Patent Application: US-201514869802-A

Abstract:
an antibacterial composition comprising arginine bicarbonate , zinc carbonate , preferably arginine bicarbonate and zinc carbonate , in combination , plus one or more physiologically acceptable excipients , administered for the modification of cutaneous microfloras , generally to inhibit the growth of pathogenic staphylococcus aureus bacteria by promoting the growth of non - pathogenic staphylococcus epidermidis bacteria .

Description:
corynebacteria , staphylococci and proprionibacteria are among the main microorganisms present in the cutaneous microbiome , with staph . epidermidis , c . striatum and p . avidum as prominent representative bacteria unexpectedly , it has been discovered that certain compositions including a zinc salt and arginine and / or its salt are useful as antibacterial compositions , inhibiting staph . aureus growth while favoring staph . epidermidis growth . this ability to select between staph . aureus and staph . epidermidis allows the treatment of significant physiological and health - related disease conditions caused by aberrant or excessive growth of staph . aureus ( see , e . g ., peacock et al ., 2001 , uehara et al ., 2000 ). although staph . aureus is capable of wide infection and fatalities ( see , e . g ., david et al ., 2010 , mainous iii et al ., 2006 , klevens et al ., 2007 ), current antibiotic treatments have achieved limited success due to the emergence of resistant staph . aureus strains , e . g ., mrsa ( see , e . g ., david et al 2010 , chen et al 2006 , centers for disease control and prevention 2003 ). a recent discovery has shown that firmicidin ( gallo et al ., 2013 , nakatsuji et al ., 2012 ), a newly discovered antibiotic generated by staph . epidermidis , can reduce staph . aureus , but it is not known whether this will , like other antibiotics , succumb to adaptation and loss of effectiveness . from a commercial stand - point , this approach is likely to be costly . unlike traditional antibacterial treatments , the compositions of the present invention are aimed at modulating natural interactions between staph . aureus and other prominent members of the cutaneous microflora , e . g ., staph . epidermidis ( see , e . g ., frank et al ., 2010 , vehara et al ., 2000 , wertheim et al ., 2005 ). these bacteria naturally compete , e . g ., for local resources and attachment to mucosal sites ( frank et al ., 2010 ). the compositions of the invention , rather than merely targeting staph . aureus , render an ecological change that favors selection of desirable staph . epidermidis over non - desirable staph . aureus bacteria . because the compositions of the present invention derive their antibacterial effectiveness not only by targeting staph . aureus directly , but also by enhancing the ability of other , non - pathogenic bacteria ( e . g ., staph . epidermidis ) to out - compete staph . aureus . the compositions disclosed here are less likely to be susceptible to the emergence of resistant strains ( e . g ., mrsa ) than traditional antibacterial treatments . a further advantage of the present invention is that the compositions disclosed herein are effective in reducing cutaneous odor production . thus , a single topical composition may be used as both deodorant and antibacterial treatment . antibacterial compositions as described herein are administered , preferably topically , for the treatment of any one or more symptoms desirable of change , e . g ., staph . aureus growth . dosage forms are solid or free - flowing . dosage forms include , but are not limited to , soaps , sprays , drops , aerosols , powders , roll - ons , lotions , creams , sticks , solutions , sachets , colloidal suspensions , films , patches and ointments . antibacterial compositions as described herein may have a ph of at least 6 . 0 , or at least 7 . 0 , or at least 8 . 0 , or at least 9 . 0 upon topical administration . antibacterial compositions as described herein may optionally include one or more physiologically acceptable buffers sufficient to maintain the ph of said composition , e . g ., at 6 . 0 or greater , at 7 . 0 or greater , at 8 . 0 or greater , or at 9 . 0 or greater upon topical application . such buffers are generally known in the art , and may include , e . g ., aces , acetic acid , ada , amp , ampd , bicine , bis - tris , bis - tris propane , bes , boric acid , cacodylate , cabs , caps , capso , ches , citric acid , diethanolamine , dipso , epps / hepps , ethanolamine , formic acid , glycine , glycylglycine , hepes , heppso , histidine , imidazole , lactic acid , maleic acid , malic acid , mes , mops , mopso , morpholine , phosphate , phosphoric acid , picolinic acid , pipes , piperazine , piperidine , pivalic acid , popso , pyridine , succinic acid , taps , tapso , tea , tes , tricine , and / or tris . except where otherwise noted , the terms “ axillary odor ” and “ foot odor ” are used interchangeably herein , the terms “ microbiome ,” “ microbiota ,” and “ microflora ” are used interchangeably herein , the terms “ foot ,” “ foot web ,” “ foot - web ,” “ toe ,” “ toe web ” and “ toe - web ” are used interchangeably herein , and the terms “ odor ” and “ malodor ” are used interchangeably herein . the terms “ cutaneous ” and “ skin ” refer , in the context of the present invention , regions of the human body including , e . g ., the axilla , foot - webs and nasal atrium . the terms “ physiologically acceptable ” and “ physiologically - acceptable ” denote , in the context of the present invention , “ safe and effective when administered to humans and / or mammals in need thereof ,” e . g ., to reduce axillary odor , promote the growth of staphylococcus epidermidis bacteria , inhibit the growth of staphylococcus aureus bacteria , or any or all of the preceding . the following examples are intended to illustrate , but not limit , the present disclosure . growth of staph . aureus ( mssa or mrsa ) and staph . epidermidis when one or the other or a mixture of the two bacteria were incubated in the presence of ( i ) cysteine and ( ii ) isoleucine , leucine , phenylalanine . zinc carbonate was also provided with and without arginine bicarbonate at 37 ° c . for 72 hours and with additional above ingredients adding into the cultural media in 37 ° c . water bath in 24 and 48 hours . materials and methods for growth comparison experiments between staph . epidermidis and staph . aureus ( a ) preparation of agar plates containing various bacterial growth media . preparation included ( i ) bhi blood agar ( fisher scientific , springfield , n . j . usa ) and ( ii ) chromagar staph . aureus agar ( chromagar , paris , france ), especially prepared for the isolation and identification of staph . aureus ; if present , it results in colonies that show a characteristic mauve color that enables ease of identification ( french , 2009 , han et al ., 2007 ). ( b ) stock solutions of cil amino acids . these amino acids include cysteine , isoleucine and leucine with each present at a concentration of 72 mm . aqueous solutions of each were sterilized by syringe filtering as described earlier ( zhang and kleinberg , 2014 ). ( c ) stock aqueous solutions of arginine bicarbonate at 144 mm and zinc carbonate at 72 mm . stock solutions of 144 mm arginine bicarbonate were sterilized together with 72 mm zinc carbonate by syringe filtering . zinc carbonate has a limited solubility and hence is sterilized by first autoclaving as a powder and then dissolving it until saturation in sterile distilled water is achieved . this means that at 72 mm and above , it may have to be used as a zinc carbonate suspension . ( d ) rabbit coagulase plasma ( pl 850 ) and prolex staph xtra latex kits ( pl . 1080 ). both of these items are provided as a kit and are obtained from pro - lab diagnostics , austin , tex . they are prepared for the identification of pathogenic staphylococci ( e . g ., staph . aureus ). ( e ) experimental and control incubation mixtures containing staph . epidermidis ( atcc 12228 ) and staph . aureus ( mssa and / or mrsa ). these incubation mixtures were prepared for comparison purposes and included mssa ( atcc 25923 ) or mrsa ( atcc 33591 ) bacterial species mixed with the microorganism staph . epidermidis . pure cultures of staph . epidermidis and staph . aureus ( mssa or mrsa ) were each prepared as 25 % ( v / v ) bacterial suspensions in sterile distilled water . as above and as much as possible , bacterial pellets were broken up into fine particles , by stirring with a sterile tb syringe and a 25 - 27 gauge needle , if and when needed . as a preparatory step , the resulting suspensions obtained were incubated in a shaking water bath at 37 ° c . for one hour , in order to deplete stored substrates acquired by some bacteria , during their preparatory growth period ( wijeyeweera and kleinberg , 1989 ). the ph of each of the above bacterial suspensions was then measured by transferring 0 . 25 ml of such to a small sterile test - tube and measuring its ph . this made it easier to avoid any bacterial contamination during handling . samples were then stored at 4 ° c . until time of inoculation of agar plates . table 1 . 1 experimental ( a and b ) and negative control ( c ) samples were prepared according to the following abc composition tables : a . experimental samples ( ml ) composition i ii iii iv v vi final concentrations amino acids cys 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm ieu 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm ileu 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm zinc carbonate ( 72 mm ) 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 12 mm arg . bicarbonate ( 144 mm ) 0 . 45 0 . 45 0 . 45 — — — 24 mm ( iv , v , vi = 0 mm ) staph . epidermidis ( 25 %) 0 . 45 — 0 . 90 0 . 45 — 0 . 90 8 . 3 % mixture 4 . 15 % staph . aureus 25 % ( mssa or mrsa ) 0 . 45 0 . 90 — 0 . 45 0 . 90 — 8 . 3 % 4 . 15 % d - water 0 . 225 0 . 225 0 . 225 0 . 675 0 . 675 0 . 675 total volume ( ml ) 2 . 70 2 . 70 2 . 70 2 . 70 2 . 70 2 . 70 b . experimental samples ( ml ) composition ia iia iiia ib iib iiib final concentrations amino acids cys 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm ieu 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm ileu 72 mm 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 6 mm zinc carbonate ( 72 mm ) 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 12 mm arg . bicarbonate ( 44 mm ) 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 0 . 45 24 mm staph . epidermidis ( 25 %) 0 . 45 — 0 . 90 0 . 45 — 0 . 90 8 . 3 % mixture 4 . 15 % staph . aureus 25 % ( mrsa ) 0 . 45 0 . 90 — 0 . 45 0 . 90 — 8 . 3 % 4 . 15 % d - water 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 0 . 225 total volume ( ml ) 2 . 70 2 . 70 2 . 70 2 . 70 2 . 70 2 . 70 c . negative controls composition 1 2 3 final concentrations amino acids cys 72 mm — — — — ieu 72 mm — — — — ileu 72 mm — — — — zinc carbonate ( 72 mm ) — — — — arg . bicarbonate ( 144 mm ) — — — — staph . epidermidis ( 25 %) 0 . 45 — 0 . 90 8 . 3 % mixture 4 . 15 % staph . aureus 25 % ( mssa or mrsa ) 0 . 45 0 . 90 — 8 . 3 % 4 . 15 % d - water 1 . 80 1 . 80 1 . 80 total volume ( ml ) 2 . 70 2 . 70 2 . 70 arginine bicarbonate is absent in iv , v and vi dilutions of experimental and negative control samples and inoculations of bhi blood agar and chromagar staph . aureus plates serial dilutions from 10 1 to 10 10 of each of experimental samples i , ii , iii , iv , v , vi and control samples 1 , 2 , 3 ( see table 1 ) were prepared with sterile distilled water . each dilution contained 0 . 1 ml of serial diluted sample and 0 . 9 ml of sterile distilled water . bhi blood agar plates were then inoculated with a mixture of 100 μl of a 10 4 to 10 10 concentration of staph . epidermidis bacteria and 100 μl of a 10 4 to 10 10 sample of staph . aureus ( mssa or mrsa ) mixture ( samples i , iv and negative control 1 ) onto chromagar staph . aureus plates using sterile glass bars on a turning table , respectively . as a first precautionary step , all agar plates were incubated for 24 hours in a 37 ° c . incubator and examined thereafter for bacterial growth to ensure initial agar plate sterility . plates were then inoculated with samples taken at times 0 , 24 , 48 and 72 hours in succession throughout the 4 days of incubation . successive inoculations consisted of the transfer of bacterial samples from a prior incubation to a subsequent fresh sterile plate , followed by incubation at 37 ° c . for 24 - 48 hours and subsequently repeating the process . colony density was scored for each of the plates as follows : between 0 and 10 as 0 — no colonies ; 1 —& lt ; 10 colonies ; 2 — 10 to 20 colonies ; 3 — 20 to 30 colonies ; 4 — 30 to 50 colonies : 5 — 50 to 100 colonies ; 6 — 100 to 250 colonies ; 7 — 250 to 500 colonies ; 8 —& gt ; 500 colonics ; 9 — colonies almost fused to form a layer ; 10 — colonies forming a bacterial layer . differentiation of colonies of staph . aureus and staph . epidermidis derived from growth on bhi blood and chromagar sa plates of samples from incubation mixtures with staph . aureus and staph . epidermidis staph . aureus colonies are usually a golden yellow color and show large and complete blood hemolytic rings around the colonies that grow on bhi blood agar plates . use of the coagulase serum test ( test procedure of rabbit coagulase plasma provided by pro - lab diagnostics , austin , tex . usa ) and prolex staph xtra latex test ( test protocol of prolix ™ staph xtra latex kit provided by pro lab diagnostics , austin , tex . usa ) showed positive results . on chromagar staph . aureus plates , where staph . aureus colonies readily grow , they show , as pointed out above , a mauve color . in contrast , their counterpart , staph . epidermidis colonies , are white and have no or small hemolytic rings around the colonies , when grown on bhi blood agar plates . on chromagar staph . aureus plates , staph . epidermidis is unable to grow or able to form tiny white colonies . coagulase serum and prolex staph xtra latex testing proved negative ( i . e . no coagulation ). inoculation of samples incubated in a water bath at 37 ° c . for 24 hours and then inoculated onto ( i ) bhi blood agar plates and ( ii ) chromagar staph . aureus plates following the same serial dilution procedures , as done for the day i incubation period , samples i , ii , iii , iv , v , vi and 1 , 2 , 3 were diluted serially 10 4 to 10 10 on bhi blood agar plates . similarly , samples of a mixture of staph . epidermidis and staph . aureus ( i , iv and negative control 1 ) were prepared on chromagar staph . aureus plates and incubated using the same procedures , as were used on day 1 , i . e . incubation at 37 ° c . for 24 - 48 hours . addition of extra ingredients to samples , ia , iia , iiia and ib , iib , iiib incubated as on day 1 , in a water bath at 37 ° c . for 24 hours under aseptic conditions , samples , ia , iia , iiia and ib , iib , iiib were each centrifuged and 1 . 35 ml of supernatant was removed from each of samples , ia , iia , iiia , and 1 . 125 ml of supernatant from samples , ib , iib , iiib , respectively . incubation of all experimental and control samples in a 37 ° c . water bath was continued for another 24 hours . total incubation time to this point was 48 hours . day 3 in the experimental protocol ( i . e ., the 48 - 72 hour time period ). this period consisted of bacterial growth on the medium agar plates inoculated on day 2 and incubated at 37 ° c ., ( as above ), on medium agar plates for another 24 hours and preparation of samples for incubation continuation for another 24 hours . bacterial growth on bhi blood agar and chromagar staph . aureus plates was then determined as before . the next step was inoculation of samples incubated in a 37 ° c . water bath for a total of 48 hours on the bhi blood agar plates and chromagar staph . aureus plates . the same procedures of serial dilutions , as was done on day 1 , was carried out here ; i . e . all samples ( i , ii , iii , iv , v , vi , 1 , 2 , 3 and ia , iia , iiia , ib , iib , iiib ). inoculated 10 4 to 10 10 serial dilutions of samples on bhi blood agar plates and the samples of the mixture of se and sa ( i , ia , ib , iv and negative control 1 ) on chromagar staph . aureus plates were tested by following the same procedures as was done on day 1 . plates were incubated as before at 37 ° c . between and for 24 and 48 hours . preparation of samples for incubation in a water bath at 37 ° c . for 48 hours and followed then for a further 24 hours additional ingredients were added to samples of ia , iia , iiia and ib , iib , iiib , which were each incubated in a 37 ° c . water bath for a total period of 48 hours . samples ia , iia , iiia and samples ib , iib , iiib were centrifuged as before and 1 . 35 ml of supernatant was removed from samples , ia , iia , iiia ; and 1 . 125 ml of supernatant was also removed from samples , ib , iib , and iiib , respectively . table 1 . 3 , below , was followed in order to serve as a guide for adding additional ingredients into the samples : incubation of all experimental and control samples in the water bath at 37 ° c . was extended for another 24 hours ( i . e . 72 hours total ). day 4 ( 72 - 96 hours , i . e ., the last part of the instant experimental protocol ) bacterial growth on medium agar plates inoculated on day 3 was examined and then incubated in a water bath at 37 ° c . for a total of 72 hours . examination of bacterial growth on bhi blood agar and chromagar staph . aureus plates inoculated on day 3 the same methods were followed as was done on day 4 . inoculation of samples incubated at 37 ° c . for a total of 72 hours on bhi blood agar plates and chromagar staph . aureus plates the same procedures of serial dilution were followed as was done on day 1 for all samples ( i , ii , iii , iv , v , vi , 1 , 2 , 3 and ia , iia , iiia , ib , iib , iiib ). inoculation of 10 4 to 10 10 serial dilutions of samples on bhi blood agar plates and the samples of the mixture of se and sa ( i , ia , ib , iv and 1 ) on chromagar staph . aureus plates were the same as the procedures carried out on day 1 . plates were then incubated at 37 ° c . for 24 - 48 hours . examination of bacterial growth on media agar plates inoculated on day 4 and a review of the entire experiment was performed . examination of bacterial growth on bhi blood agar and chromagar staph . aureus plates inoculated was performed on day 4 by following the same methods as was done on day 1 . overview of the bacterial growth of all samples on the bhi blood agar plates and on the chromagar staph . aureus plates in the 72 hour experiments reported herein are shown in tables 1 . 4 , 1 . 5 and 1 . 6 . fig1 - 15 depict the effect of different media on bacterial growth . photographs showing colony growth data from which the figures were derived are set forth as fig1 - 23 . ( mssa ) when incubated in media comprised of 6 mm cysteine , 6 mm isoleucine , 6 mm leucine ( i . e ., 6 mm cil ) and 12 mm zinc carbonate , with or without 24 mm arginine bicarbonate at 37 ° c . for 72 hours , compared with negative control ( water only ) chro , chromagar medium plate selective for staph . aureus , x , contamination * scale ( 0 - 10 ): 0 , no colony ; 1 , & lt ; 10 ; 2 , 10 - 20 ; 3 , 20 - 30 ; 4 , 30 - 50 ; 5 , 50 - 100 ; 6 , 100 - 250 ; 7 , 250 - 500 ; 8 , & gt ; 500 ; 9 , colonies almost form a layer and are unable to count ; 10 , colonies form a layer density ( 1 - 10 *) of colonies of staphylococcus epidermidis ( se ) and staphyloccus aureus ( mrsa ) when incubated in media comprised of 6 mm cysteine , 6 mm isoleucine , 6 mm leucine ( i . e ., 6 mm cil ) and 12 mm zinc carbonate , with or without 24 mm arginine bicarbonate at 37 ° c . for 72 hours , compared with negative control ( water only ) chro , chromagar medium plate selective for staph . aureus , x , contamination * scale ( 0 - 10 ): 0 , no colony ; 1 , & lt ; 10 ; 2 , 10 - 20 ; 3 , 20 - 30 ; 4 , 30 - 50 ; 5 , 50 - 100 ; 6 , 100 - 250 ; 7 , 250 - 500 ; 8 , & gt ; 500 ; 9 , colonies almost form a layer and are unable to count ; 10 , colonies form a layer density ( 1 - 10 *) of colonies of staphylococcus epidermidis ( se ) and staphyloccus aureus ( mrsa ) when incubated in media comprised of 6 mm cysteine , 6 mm isoleucine , 6 mm leucine ( i . e ., 6 mm cil ) and 12 mm zinc carbonate , with or without 24 mm arginine bicarbonate at 37 ° c . for 72 hours , compared with negative control ( water only ) chro , chromagar medium plate selective for staph . aureus , x , contamination * scale ( 0 - 10 ): 0 , no colony ; 1 , & lt ; 10 ; 2 , 10 - 20 ; 3 , 20 - 30 ; 4 , 30 - 50 ; 5 , 50 - 100 ; 6 , 100 - 250 ; 7 , 250 - 500 ; 8 , & gt ; 500 ; 9 , colonies almost form a layer and are unable to count ; 10 , colonies form a layer ( a ) incubation of staph . epidermidis and staph . aureus and their combinations in the medium containing 12 mm zinc carbonate , 24 mm arginine bicarbonate , the cil amino acids and their controls , showed : ( i ) staph . aureus ( mssa or mrsa ) quickly decreased , when incubated in the presence of arginine bicarbonate for 24 to 48 hours ; all staph . aureus organisms completely disappeared by 72 hours ( see supporting fig1 , 16 and 19 ). ( ii ) staph . epidermidis on the other hand decreased only slightly , when incubated with the medium containing arginine bicarbonate during the first 24 hours of incubation and decreased moderately or rapidly in the 48 to 72 hours thereafter ( see relevant fig1 , 8 , 9 , 16 and 19 ). ( iii ) the mixture of staph . aureus ( mssa or mrsa ) and staph . epidermidis also showed decreases , albeit only moderately , while being incubated in the medium containing arginine bicarbonate for 24 hours and where approximately 60 - 80 % of survivors were staph . epidermidis . the staph . aureus / staph . epidermidis mixture decreased quickly after 24 hours of incubation and almost all of the bacteria had disappeared by 72 hours ( see fig3 , 18 and 22 ). ( iv ) in the negative control , both staph . aureus ( mssa or mrsa ) and staph . epidermidis and the mixtures thereof incubated in sterile d - water , showed almost no reduction in 24 to 48 hours and very slight reduction in 48 to 72 hours ( see fig1 , 16 and 19 ). ( b ) incubating staph . aureus ( mssa or mrsa ), staph . epidermidis and their combinations in a medium containing the cil amino acids , and zinc carbonate without arginine bicarbonate exhibited : ( i ) staph . aureus ( mssa or mrsa ) that showed no or slight reduction , while incubating for 24 to 48 hours and then decreased slightly or moderately thereafter . staph . aureus showed much slower reduction of its numbers in the medium without arginine bicarbonate than when incubated in medium containing arginine bicarbonate ( see fig2 , 17 and 20 ). ( ii ) staph . epidermidis showed moderate to rapid reduction in numbers during incubation for 24 hours and disappeared after 48 hours ( see fig2 , 17 and 20 ). ( iii ) within 72 hours , the mixture of staph . aureus ( mssa or mrsa ) and staph . epidermidis decreased moderately , while incubating in medium without arginine bicarbonate . also , within 72 hours , approximately 70 - 90 % of survivors were staph . aureus , whereas in the mixture incubated in the medium containing arginine , bacteria correspondingly decreased slowly in 24 hours . about 70 - 75 % of survivors were staph . epidermidis and the mixture rapidly decreased in 48 to 72 hours . almost all bacteria disappeared by 72 hours ( see fig3 , 18 , 22 and 23 ). ( c ) the results of staph . aureus ( mrsa ) and staph . epidermidis being incubated in the medium including 12 mm zinc carbonate , 24 mm arginine bicarbonate , the cil amino acids , and additional same medium or 24 mm arginine bicarbonate being added in 24 and 48 hours during 72 hours of incubation at 37 ° c . showed : ( i ) slow staph . epidermidis reduction during the first 24 hours and slower reduction after 48 to 72 hours , when additional same medium was added , at 24 and 48 hours . staph . epidermidis even decreased , albeit more slowly , when additional 24 mm arginine bicarbonate was added after 24 and 48 hours , whereas staph . aureus ( mrsa ) decreased , moderately to rapidly , after 48 hours with no microbial survivors after 72 hours . there were no differences among the incubation media and additional medium , whether arginine bicarbonate was or was not added ( see fig1 and photo 21 ). ( ii ) the mixture of staph . aureus ( mrsa ) and staph . epidermidis decreased in a similar pattern , as did staph . epidermidis with 60 % of survivors being staph . epidermidis after 24 hours of incubation and more than 90 % staph . epidermidis survivors after 48 to 72 hours of incubation ( see fig1 and photo 23 ). ( d ) staph . aureus ( mssa or mrsa ) was incubated with 12 mm zinc carbonate , 24 mm arginine bicarbonate and the cil amino acids and decreased more and faster than being incubated in medium without arginine bicarbonate . this occurred within 72 hours of incubation , especially after 24 hours of incubation , when compared to samples diluted 10 4 to 10 6 ( see fig4 and 12 ). in contrast , staph . epidermidis decreased much less and more slowly in media containing arginine bicarbonate than being incubated in media without arginine bicarbonate , especially during 72 hours of incubation ( see fig5 and 13 ). ( e ) the ph values of staph . epidermidis , staph . aureus ( mssa or mrsa ) and mixtures thereof , when incubated with zinc carbonate , cil and with or without arginine bicarbonate , and additional same medium or 24 mm arginine bicarbonate being added at 24 and 48 hours during 72 hours of incubation at 37 ° c ., in comparison to a negative control ( see fig7 and 15 ). ( i ) ph values of se , sa and their mixture incubated in media containing arginine bicarbonate were stable at ph 8 . 3 to 8 . 6 . ( ii ) ph values of se , sa and their mixture incubated in media without arginine bicarbonate stayed at lower ph levels i . e . 6 . 1 to 6 . 8 . ( iii ) bacteria incubated in sterile distilled water that served as negative controls , had similar ph values , as counterpart bacteria incubated in media without arginine bicarbonate at ph 6 . 0 to 6 . 4 . the results obtained in the experiments above demonstrated that a medium of 12 mm zinc carbonate , 24 mm arginine bicarbonate and 6 mm cil ( i . e ., 6 mm of each of cysteine , isoleucine and leucine ), when incubated in a water bath at 37 ° c . for 72 hours , was able to bring about a decrease in both staph . epidermidis ( se ) and staph . aureus ( mssa or mrsa ) levels ( fig1 and 8 ). however , such a medium favored much of a reduction of staph . aureus ( mssa or mrsa ) and did so significantly more rapidly than reduction of staph . epidermidis ( fig2 and 9 ). the number of both bacteria decreased sharply after 24 hours of incubation ( fig2 and 9 ). this appeared to be due to substrate depletion , since addition of arginine bicarbonate to the medium during the staph . epidermidis incubation only decreased its numbers slightly ( fig1 ). to be noted , staph . aureus ( mrsa ) showed no positive selection at all . almost all of the staph . aureus ( mrsa ) bacteria involved had disappeared after 48 to 72 hours ( fig1 ). in contrast ( see fig4 , 12 and 13 ), when staph . epidermidis was incubated without arginine bicarbonate present , its numbers decreased much sooner than when the medium contained arginine bicarbonate . staph . aureus ( mssa or mrsa ) showed opposite results . this implies that the medium containing 12 . 0 mm zinc carbonate , 24 . 0 mm arginine bicarbonate and 6 . 0 mm cil amino acids was able to inhibit the growth of staph . aureus ( mssa or mrsa ), while maintaining growth of staph . epidermidis . in other words and needing emphasis is that arginine bicarbonate was able to support the growth of staph . epidermidis , while not similarly benefiting staph . aureus ( mssa or mrsa ) at all . ( 1 ) media containing arginine bicarbonate was able to maintain the media ph at a constant 8 . 3 - 8 . 6 ph level during 72 hours of incubation ( see fig7 and 15 ). this was beneficial for the growth of staph . epidermidis , which has proven herein to be a major bacterium for maintenance of a normal skin microflora and for suppressing staph . aureus ( mssa or mrsa ), i . e . pathogens of considerable concern . the medium containing zinc carbonate and cil , but with no arginine bicarbonate present , had a ph between 6 . 1 and 6 . 8 ( see fig7 and 15 ), which evidently was able to inhibit the growth of staph . aureus ( mssa or mrsa ) slightly to moderately ( see fig6 and 14 ). but , it was not able to strongly inhibit staph . aureus ( mssa or mrsa ), in a medium containing arginine bicarbonate ( see fig6 and 14 vs . 4 and 12 ). in contrast , staph . epidermidis was quickly reduced in this medium ( fig5 and 13 ). this would most importantly imply that a reason for this is that the alkaline ph ( 8 . 3 - 8 . 6 ), which promoted the growth of staph . epidermidis , and its anti - staph . aureus effectiveness , resulting in reduction of the growth of staph . aureus ( mssa or mrsa ). ( 2 ) evidently , as explanation , the ph may not have been the only factor to affect the survival of staph . epidermidis and staph . aureus . although the overall ph of the medium ( zinc carbonate , arginine bicarbonate and cil ) and additional same medium or 24 mm arginine bicarbonate being added at 24 and 48 hours during 72 hours of incubation , was maintained at ph 8 . 3 - 8 . 6 ; it showed remarkably well that as more arginine bicarbonate was added to the medium , the density of staph . epidermidis that was ultimately obtained was increased . nonetheless and most importantly , this indicated that arginine bicarbonate can play a significant enhancement role in the growth of staph . epidermidis and that this effect may be largely but not solely due to the elevated and sustained ph favored by the presence of arginine bicarbonate . in contrast , staph . aureus ( mssa or mrsa ) incubated in the medium containing zinc carbonate , cil and no arginine bicarbonate or in a sterile distilled water negative control ( both of which show a ph in the range of 6 . 0 - 6 . 8 ) showed almost no reduction in growth after 72 hours of incubation in distilled water ( see fig1 , 8 and 15 ). however , there was moderate reduction during incubation for 72 hours in a medium containing zinc carbonate , and cil without arginine bicarbonate ( see fig6 and 14 ). accordingly , one can conclude that zinc carbonate is an important ingredient for suppression of staph . aureus ( mssa and mrsa ) growth , and plays thereof a significant inhibitory role as well . the present invention is not limited in scope by the specific embodiments described herein . indeed , various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures . such modifications are intended to fall within the scope of the appended claims . it is further to be understood that all values are approximate , and are provided for description . patents , patent applications , publications , product descriptions , and protocols are cited throughout this application , the disclosures of which are incorporated herein by reference in their entireties for all purposes . 1 . centers for disease control and prevention : public health dispatch : outbreaks of community - 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