumannii isolate were tested and the results can be seen in Table 3. In all cases MIC values of the ceftazidime or ciprofloxacin in the antibiotic combinations were reduced by various concentrations of alginate oligomer.

Example 2

(187) The study described in Example 1 was repeated with the following strains of bacteria and antibiotics as detailed in Tables 4, 5 and 6.

(188) Bacterial Strains PA01 Pseudomonas aeruginosa ATCC 15692 (E77) R79* Mucoid Pseudomonas aeruginosa ATCC 39324 ISOLATION: sputum from a cystic fibrosis patient, Boston, Mass. R80* Mucoid Pseudomonas aeruginosa CFA 24-1 (CLINICAL ISOLATE from a CF patient) V1* R22 PSA (China) Pseudomonas aeruginosa V2* MDR 301 PSA (Poland) Pseudomonas aeruginosa V3* KP05 506 (India) Klebsiella pneumoniae V4* MDR ACB (Libya) Acinetobacter baumannii V5* AIM-1 E. coli V9* (Egypt) Acinetobacter baumannii V10* (Egypt) Acinetobacter lwoffii V11* 5702 (Wales) E. coli V12* 5725 (Wales) Klebsiella pneumoniae V22* 6056 Acinetobacter V23* 1322 Burkholderia cepacia
*Non-official labels assigned for internal identification purposes only.

(189) TABLE-US-00004 TABLE 4 Minimum inhibitory concentration (MICs) of different macrolide antibiotics for various strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and E. coli displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1). Strain R79 R80 PA01 non non V1 V2 V3 V4 non MDR MDR MDR MDR MDR MDR Antibiotic and MDR Pseud Pseud Pseud Pseud Kleb Acin V5 MIC value Pseud aerug aerug aerug aerug pneum baum MDR μg/ml % G aerug (muc.) (muc.) (China) (Pol.) (India) (Libya) E. coli Erythromycin 0G 128.sup.† 128.sup.† 512.sup.† 128.sup.† 128.sup.† 1024 8.sup.† 4.sup.† +2% G 64.sup.† 64.sup.† 512 128 128 1024 2.sup.† 4 +6% G 64.sup.† 32.sup.† 128.sup.† 64.sup.† 64.sup.† 1024 ≦1.sup.† ≦1.sup.† +10% G  16.sup.† 2.sup.† 32.sup.† 32.sup.† 16.sup.† 1024 ≦1.sup.† ≦1.sup.† Clarithromycin 0G 256.sup.† 256.sup.† 1024.sup.† 256.sup.† 512.sup.† 256.sup.† 8.sup.† 4.sup.† +2% G 128.sup.† 128.sup.† 512.sup.† 128.sup.† 256.sup.† 256 4.sup.† 4 +6% G 64.sup.† 32.sup.† 256.sup.† 64.sup.† 128.sup.† 256 ≦1.sup.† ≦1.sup.† +10% G  32.sup.† 4.sup.† 64.sup.† 32.sup.† 32.sup.† 128.sup.† ≦1.sup.† ≦1.sup.† Spiramycin 0G >1024.sup.† >1024.sup.† >1024.sup.† >1024.sup.† >1024.sup.† 1024.sup.† 512.sup.† 32.sup.† +2% G >1024 >1024 >1024 >1024 >1024 1024 64.sup.† 32 +6% G 1024.sup.† 1024.sup.† 1024.sup.† 1024.sup.† >1024 1024 64.sup.† 16.sup.† +10% G  512.sup.† 256.sup.† 1024.sup.† 512.sup.† 1024.sup.† 512.sup.† 32.sup.† 8† .sup.†Indicates decreasing MIC values with increase in G-fragment concentration .sup.‡Indicates increasing MIC values with increase in G-fragment concentration

(190) TABLE-US-00005 TABLE 5 Minimum inhibitory concentration (MICs) of different antibiotics for strains of Burkholderia cepacia, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter Iwoffii and E. coli displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1). V23 V22 V9 V10 V12 MDR MDR MDR MDR V11 MDR Antibiotic and Burk Acin. Acin. Acin. MDR Kleb MIC value μg/ml % G cep Iwoff baum Iwoff E.coli pneu Oxytetracycline 0G 256.sup.† 2.sup.‡ 2.sup.‡ 0.5.sup.† 0.5.sup.‡ 1 +2% G 128.sup.† 2 2 0.5 1.sup.‡ 1 +6% G 128.sup.† 2 1.sup.† 0.5 0.5 1 +10% G  32.sup.† 8.sup.‡ 4.sup.‡ 0.25.sup.† 0.5 1 AZACTAM 0G >512.sup.† 256.sup.† >512.sup.† 32.sup.† 256 256.sup.† (Aztreonam) +2% G >512 128.sup.† 512.sup.† 16.sup.† 256 512.sup.‡ +6% G 512.sup.† 64.sup.† 256.sup.† 4.sup.† 256 128.sup.† +10% G  128.sup.† 32.sup.† 128.sup.† 1.sup.† 256 64.sup.† Ciprofloxacin 0G 64.sup.† <0.25 -64.sup.† 0.5.sup.† 128.sup.† 64.sup.‡ +2% G 64 <0.25 32.sup.† 0.5 64.sup.† 64 +6% G 64 <0.25 32.sup.† 0.25.sup.† 128 256.sup.‡ +10% G  32.sup.† <0.25 32.sup.† 0.25.sup.† 128 256.sup.‡ PRIMAXIN 0G 32.sup.† 8.sup.† 1.sup.† <0.5 <0.5 <0.5 (Imipenem/ +2% G 32 8 2 <0.5 <0.5 <0.5 Cilastatin +6% G 32 8 <0.5.sup.† <0.5 <0.5 <0.5 +10% G  8.sup.† 4.sup.† <0.5.sup.† <0.5 <0.5 <0.5 Meropenem 0G 64.sup.† 256.sup.† 16.sup.† 1.sup.† <0.25 <0.25 +2% G 64 128.sup.† 8.sup.† 0.5.sup.† <0.25 <0.25 +6% G 32.sup.† 128.sup.† 8.sup.† <0.25.sup.† <0.25 <0.25 +10% G  8.sup.† 64.sup.† 4.sup.† <0.25.sup.† <0.25 <0.25 Ceftazidime 0G 64.sup.† 16.sup.† >512.sup.† 2.sup.† 128.sup.† 64.sup.† +2% G 32.sup.† 16 512.sup.† 2 64.sup.† 64 +6% G 8 4.sup.† 512.sup.† <0.5.sup.† 32.sup.† 16.sup.† +10% G  <0.5.sup.† 2.sup.† 256.sup.† <0.5.sup.† 64.sup.† 16.sup.† Azithromycin 0G 128.sup.† <0.25 16.sup.† <0.25 32.sup.† 32.sup.† +2% G 64.sup.† <0.25 4.sup.† <0.25 16.sup.† 8 +6% G 16.sup.† <0.25 0.5.sup.† <0.25 16.sup.† 32 +10% G  4 <0.25 <0.25 <0.25 32 32 Erythromycin 0G 512.sup.† <0.5 8.sup.† <0.5 512.sup.† 512.sup.† +2% G 256.sup.† <0.5 4.sup.† <0.5 256.sup.† 256.sup.† +6% G 128.sup.† <0.5 1.sup.† <0.5 256.sup.† 512 +10% G  16.sup.† <0.5 <0.5.sup.† <0.5 256.sup.† 512 Clarithromycin 0G 512.sup.† — 16.sup.† <0.5 512.sup.† 512.sup.† +2% G 256.sup.† — 4.sup.† <0 5 128.sup.† 256.sup.† +6% G 128.sup.† — 2.sup.† <0.5 256.sup.† 256.sup.† +10% G  32.sup.† — 1.sup.† <0.5 256.sup.† 512 Spiramycin 0G >4096.sup.† <4 256.sup.† <4 128.sup.† 67.sup.† +2% G 2048.sup.† <4 64.sup.† <4 64.sup.† 64 +6% G 2048.sup.† <4 32.sup.† <4 32.sup.† 32.sup.† +10% G  1024.sup.† <4 16.sup.† <4 32.sup.† 16† †Indicates decreasing MIC values with increase in G-fragment concentration .sup.‡Indicates increasing MIC values with increase in G-fragment concentration

(191) TABLE-US-00006 TABLE 6 Minimum inhibitory concentration (MICs) of different antibiotics for a strain (V23) of Burkholderia cepacia in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1). Results from three separate experiments. Strain Experiment 1 Experiment 2 Experiment 3 Antibiotic and V23 V23 V23 MIC value μg/ml % G (18-03-10) (18-03-10) (19-03-10) Oxytetracycline 0G >256.sup.† >256.sup.† >256.sup.† +2% G 256.sup.† >256 256.sup.† +6% G 256.sup.† 256.sup.† 256.sup.† +10% G  128.sup.† 128.sup.† 256.sup.† AZACTAM 0G >4096.sup.† >4096.sup.† >4096.sup.† (Aztreonam) +2% G >4096 >4096 >4096 +6% G 1024.sup.† 1024.sup.† 1024.sup.† +10% G  256.sup.† 512.sup.† 128.sup.† PRIMAXIN 0G 128.sup.† 256.sup.† 256.sup.† (Imipenem/ +2% G 128 128.sup.† 256 Cilastatin +6% G 64.sup.† 128.sup.† 256 +10% G  32.sup.† 64.sup.† 128.sup.† Meropenem 0G 128.sup.† 128 128.sup.† +2% G 128 128 64.sup.† +6% G 64.sup.† 128 128 +10% G  64.sup.† 128 64.sup.† Ceftazidime 0G 128.sup.† 64.sup.† 64.sup.† +2% G 64.sup.† 64 32.sup.† +6% G 16.sup.† 32.sup.† 16.sup.† +10% G  8.sup.† 32.sup.† 4.sup.† Azithromycin 0G 64.sup.† 32.sup.† 16 +2% G 64 32 16 +6% G 64 16.sup.† 16 +10% G  32.sup.† 16.sup.† 16 Erythromycin 0G 512.sup.† 256.sup.† >512.sup.† +2% G 256.sup.† 256 128.sup.† +6% G 128.sup.† 64.sup.† 64.sup.† +10% G  64.sup.† 64.sup.† 16.sup.† Clarithromycin 0G 256.sup.† 128.sup.† 64.sup.‡ +2% G 256 128 512.sup.‡ +6% G 128.sup.† 32.sup.† 512.sup.‡ +10% G  128.sup.† 16.sup.† 512‡ .sup.†Indicates decreasing MIC values with increase in G-fragment concentration .sup.‡Indicates increasing MIC values with increase in G-fragment concentration

(192) In general, Table 4 validates the results disclosed in Tables 1 and 2 in relation to the effects of OligoG CF-5/20 on the MIC's of macrolide antibiotics in a variety of planktonically growing bacteria. In virtually every combination of bacteria and macrolide, MIC values are reduced by increasing concentrations of OligoG CF-5/20. The results also show alginate oligomers potentiate the effects of the macrolide antibiotics with all bacteria tested. Such an effect may be seen with azithromycin alone or in combination with other antibiotics.

(193) From data presented in Tables 4, 5 and 6 it can been seen that in general increasing concentrations of OligoG CF-5/20 lowered the MIC values of the antibiotics used against MDR strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Burkholderia cepacia, Acinetobacter lwoffii, Acinetobacter baumannii and E. coli. The antibiotics tested include antibiotics common in the treatment of cystic fibrosis. Aztreonam, primaxin (a combination of imipenem and cilastatin), ciprofloxacin, meropenem, ceftazidime, azithromycin, erythromycin, clarithromycin, and spiramycin were all shown to have decreasing MICs with increasing amounts of OligoG CF-5/20 used. Thus, in the case of these antibiotics, the data appear to show that alginate oligomers may potentiate their effects. The macrolides display the greatest reduction in MICs with increasing amounts of OligoG CF-5/20 used. The magnitude of the effect was most pronounced for the Burkholderia tested and Acinetobacter baumannii strain V9 and in these strains every antibiotic tested showed a reduction in MIC values with increasing concentrations of alginate oligomer.

(194) Table 6 further validates the results with Burkholderia presented in Table 5. This antibiotic potentiating effect seen with alginate oligomers and Burkholderia is of clinical significance as these organisms are associated with human and animal disease and are difficult to treat on account of their tendency to display antibiotic resistance.

Example 3

(195) The study described in Example 1 was repeated with the following strains of Acinetobacter baumannii, antibiotics and M-block alginate oligomer in place of OligoG CF-5/20 as detailed in Table 7. The M-block oligomer is 100% M with a DPn of 15 to 18,

(196) TABLE-US-00007 TABLE 7 Minimum inhibitory concentration (MICs) of different antibiotics for a strain of Acinetobacter baumannii displaying an MDR phenotype and a strain of Acinetobacter baumannii displaying an non-MDR phenotype in the presence of varying concentrations of M-block oligomer (0-10%). (MIC values are expressed in μg ml.sup.−1). Strain V4 V19 MDR non Antibiotic and MIC value μg/ml Acin baum MDR concentration M block (Libya) Acin. baum Aztreonam 0M 2048.sup.† 64.sup.† +2% M 512.sup.† 32.sup.† +6% M 256.sup.† 8.sup.† +10% M  64.sup.† 8.sup.† Ciprofloxacin 0M 64.sup.‡ 64.sup.† +2% M 64 32.sup.† +6% M 64 16.sup.† +10% M  128.sup.‡ 128.sup.‡ Meropenem 0M 16.sup.† 8.sup.† +2% M 32.sup.‡ 4.sup.† +6% M 16 2.sup.† +10% M  8.sup.† 1.sup.† Azithromycin 0M 8.sup.† 32.sup.† +2% M 8 16.sup.† +6% M 8 16.sup.† +10% M  2.sup.† 16† .sup.†Indicates decreasing MIC values with increase in G-fragment concentration .sup.‡Indicates increasing MIC values with increase in G-fragment concentration

(197) The results displayed in Table 7 show that M-block oligomers are, like OligoG CF-5/20, also effective in lowering MIC values for a number of different antibiotics (including a macrolide) in MDR and non-MDR strains of Acinetobacter baumannii.

Example 4

(198) Further MIC assays were conducted with the various strains and antibiotics recited in Tables 8 to 11 using the following protocol.

(199) MIC-Assay

(200) G-block alginates (OligoG CF-5/20) were dissolved in Mueller-Hinton broth (Lab M limited, LAB114 Mueller-Hinton broth) to 1.25 times of the desired assay concentrations (2, 6 and 10%). Antibiotics were dissolved in Mueller-Hinton broth and Mueller-Hinton broth with G-block alginate at a concentration of 1.25 times the highest desired assay concentrations. Antibiotics were pharmaceutical grade purchased from Sigma-Aldrich. OligoG CF-5/20 G-fragments were provided by Algipharma AS, Norway.

(201) Two-fold serial dilutions of antibiotics were made in Mueller-Hinton with different concentrations of G-block alginate, and the solutions were placed in four parallel wells in Nunc 384-well micro plates (30 μl per well in Nunc 242757 microplates). A group of 8 wells with no addition of antibiotics for each G-block concentration was included on each micro plate as growth reference.

(202) Frozen stock cultures were made from over night cultures in TSB-broth for all strains by addition of glycerol to 15% concentration prior to freezing at −80° C. At the day of analysis, overnight TSB cultures (6 ml in 50 ml tube tilted to 45-degrees angle, 200 rpm, 2.5 cm amplitude, 37° C.) were diluted in TSB until the OD600 was 0.10, and further diluted 1:40 in Mueller-Hinton broth. Each well in the 384-well assay plates was inoculated with 7.5 μl of the diluted culture. The microplates were placed in plastic bags and incubated at 37° C. The optical density at 600 nm in the microwells was measured after approximately 18 hours of incubation, and the relative growth yield in each well was calculated based on the growth in the reference groups. The MIC value was set to the highest concentration giving less than 30% growth in all 4 parallel wells within the sample groups. The microplates were further incubated for 8 hours, and optical density in the cultures was measured once more for confirmation of the estimated MIC-values.

(203) Results

(204) In each of Tables 8 to 11 there is a main table of basic data, and a secondary table which is a representation of the overall effect of the OligoCF-5/20 on the MIC value for each particular bacteria and antibiotic combination. In the secondary table a dark shaded box represents an overall reduction in the MIC value; a hatched boxed represents an overall increase in the MIC value; M indicates that all of the MIC values were greater than the maximum concentration of antibiotic used; L indicates that all of the MIC values were less than the minimum concentration of antibiotic used; NE indicates no effect on the MIC values was observed; ND indicates that the particular combination of antibiotic and bacteria was not tested.

(205) Table 8.

(206) Minimum inhibitory concentration (MICs) of different antibiotics for strains of Burkholderia cepacia and Pseudomonas aeruginosa displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1).

(207) Table 9.

(208) Minimum inhibitory concentration (MICs) of different antibiotics for strains of Acinetobacter baumannii and Acinetobacter lwoffii displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1).

(209) Table 10.

(210) Minimum inhibitory concentration (MICs) of different antibiotics for strains of Klebsiella pneumoniae displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1).

(211) Table 11.

(212) Minimum inhibitory concentration (MICs) of different antibiotics for strains of E. coli and Providencia stuartii displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1).

(213) Table 12.

(214) Minimum inhibitory concentration (MICs) of different antibiotics for strains of Streptococcus oralis and Staphylococcus aureus (MRSA) displaying MDR phenotypes in the presence of varying concentrations of OligoCF-5/20 (0-10%). (MIC values are expressed in μg ml.sup.−1).

(215) TABLE-US-00008 TABLE 8 G- Azithro- Erythro- Roxithro- Dirithro- Aztre- Cipro- Oxytetra- Strain block mycin mycin mycin mycin onam Ceftazidime Imipenem floxacin cycline Pseudomonas aeruginosa  0% 128 >512 >512 >512 0.125 0.5 256 1 16 (MDR 301, PSA, V2)  2% 64 >512 >512 >512 0.125 0.125 256 0.5 16 MDR  6% 64 512 >512 512 0.125 0.25 128 0.5 8 10% 32 512 >512 512 0.0625 0.125 128 0.25 8 Pseudomonas aeruginosa  0% 128 >512 >512 >512 0.125 >16 128 >8 >128 (R22, PSA, V1)  2% 32 512 >512 >512 0.125 >16 128 >8 >128 MDR  6% 32 512 512 512 0.0625 >16 64 >8 >128 10% 8 256 256 256 0.03125 >16 16 >8 >128 Burkholderia cepacia  0% 64 256 512 256 >16 >16 >16 >8 128 (1322, V23)  2% 32 128 512 256 >16 >16 16 >8 128 MDR  6% 32 128 256 128 >16 16 16 >8 64 10% 8 64 64 64 >16 16 16 >8 64 Burkholderia cepacia  0% 64 128 256 512 >16 >16 >16 >8 32 (LMG18941, ATCC-BAA-246)  2% 32 128 256 256 >16 16 >16 >8 16 MDR  6% 16 64 256 256 >16 16 >16 >8 32 10% 16 64 128 256 >16 16 >16 >8 32 Pseudomonas aeruginosa (V2) M Pseudomonas aeruginosa (V1) M M M Burkholderia cepacia (V23) M M Burkholderia cepacia M M M NE (LMG18941)

(216) TABLE-US-00009 TABLE 9 G- Azithro- Erythro- Roxithro- Dirithro- Cefta- Cipro- Oxytetra- Strain block mycin mycin mycin mycin Aztreonam zidime Imipenem floxacin cycline Acinetobacter Iwoffii (6056, V22)  0% <2 <2 4 <2 64 8 2 <2 <2 MDR  2% <2 <2 <2 <2 32 4 2 <2 <2  6% <2 <2 <2 <2 16 2 2 <2 <2 10% <2 <2 <2 <2 4 2 <2 <2 <2 Acinetobacter baumannii (Egypt, V9)  0% 4 8 8 4 >16 >16 2 >8 1 MDR  2% <1 2 4 <1 >16 >16 <2 >8 0.5  6% <1 1 4 <1 >16 >16 2 8 1 10% <1 <1 2 <1 >16 >16 <2 8 0.5 Acinetobacter baumannii  0% 16 32 128 64 4 16 2 4 2 (MDR ACB, Libya, V4)  2% 2 8 32 8 4 8 2 1 2  6% <1 4 16 2 1 8 2 1 2 10% <1 2 16 <1 0.5 8 <2 0.5 1 Acinetobacter Iwoffii (Egypt, V10)  0% <1 1 4 <1 8 2 <2 0.063 0.5 MDR  2% <1 <1 <1 <1 4 1 <2 0.031 0.5  6% <1 <1 <1 <1 2 0.25 <2 0.031 0.5 10% <1 <1 <1 <1 1 0.25 <2 0.031 0.5 Acinetobacter Iwoffii (V22) L L L L L Acinetobacter baumannii (V9) M M NE NE Acinetobacter baumannii (V4) Acinetobacter Iwoffii (V10) L L L L NE

(217) TABLE-US-00010 TABLE 10 G- Azithro- Erythro- Roxithro- Dirithro- Cefta- Cipro- Oxytetra- Strain block mycin mycin mycin mycin Aztreonam zidime Imipenem floxacin cycline Klebsiella pneumoniae  0% 128 512 1024 1024 512 >1024 <2 128 2 (IR25, India, V6)  2% 64 512 >1024 1024 256 1024 2 64 2 MDR  6% 32 256 1024 512 128 >1024 2 64 2 10% 16 256 >1024 256 64 1024 2 64 2 Klebsiella pneumoniae  0% 16 512 256 128 4 >16 <2 >8 1 (5712, Wales, V12)  2% 16 256 512 64 4 8 <2 >8 1 MDR  6% 16 256 512 64 4 >16 <2 >8 1 10% 16 256 512 32 4 >16 <2 >8 1 Klebsiella pneumoniae  0% 16 128 512 256 512 >1024 8 32 512 (K3, India, V8)  2% 8 128 256 128 256 >1024 16 32 512 MDR  6% 16 128 >1024 128 128 >1024 16 >1024 512 10% 16 128 >1024 128 128 >1024 8 >1024 1024 Klebsiella pneumoniae (V6) M M NE Klebsiella pneumoniae NE NE M L M NE (V12) Klebsiella pneumoniae (V8) NE NE M M NE M

(218) TABLE-US-00011 TABLE 11 G- Azithro- Erythro- Roxithro- Dirithro- Aztre- Cefta- Cipro- Oxytetra- Strain block mycin mycin mycin mycin onam zidime Imipenem floxacin cycline Escherichia coli (5702, Wales, V11)  0% 16 512 512 128 256 >16 <2 >8 0.5 MDR  2% 16 256 128 64 256 >16 <2 >8 0.5  6% 8 256 256 32 128 >16 <2 >8 0.5 10% 16 256 512 64 64 >16 <2 >8 0.25 Providencia stuartii  0% <2 32 64 8 >1024 >1024 16 128 128 (IR57 India, V7)  2% <2 16 32 4 >1024 >1024 16 128 128 MDR  6% <2 16 32 4 >1024 >1024 16 128 128 10% <2 16 32 2 >1024 >1024 8 128 128 Escherichia coli (V11) NE NE M L M Providencia stuartii (V7) L M M NE NE

(219) TABLE-US-00012 TABLE 12 G- Azithro- Erythro- Roxithro- Dirithro- Aztre- Cefta- Cipro- Oxytetra- Strain block mycin mycin mycin mycin onam zidime Imipenem floxacin cycline Streptococcus oralis  0% 2 8 4 8 <0.03125 32 <0.25 (5610, V17)   MDR  2% 16 8 4 4 16 <0.0625 <0.25  6% 0.03125 0.03125 <0.03125 0.03125 1 2 0.5 10% 0.0625 16 0.03125 1 <0.03125 2 <0.25 MRSA 1040s, U50  0% 512 >1024 >1024 1024 1024 >16 0.0625 32 2 MDR  2% 256 >1024 1024 512 1024 16 0.0625 32 2  6% 256 1024 512 512 512 8 0.03125 >1024 1 10% 256 512 256 256 512 2 <0.03125 >1024 0.5 Streptococcus oralis ND ND NE (5610, V17) MRSA 1040s, U50 M

(220) The data presented in Tables 8 to 12 generally show that increasing concentrations of OligoCF-5/20 (0-10%) decreases MIC values for all antibiotics tested (azithromycin, erythromycin, roxithromycin, dirithromycin (macrolides) aztreonam (monobactam) ceftazidime (cephalosporin) imipenem (carbapenem), ciprofloxacin (quinolone) and oxytetracycline (tetracycline)) in one bacterial strain or another). Notably, Table 12 shows that OligoCF-5/20 reduces MIC values in Gram positive organisms (MRSA U50 and Streptococcus oralis). The effect is particularly pronounced with the MRSA strain tested. This highlights the general applicability of the use of alginate oligomers alongside antibiotics in the treatment of all MDR bacteria (whether Gram negative, Gram positive, or Gram test non-responsive) e.g. by overcoming the resistance of MDR bacteria to antibiotic treatments or enhancing the efficacy of those antibiotics.

(221) The effect was most consistently observed across the antibiotics tested in the Pseudomonas, Acinetobacter, Burkholderia and MRSA species tested, and strains V1, V2, V23, V4 and V9 in particular. Interestingly, in this Example strains V23 and V9 showed five instances of NE (no effect) or M (MICs were above the maximum concentration of antibiotic used), however in Example 2, data shows that these five combinations of bacteria and antibiotic do in fact display reductions in MIC with increasing concentrations of OligoCF-5/20. This highlights the more specific applicability of the use of alginate oligomers alongside antibiotics in the treatment of MDR Pseudomonas, Acinetobacter, Burkholderia and MRSA, e.g. by overcoming the resistance these bacteria have to antibiotic treatments or enhancing the efficacy of those antibiotics against these bacteria in particular.

(222) The effect is most consistently observed across the strains tested with the macrolides (azithromycin, erythromycin, roxithromycin, dirithromycin) and to a slightly lesser extent aztreonam, ceftazidime and ciprofloxacin. This highlights the more specific applicability of alginate oligomers to the treatment of bacteria in general, including MDR bacteria with macrolides (e.g. azithromycin, erythromycin, roxithromycin, dirithromycin) in particular, but also quinolones (e.g. ciprofloxacin), monobactams (e.g. aztreonam) and cephalosporins (e.g. ceftazidime), e.g. by overcoming the resistance in bacteria to these antibiotic treatments or by enhancing the efficacy of these antibiotics against bacteria.

(223) Also of significant note is the evidence provided in Table 11 that shows that OligoCF-5/20 can lower MIC values for a β-lactam (imipenem, a carbapenem) in MDR Providencia stuartii, β-lactam resistance in Providencia populations is rising and so alginate oligomers may represent a new approach to the treatment of Providencia infections