Abstract:
A method of controlling protozoa trophozoites and cysts is disclosed. The method comprises the addition of guanidine or biguanidine salts to aqueous systems harboring protozoa trophozoites and cysts. Addition of such materials to aqueous systems was found to be effective at controlling both the protozoa trophozoites and cysts. By effectively controlling both the protozoa trophozoites and cysts, organism such as Legionella cells harbored in both the trophozoites and cysts are in turn controlled.

Description:
[0001]     This application is a continuation of Ser. No. 10/461,114, filed June 13, 2003.  
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates to methods for controlling  Legionella  harboring protozoa trophozoites and cysts in aqueous systems. More particularly, the present invention relates to methods for controlling  Legionella  type bacteria engulphed within a protozoa in the trophozoite form or in  Acanthamoeba  in the trophozoite and cyst form.  
       BACKGROUND OF THE INVENTION  
       [0003]     Intracellular bacterial pathogens are a major cause of human morbidity and mortality. Evading hostile intracellular environments is one of the ways pathogens can live within a host cell, even grow within host cells, and yet not be killed or inhibited by the host cell. These parasites have developed ways of interacting and overcoming the host cell&#39;s natural defense mechanisms.  Legionella pneumophila,  a bacterium known to cause Legionnaire&#39;s Disease and Pontiac fever in humans is a parasite of this type. While the  Legionella  cells can be killed while readily exposed to certain chemical agents and antibiotics,  Legionella  can also be found engulphed (phagocitized) within certain protozoa hosts.  Legionella  are often found in biofilms adsorbed to solid surfaces in water distribution systems, cooling towers, showers, aquaria, sprinklers, spas, and cleaning baths. Protozoa are natural grazers on surfaces and engulph and digest bacteria as part of their natural life cycle. In most cases, the protozoa digest these bacteria through the use of digestive enzymes in their phagosomes (digestive vacuoles). In the case of  Legionella,  however, this is not the case. The protozoa are not readily capable of degrading the engulphed  Legionella  cells, and in fact, the  Legionella  grow and increase their numbers while protected within protozoa phagosomes. Legionellosis in humans can be contracted by breathing  Legionella  aerosols containing either the free-living bacterial cells or by inhaling aerosols of  Legionella  concentrated within susceptible protozoa. A  Legionella  control agent, therefore, must be capable of killing free living  Legionella, Legionella  within protozoa, or the protozoa themselves. The agents described in this invention are capable of killing the free-living  Legionella  and the host protozoa. Two protozoa species capable of harboring infectious  Legionella  are  Acanthamoeba  and  Tetrahymena.    
         [0004]     In order to effectively control  Legionella,  in addition to killing the free-living  Legionella  or protozoa an additional factor must be taken into account. Certain protozoa, particularly amoeboid forms, have evolved mechanisms for surviving in hostile environments. Examples of hostile environments are high temperature, desiccation, presence of chemical agents/antibiotics, lack of food sources, etc. Upon introduction of a hostile environment, these protozoa revert to a cyst form which is very difficult to kill. The cyst form becomes much less susceptible to chemical agents which readily kill the same organism when in it is in a non-cyst (trophozoite) form. Introduction of a chemical control agent to eliminate  Acanthamoeba  can actually provide the hostile environment to which the protozoa responds by reverting to a cyst form, thereby rendering it invulnerable to the chemical agent. When the cyst contains the pathogen  Legionella,  the chemical agent can no longer reach the engulphed bacteria, and the chemical treatment is rendered ineffective. As an example, chlorination or bleach is considered essential to control  Legionella  in water distribution systems. Exposed  Legionella  are readily killed by low levels of free chlorine (0.2-0.5 μg/ml).  Legionella  can also be contained in  Acanthamoeba  phagosomes if those protozoa are present. The  Acanthamoeba  sensing the chlorine presence reverts to a cyst form, inadvertently preserving and protecting the  Legionella  parasites engulphed within it.  Acanthamoeba  cysts treated with &gt;500 times (&gt;100 μg/ml ‘free’ chlorine) the concentration needed to kill the trophozoite forms do not kill these cysts. The cysts can revert to the active trophozoite form upon removal of the oxidant. Currently there are no cyst deactivating (killing) agents in commercial use. Control agents that kill the  Legionella  harboring protozoa cysts would provide a much needed additional tool to safeguard the health of workers and the public against the respiratory pneumonias which can result from inhalation of  Legionella  or  Legionella  containing protozoan cysts.  
       SUMMARY OF THE INVENTION  
       [0005]     The present invention relates to methods for controlling  Legionella  harboring protozoa trophozoites and cysts in aqueous systems. More particularly, the present invention relates to methods for controlling  Legionella  type bacteria engulphed within a protozoa in the trophozoite form or in  Acanthamoeba  in the trophozoite and cyst form. The methods of the present invention involve exposing the protozoa to guanidine or biguanidine salts of the general formulas:  
                         
 
 wherein R, R 1 , R 2  are independently H, C 1 -C 20  substituted or non-substituted alkyl (linear or branched) or aryl, X is an organic or inorganic acid, n is 0-20 and z is 1-12.
 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0007]     It has been discovered that unique guanidine and biguanidine salts are effective at controlling  Legionella  type bacteria in the free living state as well as when engulphed in protozoa in the trophozoite form or  Acanthamoeba  in cyst form. The ability to control materials in the cyst form as well as the trophozoite form at comparable treatment levels is an unexpected feature of the treatment of the present invention.  
         [0008]     It was discovered that the guanidine and biguanidine salts of the general formula are especially effective:  
                         
 
 wherein R, R 1 , R 2  are independently H, C 1 -C 20  substituted or non-substituted alkyl (linear or branched) or aryl, X is an organic or inorganic acid, n is 0-20 and z is 1-12. In the formulas above, X is e.g., hydrochloric, sulfuric or acetic acid. 
 
         [0010]     The efficacy of the present invention was determined by evaluating the effect of a variety of treatments on the mortality of  Tetrahymena, Acanthamoeba  trophozoite, and  Acanthamoeba  cysts according to the following procedures.  
         [heading-0011]      Tetrahymena  Toxicity Test Procedure  
         [0012]      Tetrahymena  cells from a commercial source were grown in PCB broth in a tissue culture flask. The cells were removed from the broth via centrifuge and suspended in Osterhout-tris buffer at a concentration of no greater than 60 cells per 10 microliters. A standard 96 well test plate comprising successive 50% dilutions of this cellular solution per row was prepared. Chemicals to be tested were added to 3 adjacent wells. Organism viability was tested via observation through an inverted microscope at time zero and every 24 hours thereafter.  Tetrahymena  were judged viable if they were motile or had active contractile vacuoles. All organisms in a well had to be dead to have a negative reading. A positive reading indicated all or some viable organisms in a well. The minimal lethal concentration (MLC) of the test materials to  Tetrahymena  was the lowest toxicant concentration in which all  Tetrahymena  were dead in all replicate wells.  
         [heading-0013]      Acanthamoeba  Toxicity Test Procedure  
         [0014]      E. coli  (ATCC #25922) grown in nutrient agar and killed via UV light were used as nutrient for the  Acanthamoeba.  The killed  E. coli  were placed on a non-nutrient agar plate. 1-2 drops of washed  Acanthamoeba  Trophozoite (from Tennessee Technological University) were placed on the plate and incubated for 2-3 days at 30° C. An inoculum was prepared by placing about 2 ml of Osterhout-tris buffer onto the 2-3 day old plates. A sterile loop was used to dislodge the Trophozoites from the agar surface. The liquid was transferred to a sterile tube and diluted 1 :10. 10 microliters were placed on a slide and counted to confirm about 90  Acanthamoeba  per 10 micro liters for the test. This solution was placed in a standard 96 well test plate with successive 50% dilutions per row. A 400 ppm solution of toxicants in Osterhout-tris buffer was prepared. Toxicants were added to 3 adjacent wells for testing. To avoid cross contamination, a well was skipped between each 3 replicate wells in every row and every other row skipped on the plate. The plate was incubated at 30° for 24 hours. An inverted microscope was used to observe the organisms in the wells. Cytoplasm will move in live amoeba and/or the contractile vacuoles will remain active. All organisms had to be dead in a well to have a negative reading. The minimal lethal concentration (MLC) of the test toxicant was the toxicant concentration in which all organisms died in all replicate wells.  
         [heading-0015]      Acanthamoeba  Cyst Toxicity Test Procedure  
         [0016]      E. coli  (ATCC#25922) were grown in Difco Bacto nutrient agar and killed via UV light for use as nutrient for the  Acanthamoeba  cysts. The killed  E. coli  were placed on a non-nutrient agar plate. 1-2 drops of washed  Acanthamoeba  (from Tennessee Technological University) from a 2-3 day old plate were placed on the plate and incubated for 2-3 days at 30° C. A biofilm was prepared by placing approximately 9 milliliters of the active  E. coli  culture in sterile coplin jars containing 4 cover slips and incubating over night. The cover slips were rinsed in Osterhout-tris buffer and placed on 2-3 day old  Acanthamoeba  trophozoite plates and incubated for 7 days. In 7 days, the trophozoites will exhaust the  E. coli  nutrients and form cysts. The cover slips were soaked in approximately 9 milliliters of Osterhout-tris buffer and the cover slips placed in coplin jars. 50 ppm dilutions of the biocides to be tested were added to the coplin jars containing the cover slips with cysts and the coplin jars were incubated at 30° C for 24 hours. After 24 hours, the test solutions were removed and the cover slips soaked in Osterhout-tris buffer for 30 minutes. The cover slips were placed on non-nutrient agar plates with live  E. coli.  The plates were observed using an inverted microscope every day for 6 days to see if trophozoites were present. If trophozoites appeared, the test was positive. If no trophozoites appeared after 6 days, the test is negative (all cysts were killed). The test was repeated at different concentrations of treatment if the 50 ppm dilution was effective to determine the lower limit of efficacy.  
                                                   TABLE I                           Minimal Lethal Concentration (μg/ml as 100% active)                Tetrahymena   Acanthamoeba   Acanthamoeba       Compound   (Trophozoite)   (Trophozoite)   (Cyst)                    Chlorhexidine   15   10   50       PHMB*   10   5   50       Dodecylguanidine   10   25   40       Guanidine   400   500   500                 *Polyhexamethylene Biguanide               
         [0017]     The test results summarized in Table I show the minimal lethal concentration (MLC) in micrograms per milliliters (μg/ml) for tests of the guanidine salts: polyhexamethylene biguanide and dodecylguanidine for the  Tetrahymena  and  Acanthamoeba  in the trophozoite stage and  Acanthamoeba  in the cyst stage. The data shows that the guanidine salts having a long chain substituent group were effective at killing the protozoa in both the trophozoite and the cyst stage.  
         [0018]     While the present invention has been described with respect to particular embodiments thereof, it is apparent that numerous other forms and modifications of the invention will be obvious to those skilled in the art. The appended claims and the present invention generally should be construed to cover all such obvious forms and modifications which are within the true spirit and scope of the present invention.