US3595956A - Compositions containing 2,4,5-trichlorophenol and polymyxin b and neomycin - Google Patents

Abstract

ANTIMICROBIAL COMPOSITIONS COMPRISING MIXTURES OF 2, 4,5-TRICHLOROPHENOL AND NEOMYCIN OR POLYMYXIN B AS THE ESSENTIAL ACTIVE INGREDIENTS THEREOF ARE DISCLOSED. SUCH COMPOSITIONS COMPRISING 2,4,5-TRICHLOROPHENOL AND POLYMYXIN B OR NEOMYCIN POSSESS GREATER ANTIMICROBIAL ACTIVITY THAN THE INDIVIDUAL COMPONENTS OF THE COMPOSITIONS EMPLOYED SEPARATELY.

Description

United States Patent 3,595,956 COMPOSITIONS CONTAINKNG 2,4,5-TRICHLORO- PHENOL AND POLYMYXIN B AND NEOMYCIN Herbert J. Florestano, Indianapolis, Ind, assignor to The Dow Chemical Company, Midland, Mich.

No Drawing. Continuation-impart of applications Ser. No. 604,608 and Ser. No. 604,631, both Dec. 27, 1966. This application June 6, 1968, Ser. No. 734,850

Int. Cl. A61k 27/00 US. Cl. 424-177 8 Claims ABSTRACT OF THE DISCLOSURE Antimicrobial compositions comprising mixtures of 2, 4,5-trichlorophenol and neomycin or polymyxin B as the essential active ingredients thereof are disclosed. Such compositions comprising 2,4,5-trichlorophenol and polymyxin B or neomycin possess greater antimicrobial activity than the individual components of the compositions employed separately.

CROSS-REFERENCES TO RELATED APPLICATIONS This is a continuation-in-part of my copending prior application, Ser. No. 604,608, filed Dec. 27, 1966 now abandoned, and of my copending prior application, Ser. No. 604,631, filed Dec. 27, 1966 now abandoned.

SUMMARY OF THE INVENTION This invention relates to compositions comprising 2,4,5-

, trichlorophenol and an antibiotic selected from neomycin and polymyxin B. More particularly, this invention is concerned with antimicrobial preparations comprising 2, 4,5-trichlorophenol and neomycin or polymyxin B, or both. Such preparations possess antimicrobial action that is greater than that possessed by either 2,4,5-trichlorophenol, neomycin or polymyxin B when employed alone.

2,4,5-trichlorophenol has been employed for many years as a fungicide. In addition to its antifungal properties, the compound also has significant antibacterial activity and antiyeast activity.

Polymyxin B is a basic polypeptide with selective antimicrobial activity against gram-negative bacteria. The antibiotic is predominantly bactericidal in action. The characteristics of polymyxin B are described in the following publications: Jawetz, Polymyxin, Neomycin, Bacitracin, Medical Encyclopedia, Inc., New York (1956) and Florestano et al., Proc. Soc. Exp. Biol. & Med., vol. 79, pp. 141-143 (1952).

In the present specification and claims, the term polymyxin B refers to polymyxin B and to its antimicrobial derivatives such as the hydrochloride, sulfate, palmitate, methanesulfonate and oxalate salts, The anionic moieties of such salts do not substantially alter the synergistic antimicrobial activity of the compositions of the invention, and such salts can be employed advantageously therein. Polymyxin B sulfate is preferred for use in the compositions of the invention.

Neomycin is an amorphous polybasic material that readily forms salts with a variety of acids. Neomycin has significant activity in vitro against various bacteria, a slight degree of activity against fungi and protozoa and very slight activity against yeasts. The properties of neomycin are described by Jawetz in Polymyxin, Neomycin, Bacitracin, Medical Encyclopedia, Inc., New York (1956). In the present specification and claims, the term neomycin is intended to include the various forms of neomycin (such as the B and C forms) and their antimicrobial derivatives such as the sulfate and the hydrochloride salts. The anionic moieties of such salts have no substantial effect on the synergistic antimicrobial activity of the compositions of the invention, and such salts can be employed in the invention. Representative and suitable derivatives of neomycin which can be employed in the compositions of the invention include the antimicrobial inorganic acid salts such as neomycin hydrochloride and neomycin sulfate, and the antimicrobial organic acids such as neomycin acetate, neomycin succinate, neomycin methanesulfonate, neomycin palmitate and neomycin undecylenate. The preferred derivative is neomycin sulfate.

It has been discovered that compositions containing an antimicrobial amount of a mixture of 2,4,5-trichlorophe- 1101 with one or both of neomycin and polymyxin B in a ratio of from about 99 parts of 2,4,5-trichlorophenol to about 1 part of neomycin or polymyxin B to a ratio of about 3 parts of 2,4,5-trichlorophenol to about 97 parts of neomycin or polymyxin B, as well as the other intermediate ranges of proportions such as from 98.5:1.5, to 97:3. to 94:6, to 89:11, to :20, to 74:26, to 67:33, to 50:50, to 33:67, to 25:75, to 20:80, to 15:85 to 11:89, to 6:94, to 3:97 of 2,4,5-trichlorophenol to neomycin or polymyxin B- have greater antimicrobial activity than the separate components of the compositions when employed alone. Such compositions exhibit synergistic antimicrobial properties both in killing of microorganisms and in the inhibition of their growth. The term antimicrobial, as employed herein, refers both to the killing and to the inhibition of the growth of microorganisms. The term is thus inclusive of bactericidal, fungicidal, bacteriostatic and fungistatic properties.

The novel compositions are useful as disinfectants and as preservatives. They can be employed by conventional procedures to control bacteria and fungi in various substrates, and can be applied to bacterial or fungal organisms or their substrates in antimicrobial amounts by conventional procedures including spraying, dipping, drenching, impregnation and the like.

Preferred synergistic antimicrobial compositions of the invention include those comprising 2,4,5-trichlorophenol and one of neomycin or polymyxin B in the proportions of from about 99 to 11 parts by weight of the phenol ingredient and from about 1 to about 89 parts by weight of the antibiotic ingredient. Other preferred compositions are those wherein the phenol is present in an amount of from about 11 to about 94 parts by Weight and the antibiotic is present in amounts of from about 89 to about 6 parts by weight. such preferred compositions include other preferred compositions such as those containing from about 11 to about 67, to about 80 and from about 89 to about 94 parts by weight of the phenol ingredient and from about 89 to about 33, to about 20 and from about 11 to about 6 parts by weight of the antibiotic. Other preferred compositions include those in the above ranges of proportions wherein the polymyxin B or neomycin antibiotic ingredient is in the form of the sulfate salt.

Other preferred compositions of the invention comprise from about 99 parts 'by weight to about 3 parts by weight of 2,4,5-trichlorophenol and from about 1 part by weight to about 97 parts by weight of polymyxin B or a mixture comprising 2,4,5-trichlorophenol and polymyxin B in the proportions of from about 80 to about 3 parts by weight of the former to from about 20 to about 97 parts by weight of the latter. Further preferred compositions are those having the above proportions wherein the polymyxin B is in the form of polymyxin B sulfate.

A preferred composition containing neomycin as the antibiotic ingredient comprises from about 97 parts by Weight to about 67 parts by weight of 2,4,5-trichlorophenol and from about 3 parts to about 33 parts of neomycin. Yet another preferred composition comprises from 98.5 to about 11 parts of 2,4,5-trichlorophenol and from 1.5 to about 89 parts of neomycin. Another preferred composition comprises from about 99 to about 20 parts of 2,4,5- trichlorophenol and from about 1 to 80 parts of neomycin. Other preferred compositions containing neomycin are those wherein the neomycin is present in the form of neomycin sulfate.

The provision of an antimicrobial amount of the mixture of 2,4,5-trichlorophenol and antibiotic in the composition of the invention is essential to the beneficial use of the compositions in the control of bacteria and fungi.

The antimicrobial amount of the active mixture to be employed can vary depending upon such factors as the particular antiobiotic ingredient employed and the proportion of the ingredients in the composition, the microbial organism or organisms to be controlled, the degree of microbial infestation, the effect to be produced, whether microbicidal effects or inhibition is desired, the manner of application and the like. Against certain organisms, antimicrobial effects of the compositions are observed at concentrations as low as about 8 parts by weight of active antimicrobial mixture per million parts by weight of total composition. In general, it is desirable to provide an antimicrobial amount of at least about 0.1 percent by weight of the active mixture of 2,4,5-trichlorophenol and antibiotic in compositions to be employed as disinfectants or preservatives. Compositions containing from about 0.5 percent to 1 percent to about 10 percent by weight of the antimicrobial mixtures are preferred. Compositions containing higher concentrations of the antimicrobial mixtures such as 1050 percent, 40-75 percent, or 60-99 percent 'by weight can be employed as concentrates. These concentrated compositions and compositions consisting of 2,4,5-trichlorophenol and neomycin or polymyxin B without any adjuvants or diluents can be diluted with inert diluents, suspending agents, surface active agents and the like.

The compositions of the invention can be conveniently prepared by mixing together the 2,4,5-trichlorophenol and the antibiotic ingredient in any order or fashion. The compositions comprising the active mixture can be employed directly to control microbial growth by contacting the bacteria or fungi to be controlled or the substrate to be protected with an antimocrobial amount of the unmodified mixture of 2,4,5-trichlorophenol and antibiotic or with a suitable treating composition comprising an antimocrobial amount of the mixture. In general, it is preferred to employ the composition of the invention in the form of treating compositions comprising an antimocrobial amount of 2,4,5-trichlorophenol and neomycin or polymyxin B and a carrier. Suitable inert carriers can be liquids such as water, liquid hydrocarbons, ethanol, isopropanol or compatible mixtures thereof; semi-solid carriers such as petrolatum, beeswax, polyethylene glyols, diesters of polyethylene glycols of from about 200 to 600 mulecular weight with fatty acids containing from 12 to 18 carbon atoms and fatty acid esters of glycerol in which the fatty acids contain from 12 to 18 carbon atoms; and finely divided solid carriers such as talc, chalk and diatomaceous earth. The compositions can thus be liquids, spreadable solids or finely divided solids such as dusts. When the compositions are employed as preservatives to protect a 4 substrate such as cloth, aqueous suspensions, leather, soap, etc. from microbial attack, the substrate can also serve as a carrier.

The compositions can also include modifying substances. Such substances include suspending agents such as hydroxypropyl methyl cellulose and other cellulose derivatives and vegetable gums. The preferred liquid compositions include at least one such suspending agent. Another preferred modifying substance is a surface active agent, including alkyl aryl sulfonates, sodium alkyl aryl polyether sulfonates, fatty alcohol sulfates, fatty acid esters of sugars such as sucrose tallowates, condensation products of ethylene oxide with esters derived from fatty acids and hexitol anhydrides and the like.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following specific examples will illustrate the invention.

Example 1 The synergistic antimicrobial action of the composi tion of the invention is demonstrated in the following representative determination. Combinations of various fractional amounts of two ingredients are prepared in brain heart infusion broth as the test medium. Tubes containing the test medium and the antimicrobial combinations are inoculated with 0.1 cubic centimeter of an 18 hour culture of Pseudomonas aeruginosa diluted so that the number of cells averages about 18,000 per cubic centimeter of broth medium in the final tubes of inoculum. The inoculated tubes are incubated at 37 C. and examined at 24 hour intervals. Other separate tubes of broth containing one of either 2,4,5-trichlorophenol or neomycin sulfate as the sole antimicrobial agent therein are similarl inoculated and incubated. Similar tubes of broth containing no antimicrobial agent are similarly inoculated and incubated to serve as checks. At the times of the observations, the check tubes are found to contain vigorous bacterial growth. Tubes showing no visible growth after 48 hours are subcultured into fresh broth to ascertain minimum lethal concentrations. When no growth is observed in a fresh broth subculture, the corresponding original tube is recorded as containing a lethal concentration of the antimicrobial agent or combination. When growth is observed in a fresh broth subculture from an original tube having no growth, the original tube is recorded as containing a bacteriostatic concentration of the antimicrobial agent or combination. The lowest concentration of an antimicrobial agent or combination which is observed to produce lethal or bacteriostatic results is recorded as the minimum lethal or minimum bacteriostatic concentration, respectively. In tubes containing lower concentrations of antimicrobial agent, bacterial growth similar to that observed in the check tubes is found.

The results of these studies indicate that the minimum lethal concentration of 2,4,5-trichlorophenol is equal to or greater than 125 micrograms per cubic centimeter. The minimum lethal concentration of neomycin sulfate is observed to be 62.5 micrograms per cubic centimeter. The minimum bacteriostatic concentrations observed for 2,4,5-trichlorophenol and neomycin sulfate are 125 and 7.8 micrograms per cubic centimeter respectively.

These data indicate that neomycin sulfate is approximately two times as potent a bactericide and 16 times as potent a bacteriostat as 2,4,5-trichlorophen0l. Using these estimated relative potencies of the compounds, the expected relative potencies of the mixtures as bacteriostats and as bactericides are calculated. These relative potencies are then employed to calculate the expected minimum lethal and minimum bacteriostatic concentrations of the mixtures. For example, an 80:20 mixture of 2,4,5-trichlorophenol and neomycin has an expected bactericidal potency of 0.8 plus 2 (0.2) or 1.2 relative to 2,4,5-trichlorophcnol alone. Such a mixture would be expected to have a minimum lethal concentration of 1/ 1.2) times the observed minimum lethal concentration of 2,4,5-trichloropl1enol alone or 104 micrograms per cubic centimeter. The relative proportions in each mixture, expected minimum lethal and bacteriostatic concentrations and observed minimum lethal and bacteriostatic concentrations are tabulated in the following table wherein concentrations are given in micrograms per cubic centimeter g. per cc.).

TABLE I Minimum lethal concentration, pg. per cc. Neomycin 2,4,5-trichlorophenol sulfate Expected Observed Minimum bacteriostatic concentration, pg. per cc.

Expected Observed Example 2 The antimicrobial action of the composition of the invention is further illustrated by a procedure similar to that employed in the preceding example. Different mixtures employing 2,4,5-trichlorophenol and neomycin sulfate in various proportions representative of the composition ranges of the invention are prepared in tubes containing a bacterial nutrient medium. Separate tubes are similarly prepared containing one of either 2,4,5-trichlorophenol or neomycin sulfate as the sole antimicrobial agent therein. The tubes are then inoculated with one of two strains of Staphylococcus aureus. The tubes are incubated at 37 C. and examined at 24 hour intervals. Similar tubes containing no antimicrobial agent are similarly inoculated and incubated to serve as checks. At all times of observation, vigorous bacterial growth is found in the check tubes. Tubes showing no growth at the end of a 48 hour incubation period are subcultured into fresh nutrient medium containing no antimicrobial agents and incubated. When no growth is observed in a fresh broth subculture from one of the original tubes, the original tube is recorded .as Containing a lethal concentration of the antimicrobial mixture. Growth in the fresh medium subculture indicates that the corresponding original tube contains a bacteriostatic concentration of the mixture. Minimum lethal and minimum bacteriostatic concentrations are recorded as described in Example 1. 2,4,5-trichloropheno1 is found to have minimum bacteriostatic concentrations of 250 micrograms per cubic centimeter against each strain of S. aureus tested and a minimum lethal concentration of 250 micrograms per cubic centimeter against S. aureus 8790. The minimum lethal concentration of 2,4,5-trichlorophenol against S. aureus H9059 is found to be equal to or greater than 250 micrograms per cubic centimeter, and such value is employed in the calculation of estimated concentrations. Neomycin sulfate is found to have minimum lethal and bacteriostatic concentrations of 1000 micrograms per cubic centimeter against S. aureus 8790 and of 1000 and 250 micrograms per cubic centimeter, respectively, against S. aureus H9059. Using these minimum lethal and bacteriostatic concentrations, the relative potencies of the two compounds are calculated. The minimum lethal and bacteriostatic concentrations which would be expected if only additive antimicrobial effect is present are calculated for the mixtures. The proportions of the antimicrobial agents in each mixture, the expected and observed minimum lethal and bacteriostatic concentions of the mixtures in micrograms per cubic centimeter the following table.

TABLE II Percentage of agent in Minimum lethal conmixture centration, pg. per cc.

2,4,5-tri- Neomycin sulfate Test organism Expected Observed 33 S. aureus H9059. 332 187. 0 50 S. aureus H9059. 400 255 S. aureus H9059. 625 312. 5 89 S. cureus H9059. 752 562. 5 33 S. GUTE'MS 8790. 332 187. 5 67 S. aureus 8790. 502 375 89 S. aureus 8790... 752 562. 5 89 S. aureus 8790. 752 562. 5

Minimum bacteriostatic concentration, g. per cc.

Expected Observed 1. 5 S. aureus H905L 250 127 8 S. aureus H9059. 250 136 67 S. aureus H9059. 250 187. 5 67 S. aureus H9059. 250 93. 7 6 S. aureus 8790 262 133 80 S. aurcus8790-.. 625 312. 5 80 S. aureus 8790. 625 312. 5 89 S. aureus 8790. 752 562. 5

Example 3 The antimicrobial action of a representative composition of the invention comprising 67 parts by Weight of 2,4,5-trichlorophenol and 33 parts by weight of polymyxin B is illustrated by a procedure similar to that employed in the preceding examples. The mixtures of the antimicrobial agents is prepared in tubes containing a bacterial nutrient medium. Separate tubes are inoculated with Staphylococcus aureus Barlow. The tubes are incubated at 37 C. and examined at 24 hour intervals. Separate tubes containing one of either 2,4,5-trichlorophenol or polymyxin B sulfate are similarly inoculated and incubated. Separate tubes containing no antimicrobial agent are similarly inoculated and incubated to serve as checks. At the times of observation, vigorous bacterial growth is observed in the check tubes. Tubes showing no growth at the end of a 48 hour incubation period are subcultured into fresh nutrient medium containing no antimicrobial agents and incubated. When no growth is observed in a fresh medium subculture, the corresponding original tube is recorded as containing a lethal concentration of the antimicrobial mixture. A tube showing no growth after 48 hours in the original medium is recorded as containing a bacteriostatic concentration of the mixture when a subculture from such a tube shows bacterial growth. Minimum lethal and minimum bacteriostatic concentrations are recorded as described in Example 1. Both 2,4,5-trichlorophenol and polymyxin B sulfate are found to have minimum lethal and bacteriostatic concentrations of 62.5 and 31.2 micrograms per cubic centimeter, respectively, against S. aureus Barlow. Minimum lethal and bacteriostatic concentrations which would be expected if only additive results were obtained are calculated as described above. The mixture of 67 parts of 2,4,5-trichlorophenol and 33 parts of polymyxin B sulfate is found to be bacteriostatic at a concentration of 23.4 micrograms per cubic centimeter as compared with an expected minimum bacteriostatic concentration of 31.2 micrograms per cubic centimeter. In two replications, the same mixture is found to be lethal at a concentration of 46.8 micrograms per cubic centimeter as compared with an expected minimum lethal concentration of 62.5 micrograms per cubic centimeter.

Example 4 The antimicrobial properties of representative compositions of the invention comprising 2,4,5-trichlorophenol and polymyxin B in the proportion ranges of about 99 parts or more of the former to about 1 part or less of the latter is illustrated by carrying out the procedure of Example 3 With Escherichia coli as the test organism. In such operations, 2,4,5-trichlorophenol is found to have minimum lethal and bacteriostatic concentrations equal to or greater than 62.5 micrograms per cubic centimeter. Polymyxin B sulfate is found to have minimum lethal and bacteriostatic concentrations of 0.12 and 0.015 micrograms per cubic centimeter, respectively, against E. coli. The proportions of the antimicrobial agents in each mixture and the expected and observed minimum lethal and minimum bacteriostatic concentrations of the mixtures in micrograms per cubic centimeter g. per cc.) are set out in Table III.

TABLE III Proportion of agents in mixture Minimum lethal concentration, g. per cc. 2,4,fi-trichlorophenol Polymyxin B sulfate Expected Observed Minimum baeteriostatie concentration, g. per cc. of agents Expected Observed 0. 0038 62 15. 6 0. 0019 62. 4 31. 2 0. 0000 62. 4 31. 2

Example 5 Representative mixtures employing different proprotions of 2,4,5-trichlorophenol and polymyxin B sulfate are prepared aseptically in tubes of Sabouraud maltose broth. The broth tubes are inoculated with a dilution of a 48 hour broth culture of Pityrosporum ovale by the introduction of 0.1 cubic centimeter of the diluted culture. The inoculum contains about 70,000 cells per cubic centimeter of broth. The tubes are incubated at 37 C. and examined at 24 hour intervals. Other separate tubes of broth containing either 2,4,5-trichlorophenol or polymyxin B sulfate as the sole antimicrobial agent therein are similarly inoculated and incubated. Other tubes containing no antimicrobial agent are similarly inoculated and incubated to serve as checks. At the times of observation, the check tubes show vigorous growth of P. ovale. Those tubes showing no growth at the end of 48 hours are subcultured into fresh Sabouraud maltose broth. When no growth is observed in the fresh broth subculture, the corresponding original tubes are recorded as containing a lethal concentration of the antimicrobial agents. The lowest concentration of an antimicrobial agent or mixture at which lethal results are observed is recorded as the minimum lethal concentration.

The results indicate that 2,4,5-trichlorophcnol is lethal to Pityrospormn ovale at a minmum lethal concentration of 15.6 micrograms per cubic centimeter. The minimum lethal concentration of polymyxin B sulfate is found to be 3.9 micrograms per cubic centimeter. These data indicate that polymyxin B sulfate is about four times as potent as 2,4,5 -trichlorophenol. Using this relative potency of the two antimicrobial compounds, the expected relative potencies of the mixtures are calculated. These relative potencies are then employed to calculate the expected minimum lethal concentrations for the mixtures. For a mixture containing 94 parts of 2,4,5-trichlorophenol and 6 parts polymyxin B sulfate, the expected minimum lethal concentration is calculated to be 13.2 micrograms per cubic centimeter, and the observed minimum lethal concentration is found to be 8.29 micrograms per cubic centimeter. For a mixture containing 89 parts 2,4,5-trichlorophenol and 11 parts of polymyxin B sulfate, the expected minimum lethal concentration is calculated to be 11.7 micrograms per cubic centimeter and the experimentally found minimum lethal concentration is observed to be 8.78 micrograms per cubic centimeter.

Example 6 Representative mixtures employing various proportions of 2,4,5-trichlorophenol and polymyxin B sulfate are prepared aseptically in tubes of Sabouraud maltose broth. The

broth tubes are inoculated with a solution of a 24 hour broth culture of Candida albicans by the introduction of 0.1 cubic centimeter of the diluted culture. The inoculated tubes contain about 11,000 cells per cubic centimeter. The tubes are incubated at 37 C. and examined at 24 hour intervals. Separate tubes each containing either 2,4,5-trichlorophenol or polymyxin B sulfate as the sole toxicant therein are similarly inoculated and incubated. Other separate tubes containing no antimicrobial agent are similarly inoculated and incubated to serve as checks. After 24 and 48 hours incubation, these check tubes show luxuriant growth of Candida albicans. Those tubes showing no growth at the end of 48 hours are subcultured into fresh Sabouraud maltose broth. When a fresh broth subculture shows no growth after 48 hours, the corresponding original tube is recorded as containing a lethal concentration of the antimicrobial agent. When an original tube shows no growth after 48 hours and a fresh broth subculture shows growth, the original tube is recorded as containing a fungistatic concentration of the antimicrobial agent. The lowest concentrations at which lethal or fungistatic effects are observed are recorded as the minimum lethal and minimum fungistatic concentrations, respectively. Tubes containing concentrations of antimicrobial agent less than the minmium lethal or minimum fungistatic concentration are observed to support vigorous growth of Candida albicans comparable to that of the checks.

The results indicate that 2,4,5-trichlorophenol is lethal to Candida albicans at a minimum lethal concentration of 31.2 micrograms per cubic centimeter. The minimum lethal concentration of polymyxin B sulfate is observed to be about micrograms per cubic centimeter. The results also indicate that 2,4,5-trichlorophenol inhibits the growth of Candida albicans at a minimum fungistatic concentration of 7.8 micrograms per cubic centimeter. The minimum fungistatic concentration of polymyxin B sulfate is found to be 62.5 micrograms per cubic centimeter. Minimum lethal and fungistatic concentrations which would be expected if the antimicrobial effects were additive are calculated. The proportions of 2,4,5-trichlorophenol and polymyxin B sulfate in the mixtures, the expected and observed minimum lethal concentrations in micrograms per cubic centimeter g. per cc.) and the expected and observed minimum fungistatic concentra tions are set forth in Table IV below.

TABLE IV Mlnimium lethal concentrat on er 0. 2,4,5-trrehlorophenol Polymyxin p c B sulfate Expected Observed Minimum iungistatic concentration, g. per cc.

Expected Ovserved Example 7 9.7 parts by weight of 2,4,5-trichlorophenol and 0.3 part by weight of neomycin sulfate are dissolved in 800 parts by weight of denatured alcohol. The mixture is diluted by the addition of 200 parts by weight of an aqueous solution containing 2 percent by weight polymyxin B sulfate. The resulting mixture is employed as a spray to disinfect floors, containers, storage bins, utensils and the like.

Example 8 parts by weight of water are heated at a temperature of about 8090 C. while 15 parts by weight of 2,4,5-trichlorophenol and 5 parts by weight of polymyxin B sulfate are added to the mixture and dispersed therein with mechanical agitation. Two parts by weight of hydroxypropylmethyl cellulose are added to the mixture and the resulting mixture is stirred for about five minutes. 328 parts of water are then added to the mixture with stirring. The mixture is cooled to room temperature to provide an aqueous suspension containing 4 percent by weight .of a 75:25 mixture of 2,4,5-trichlorophenol and polymyxin B.

In a similar procedure, 5 parts by weight of 2,4,5- trichlorophenol and 20 parts by weight of neomycin sulfate are dispersed in 500 parts of aqueous 3 percent hydroxypropylmethyl cellulose to prepare an aqueous suspension containing about-4.75 percent by Weight of 20:80 mixture of 2,4,5-trichlorophenol and neomycin sulfate.

The aqueous suspensions prepared as described above are employed to disinfect veterinary operating tables and animal cages and the like.

Example 9 Ten parts of liquid petrolatum U.S.P., 10 parts of white petrolatum U.S.P., 12 parts of Glycowax S-392 (a polyhydric alcohol fatty acid ester), 10 parts of polyethylene glycol 400 distereate and 58 parts of polyethylene glycol 400 dilaurate are placed together in a clean, dry sterile vessel. The mixture is heated to approximately 125 C. and maintained at that temperature with occasional stirring for about two hours. The mixture is cooled with stirring .to about 4045 C. to produce a solid, water-miscible composition. One .gram of 2,4,5-trichlorophenol and 0.35 gram of neomycin sulfate are thoroughly mixed with the composition base to make 100 grams of an antimicrobial preparation containing 1.35 percent of a 74:26 mixture of 2,4,5-trichlorophenol and neomycin sulfate.

Two grams of 2,4,5-trichlorophenol and 0.35 gram of neomycin hydrochloride are thoroughly mixed with a similar composition to make 100 grams of antimicrobial compositionlcontaining 2.35 percent of an 85:15 mixture of 2,4,5-trichlorophenol and neomycin hydrochloride.

Example 10 An anhydrous water-immiscible preparation is prepared by placing a purified mixture of semi-solid hydrocarbons melting at about 38-54 C. (white petrolatum U.S.P.) in a clean, dry, sterile vessel and heating to melt. The white petrolatum is cooled with stirring and 2 grams of 2,4,5-trichlorophenol and 0.35 gram of neomycin sulfate are thoroughly mixed with 97.65 grams of white petrolatum to make 100 grams of composition. This mixture is milled and then dispersed into tubes each containing 10 grams of composition representing 0.2 .gram of 2,4,5-trichlorophenol and 0.035 gram of neomycin sulfate. The composition is applied to leather materials and the like to protect the same from microbial attack.

In a similar procedure, 0.12 gram of 2,4,5-trichlorophenol and 3.88 grams of neomycin palmitate are thoroughly mixed with white petrolatum to obtain an antimicrobial composition containing 0.4 percent of a 3:97 mixture of 2,4,5-trichlorophenol and neomycin palmitate.

In a similar procedure, 5 grams of 2,4,5-trichlorophenol and 5 grams of neomycin are thoroughly mixed with 90 grams of white petrolatum and the product is milled to a uniform consistency to obtain a composition which is employed to control bacteria and fungi.

Example 11 Two parts of bleached beeswax, 18 parts of stearic acid, 5 parts of polyethylene glycol 400 monostearate, 8 parts of polyethylene glycol 1000 monostearate and 67 parts of distilled water are mixed together in a clean, sterile, dry vessel. The mixture is heated with stirring to melt the solid ingredients. The mixture is then stirred constantly while cooling to about 40-45 C. to prepare a water- 10 miscible composition. One gram of 2,4,5-trichlorophenol and 0.35 gram of neomycin sulfate are thoroughly mixed together with 98.65 grams of the composition to prepare a synergistic antimicrobial composition containing 1.35 percent of a 74:26 mixture of 2,4,5-trichlorophenol and neomycin sulfate.

In a similar procedure, 1.98 grams of 2,4,5-trichloro phenol and 0.02 gram of neomycin undecylenate are thoroughly mixed with 98 .grams of the water-miscible composition prepared as described above to prepare a synergistic antimicrobial composition containing 2 percent of a 99:1 mixture of 2,4,5-trichlorophenol and neomycin undecylenate.

Example 12 An aqueous composition is prepared by heating 30 grams of disilled water to about -90 C. and adding one gram of 2,4,5-trichlorophenol and 0.35 gram of neomycin sulfate to the Water with stirring. 0.5 gram of hydroxypropylmethyl cellulose is added to the mixture and the resulting mixture is stirred for about five minutes. An additional 68.15 grams of distilled water are added and the mixture is stirred and cooled to room temperature to provide an aqueous suspension containing 1.35 percent of a 74:26 mixture of 2,4,5-trichlorophenol and neomycin sulfate. This composition can be employed by conventional procedures to disinfect veterinary cages, floors, utensils and other articles.

Example 13 Compositions prepared according to Examples 9-12 are tested by the agar cup-plate procedure against one or more of the organisms Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The 2,4,5-trichlorophenol-neomycin mixtures of Examples 9-12 are found to be compatible with the water-miscible solid carrier of Example 9, with the water-immiscible carrier of Example 10, with the water-miscible liquid composition of Example 11 and with the aqueous composition of Example 12. Observations of zones of inhibition indicate the antimicrobial properties of each composition. Representative results are zones of inhibition on the order of 6 to 7 millimeters in radial width observed when a composition according to Example 9 and containing 2.35 percent of an :15 mixture of 2,4,5-trichlorophenol and neomycin sulfate is tested against each of the abovenamed organisms.

Example 14 55 parts by Weight of 2,4,5-trichloropheno1 and 40 parts by 'weight of polymycin B sulfate are mixed together with 20 parts by weight of a polymerized sodium salt of a substituted benzoid alkyl sulfonic acid (Daxad 27), 45 parts by weight of an alkyl aryl sulfonate (Acto 700) and 910 parts by weight of talc. The mixture is intimately blended on a hammermill to prepare a dust composition which, if desired, can be dispersed in water.

In a second operation, parts by weight of 2,4,5-trichlorophenol and 15; parts by Weight of neomycin sulfate are mixed together; The mixture is hammermilled with 400 parts by weight of attapulgite clay and parts of a sodium alkyl aryl sulfonate (Nacconal NR) to produce a concentrate composition :Which can be diluted and dispersed with Water.

What is claimed is:

1. An antimicrobial composition for controlling bacteria and fungi comprising from about 97 to about 67, inclusive, parts by weight of 2,4,5-trichlorophenol and from about 3 to about 33, inclusive, parts by weight of neomycin sulfate.

2. The composition of claim 1 further comprising an inert carrier and wherein the mixture is dispersed in the carrier in an antimicrobial amount.

3. The composition of claim 2 wherein the mixture is present in an amount of from about 0.5 to about 10 percent by weight.

1 1 1 2 4. The composition of claim 1 further comprising 8. References Cited surface'acti? disperting UNITED STATES PATENTS 5. An antlmicrobial composition for controlllng bacteria and fungi comprising 3,275,511 9/1966 Shaw from about 80 to about 3, inclusive, parts by weight of 5 OTHER REFERENCES 2,4,5-trichlorophen0l and from about 20 to about 97, inclusive, parts by weight of polymyxin B sulfate. 6. The composition of claim 5 further comprising an inert carrier and wherein the mixture is dispersed in the 833 and 1060' carrier in an antimicrobial amount 10 Florestano et al., 'Proc. Soc. Exp. B101. and Med., vol.

7. The composition of claim 6 wherein the mixture is PP- 141-143 present in an amount of from about 0.5 to about 10 per cent by weight JEROME D. GOLDBERG, Primary Examiner 8. The composition of claim 5 further comprising a U8. Cl. X.R. surface-active dispersing agent. 42418l, 347

The Merck Index of Chemicals and Drugs, Seventh Edition, Merck and Co., Inc., Rahway, .N.J., 1960, pp.