Patent Publication Number: US-4581227-A

Title: Control of microflora

Description:
The present invention relates to a method of removing undesired micro-organisms, including those of pathogenic nature, attached to surfaces, phase boundaries and interfaces. The invention also relates to a method of effecting an adhesion-stimulating result for desired micro-organisms by administering adhesion-stimulating substances. The invention also relates to preparations for achieving the abovementioned results. 
     In the following the invention will be described first as applied to the care of livestock, other applications being dealt with afterwards. 
     The present invention can be used to prevent the establishment of pathogenic bacteria flora and to eliminate such flora already established in pigs, calves and poultry. By &#34;poultry&#34; is meant chickens, turkeys, geese and ducks, for instance. 
     The mortality rate of such animals is at present high as a result of the establishment and colonization of pathogenic bacteria in the stomach and intestines. Pathogenic bacteria oust the normal bacteria flora, adhere to the bonding seats on the walls of the intestines, give rise to disease symptoms such as diarrhoea and result in increased mortality. Some farms lose large numbers of animals following an outbreak of pathogenic bacteria such as Escherichia coli, Salmonella typhimurium, Salmonella Sp., Shigella sp. and Clostridium perfringens. 
     Current methods of reducing the risk of outbreaks of the type mentioned above can be divided into two categories: 
     1. Treatment with antibiotics 
     2. Oral administration of non-pathogenic bacteria in pure or mixed cultures 
     There are a number of drawbacks in treating animals with antibiotics, which makes the method less attractive. 
     Examples are: 
     Non-selectivity, which also causes a reduction in the natural intestinal flora; 
     High cost; 
     The appearance of resistant strains; 
     It is time-consuming and impractical when the farm worker cannot be responsible for all steps of the treatment himself; 
     Legislative control of treatment with antibiotics. 
     It has been impossible to follow up successful laboratory experiments with oral administration of non-pathogenic bacteria resulting in reduced risk of diarrhoea in pigs, under full-scale conditions. Careful tests with commercial products containing, e.g. Lactobacillus acidophilus and Lactobacillus bulgaricus have not given significant positive results. Neither has it been possible to colonize the intestines of pigs with Streptococcus faecium. It has not been possible to establish Lactobacillus acidophilus in chicken intestines and there have been no successful large-scale experiments with oral administration to chickens. 
     More recent experiments with plasmid-carrying strains to increase adhesion appear to be futile due to instability of the plasmids and difficulties in maintaining suitable selection pressure for their preservation. 
     Initial studies of the adhesion of bacteria to the intestinal walls revealed surprisingly that oral administration is anyway an extremely successful method of preventing the establishment, colonization and growth of pathogenic bacteria in the stomach and intestines of pigs, calves and poultry. 
     The following points comprise the basis of the process forming the grounds of the present invention is based: 
     1. The stomach and intestines is rendered substantially bacteria-free by treating with anti-microbial substances or substances which adhere to the walls and block the adhesion of bacteria. 
     2. Substances are used which increase the degree of adhesion of the orally administered non-pathogenic bacteria. 
     3. Non-pathogenic enterobacteria are cultivated in a special way to produce specific protein-protrusions, fimbria, giving rise to greatly increased adhesion of bacteria to the bacteria surface of intestinal walls. 
     Substances which may be used to remove bacteria from surfaces include erythrosine, anti-oxidants, and organic or inorganic salts. Among the salts which can be used are magnesium, aluminum, ammonium, beryllium, lithium and calcium salts. The various bacteria removing substances may be used alone or in combination. 
     Substances which may be used to increase the degree of adhesion of the non-pathegenic bacteria include curcumin and its derivatives. Curcumin also known as 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is the coloring matter of tumeric, the rhizome of curcuma longa. Curcumin has the following structural formula: ##STR1## 
     The derivatives of curcumin which may be used as adhesion promoting substances include those of the following structural formula: ##STR2## Where N=1,2,3 . . . , X=1,2,3,4 . . . , and R 1  -R 5  may be same or different, and may be for example, H, OH, OCH 3 , OCH 2  CH 3 , CH 3 , CH 2  CH 3 , COOH, or CH 2  COOH. 
     The curcumin may be administered in its pure form, or may be administered in the form of oleo-resins and/or cleaned fractions of a plant extract containing curcumin, which also includes turmeric acid. 
    
    
     The invention is further illustrated with reference to the following non-limiting examples of its application: 
     EXAMPLE 1 
     RELATING TO PIGS AND CALVES 
     A. Erythrosine, anti-oxidants or MgSO 4  were mixed into the fodder or drinking water in concentrations expressed in g/kg bodyweight and day 0.06, 1, 0.5, respectively. Observation of laboratory animals (mice) with a scanning transmission electron microscope has revealed that the bacterial flora in the intestines were greatly reduced. Large bacteria-free areas were observed using erythrosine and anti-oxidants and the use of MgSO 4  resulted in completely bacteria-free intestines. 
     B. Faeces samples from uncontaminated animals were coated with MacConkey plates. The isolated samples were tested using a screening test, for Enterobacteriaceae in order to avoid Escherichia coli and Salmonella types. A suitable culture was selected and cultivated to produce fimbria which were checked with the yeast agglutination method. Intestinal wall was at the same time taken with caecum and large intestine of uncontaminated animals. Irreversibly bound bacteria plus intestinal wall was homogenized and frimbria-carrying bacteria were produced as described above. 
     Binding studies revealed that curcumine and similar compounds increased the degree of adhesion of Lactobacillus acidophilus to pig intestines five times. 
     The following bacteria preparations were produced for oral adminstration to pigs in skimmed milk fermented with Lactobacillus acidophilus. Capsules (sterilized) were used containing 10 10  /ml Streptococcus caecalis or S. faecium, fimbriated Enterobacteria and curcumine or analogous compounds, in sizes suited to the size of the animals. Curcumine is active if administered in amounts of 0.8 g/body-weight and day. This bacteria preparation was administered immediately after the treatment described under A. 
     EXAMPLE 2 
     RELATING TO POULTRY 
     A and B in Example 1, with the exception of the Lactobacillus acidophilus. The preparation was administered in drinking water. 
     Stages A and B can be repeated as necessary at intervals. 
     Some of the mechanisms binding micro-organisms to phase boundaries or interfaces are responsible for irreversible binding which acts over short distances (2-5 nm). This results in strong adhesion which is only mechanically broken by tearing away the components belonging to the adhered micro-organisms. Hydrophobic interaction is primarily responsible for this type of irreversible binding. 
     Successful treatment, i.e. removal of micro-organisms adhered in this manner, is effected by modifying the quantity of substances, salts or other components, able to alter the water-structure binding properties in the surfaces, phase boundaries and interfaces of the system. 
     Experiments with infectious bacteria in wounds and micro-organisms adhered to teeth showed surprisingly that the principles and substances used in the experiments with livestock described above were also extremely effective in the following widely differing fields of application. 
     The following points constitute the basis of the process used in these new fields of application. 
     1. Substances altering the degree of hydrophobic interaction between surface and micro-organisms are added to the surface/phase boundaries being treated, so that smaller or larger portions of the irreversibly adhere micro-organisms become detached. 
     2. Substances promoting adhesion of specifically supplied micro-organisms are added to the system treated under point 1. 
     The procedure is further illustrated with reference to the following non-limiting examples. 
     EXAMPLE 3 
     In both model system and during clinical testing using dentifrice or mouthwash with the addition of magnesium sulphate (MgSO 4 ) for brushing teeth or rinsing out the mouth, a significant reduction in the number of bacteria adhered was established. 
     EXAMPLE 4 
     Bathing liquids to which magnesium sulphate and/or anti-oxidants had been added were applied to a wound. Here too a significant reduction in the number of bacteria adhered to the wound surface was noted. It was also noted that cleaning the surface of a wound in this manner stimulated healing. 
     The result of the method is also illustrated by but not limited to the following experiments in a model system. 
     A number of glass tubes, all the same size, were immersed in a suspension of bacteria marked with a radioactive isotope (tritium) and the bacteria were allowed to adhere over a period of 30 minutes. The glass tubes were then removed one by one and rinsed by repeated dipping (30 times) in a buffer solution in order to remove reversibly bound bacteria. Only the irreversibly bound bacteria then remained on the glass tube. The glass tube was then immersed in a salt solution for 5 minutes. After rinsing, the quantity of bacteria adhered to the tube was determined by measuring the radiation in a liquid scintillator. 
     A comparison of the various glass tubes which had been immersed in salt solution of various concentrations with control tubes which had undergone the same treatment with the exception of immersion in the salt solution, enabled assessment of the effect of the salt solution on the irreversible bound bacteria. The table below and the drawing show the effects of the various salts on detachment of the irreversibly adhered bacteria, expressed in percentage of the quantity of irreversibly adhered bacteria before the salt treatment. The concentrations of the solutions are in multiples of a specific critical concentration (C) of the basic solution. 
     Of the salts tested in this series of experiments, magnesium sulphate (MgSO 4 ) gave the strongest detaching effect. However, magnesium chloride (MgCl) and ammonium acetate (NH 4  Ac) solutions also gave good results, whereas ammonium sulphate ((NH 4 ) 2  SO 4 ) solutions gave a slightly different pattern. 
     Several other compounds and salts have been tested besides those mentioned in this series of experiments, and the results were similar. 
     
                       TABLE                                                       
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Detachment percentage of irreversibly adhered bacteria after              
treatment.                                                                
The number of bacteria irreversibly adhered to the glass tube is          
3.0 × 10.sup.6 before treatment.                                    
       Salt concentration in multiples of basic solution C                
Salts    0.05 × C                                                   
                   0.1 × C                                          
                             0.2 × C                                
                                     0.6 × C                        
______________________________________                                    
MgSO.sub.4                                                                
         41        54        28      28                                   
MgCl.sub.2                                                                
         20        40        21      25                                   
NH.sub.4 Acetate                                                          
         8.5       41        17      24                                   
(NH.sub.4)SO.sub.4                                                        
         31        36        44      28                                   
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     FIG. 1 shows the detachment of irreversibly adhered bacteria after treatment with salt solutions of various concentrations. The salt concentration is expressed in multiples of the basic solution C. The number of irreversibly adhered bacteria is ca. 3.0×10 6  per glass tube before treatment. 
     The preparations containing curcumine described in the following claims are intended for use in performing the method according to the invention, but can also be used for other purposes, such as stimulating secretion in micro-organisms tissues and tissue cells.