Compositions and methods for altering human cutaneous microbiome to increase growth of Staphylococcus epidermidis and reduce Staphylococcus aureus proliferation

A composition including arginine or a salt thereof, a zinc salt, preferably arginine bicarbonate and zinc carbonate (ABZC), 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 and also promote the growth of non-pathogenic Staphylococcus epidermidis bacteria, and methods for using such composition.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for selectively increasing the growth ofStaphylococcus epidermidisand inhibiting the growth ofStaphylococcus aureusbacteria in the cutaneous microbiome. More particularly, the present invention relates to compositions and methods for increasing the growth ofStaphylococcus epidermidisand reducing the incidence of MRSA and MSSA by the selective inhibition ofStaphylococcus aureus.

BACKGROUND OF THE INVENTION

The cutaneous microbiome in humans is comprised of a variety of microorganisms, of which staphylococci, corynebacteria and propionibacteria are among the most prominent (Starkemann et al., 2005, Troccaz et al., 2004, Jackman, 1982). These bacteria act upon odorless precursors contained in sweat per se, producing sugars, sugar amines, amino acids, and short chain carboxylic acids (SCCAs), of which some are degraded further to products that include odorants that are associated to a major extent with cutaneous odor (Zeng et al, 1991; Jackman, 1982).

One frequent undesirable member of the cutaneous microbiome,Staphylococcus aureus(Staph. aureus, including methicillin-resistantStaph. aureus(MRSA) and methicillin-susceptibleStaph. aureus(MSSA)), has a well-known role in invasive infections in humans. It is one of the most problematic of human pathogens, because it is capable of wide infection and fatalities (see, e.g., David et al., 2010, Mainous III et al., 2006, Klevens et al., 2007). Antibiotics used against it have achieved limited success. Methicillin is effective but limited because of adaptation, which can result in the emergence of MRSA, which is representative of antibiotic failure occurring now more so with increasing frequency of use (see, e.g., David et al 2010, Chen et al 2006, Centers for Disease Control and Prevention 2003).

SUMMARY OF THE INVENTION

The present invention is directed to compositions of zinc salts and arginine and/or its salts for the selective inhibition ofStaph. aureusgrowth and the favoring of the growth ofStaph. epidermidis, and methods for using such compositions.

The present invention is directed to topical cutaneous compositions including arginine or a salt thereof, a zinc salt, and, optionally, a buffer for maintaining the pH of the composition at 6.0 or greater, and methods for using such compositions. The compositions and methods of the invention are useful in selectively inhibiting the growth ofStaphylococcus aureusand increasing the growth ofStaphylococcus epidermidisbacteria in the cutaneous microbiome.

DETAILED DESCRIPTION OF THE INVENTION

Corynebacteria, staphylococci and proprionibacteria are among the main microorganisms present in the cutaneous microbiome, withStaph. epidermidis, C. striatumandP. avidumas prominent representative bacteria

Unexpectedly, it has been discovered that certain compositions including a zinc salt and arginine and/or its salt are useful as compositions for modifying cutaneous microflora, inhibitingStaph. aureusgrowth while favoringStaph. epidermidisgrowth. This ability to select betweenStaph. aureusandStaph. epidermidisallows the treatment of significant physiological and health-related disease conditions caused by aberrant or excessive growth ofStaph. aureus(see, e.g., Peacock et al., 2001, Uehara et al., 2000). AlthoughStaph. aureusis 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 resistantStaph. aureusstrains, 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 byStaph. epidermidis, can reduceStaph. 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 betweenStaph. aureusand other prominent members of the cutaneous microflora, e.g.,Staph. epidermidis(see, e.g., Frank et al., 2010, Uehara 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 targetingStaph. aureus, render an ecological change that favors selection of desirableStaph. epidermidisover non-desirableStaph. aureusbacteria.

The compositions of the present invention targetStaph. aureusdirectly, and also enhance the ability of other, non-pathogenic bacteria (e.g.,Staph. epidermidis) to out-competeStaph. 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 for modification of the cutaneous microflora.

The 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.

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 ofStaphylococcus epidermidisbacteria, inhibit the growth ofStaphylococcus aureusbacteria, or any or all of the preceding.

EXAMPLES

The following examples are intended to illustrate, but not limit, the present disclosure.

Growth ofStaph. aureus(MSSA or MRSA) andStaph. epidermidiswhen 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 betweenStaph. epidermidisandStaph. 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) CHROMagarStaph. aureusagar (CHROMagar, Paris, France), especially prepared for the isolation and identification ofStaph. 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.(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 containingStaph. epidermidis(ATCC 12228) andStaph. 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 microorganismStaph. epidermidis. Pure cultures ofStaph. epidermidisandStaph. 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.

Preparation of Experimental and Control Samples

Preparation was performed according to information in Table 1 below.

Serial dilutions from 101to 1010of 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 104to 1010concentration ofStaph. epidermidisbacteria and 100 μl of a 104to 1010sample ofStaph. aureus(MSSA or MRSA) mixture (Samples I, IV and Negative Control 1) onto CHROMagarStaph. aureusplates using sterile glass bars on a turning table, respectively.

Incubation Procedures

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.

Differentiation of Colonies ofStaph. aureusandStaph. epidermidisDerived from Growth on BHI Blood and CHROMagar SA Plates of Samples from Incubation Mixtures withStaph. aureusandStaph. epidermidis

Staph. aureuscolonies 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 CHROMagarStaph. aureusplates, whereStaph. aureuscolonies readily grow, they show, as pointed out above, a mauve color. In contrast, their counterpart,Staph. epidermidiscolonies, are white and have no or small hemolytic rings around the colonies, when grown on BHI Blood agar plates. On CHROMagarStaph. aureusplates,Staph. epidermidisis 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) CHROMagarStaph. aureusPlates

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 104to 1010on BHI Blood agar plates. Similarly, samples of a mixture ofStaph. epidermidisandStaph. aureus(I, IV and Negative Control 1) were prepared on CHROMagarStaph. aureusplates 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.The table immediately below, lists additional ingredients introduced into samples:

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 CHROMagarStaph. aureusplates 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 CHROMagarStaph. aureusplates.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 104to 1010serial 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 CHROMagarStaph. aureusplates 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 HoursAdditional 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:

TABLE 1.3The (ml) volumes added to the experimental samplesIngredientsIAIIAIIIAIBIIBIIIBCys 72 mM0.2250.2250.2250.2250.2250.225Ieu 72 mM0.2250.2250.2250.2250.2250.225Ileu 72 mM0.2250.2250.2250.2250.2250.225Zinc Carbonate0.2250.2250.225———(72 mM)Arg. Bicarbonate0.4500.4500.4500.4500.4500.450(144 mM)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 CHROMagarStaph. aureusplates 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 CHROMagarStaph. aureusPlates

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 104to 1010serial 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 CHROMagarStaph. aureusplates were the same as the procedures carried out on Day 1.Plates were then incubated at 37° C. for 24-48 hours.
Day 5 (End of Experiment, 96 Hours Total Duration)

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 CHROMagarStaph. aureusplates inoculated was performed on Day 4 by following the same methods as was done on Day 1.

Results

Overview of the bacterial growth of all samples on the BHI Blood agar plates and on the CHROMagarStaph. aureusplates in the 72 hour experiments reported herein are shown in Tables 1.4, 1.5 and 1.6.FIGS. 1-15depict the effect of different media on bacterial growth. Photographs showing colony growth data from which the Figures were derived are set forth asFIGS. 16-23.

Tables 1.4-1.6, above, include the following elements:(a) Incubation ofStaph. epidermidisandStaph. aureusand 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; allStaph. aureusorganisms completely disappeared by 72 hours (see supportingFIGS. 1, 8, 16 and 19).(ii)Staph. epidermidison 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 relevantFIGS. 1, 2, 8, 9, 16 and 19).(iii) The mixture ofStaph. aureus(MSSA or MRSA) andStaph. epidermidisalso showed decreases, albeit only moderately, while being incubated in the medium containing arginine bicarbonate for 24 hours and where approximately 60-80% of survivors wereStaph. epidermidis. TheStaph. aureus/Staph. epidermidismixture decreased quickly after 24 hours of incubation and almost all of the bacteria had disappeared by 72 hours (seeFIGS. 3, 11, 18 and 22).(iv) In the negative control, bothStaph. aureus(MSSA or MRSA) andStaph. epidermidisand 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 (seeFIGS. 1, 8, 16 and 19).(b) IncubatingStaph. aureus(MSSA or MRSA),Staph. epidermidisand 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. aureusshowed much slower reduction of its numbers in the medium without arginine bicarbonate than when incubated in medium containing arginine bicarbonate (seeFIGS. 2, 9, 17 and 20).(ii)Staph. epidermidisshowed moderate to rapid reduction in numbers during incubation for 24 hours and disappeared after 48 hours (seeFIGS. 2, 9, 17 and 20).(iii) Within 72 hours, the mixture ofStaph. aureus(MSSA or MRSA) andStaph. epidermidisdecreased moderately, while incubating in medium without arginine bicarbonate. Also, within 72 hours, approximately 70-90% of survivors wereStaph. aureus, whereas in the mixture incubated in the medium containing arginine, bacteria correspondingly decreased slowly in 24 hours. About 70-75% of survivors wereStaph. epidermidisand the mixture rapidly decreased in 48 to 72 hours. Almost all bacteria disappeared by 72 hours (seeFIGS. 3, 11, 18, 22 and 23).(c) The results ofStaph. aureus(MRSA) andStaph. epidermidisbeing 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) SlowStaph. epidermidisreduction 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. epidermidiseven decreased, albeit more slowly, when additional 24 mM arginine bicarbonate was added after 24 and 48 hours, whereasStaph. 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 (seeFIG. 10and Photo 21).(ii) The mixture ofStaph. aureus(MRSA) andStaph. epidermidisdecreased in a similar pattern, as didStaph. epidermidiswith 60% of survivors beingStaph. epidermidisafter 24 hours of incubation and more than 90%Staph. epidermidissurvivors after 48 to 72 hours of incubation (seeFIG. 11and 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 104to 106(seeFIGS. 4 and 12). In contrast,Staph. epidermidisdecreased much less and more slowly in media containing arginine bicarbonate than being incubated in media without arginine bicarbonate, especially during 72 hours of incubation (seeFIGS. 5 and 13).(e) The pH values ofStaph. 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 (seeFIGS. 7 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.
Discussion

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 bothStaph. epidermidis(SE) andStaph. aureus(MSSA or MRSA) levels (FIGS. 1 and 8). However, such a medium favored much of a reduction ofStaph. aureus(MSSA or MRSA) and did so significantly more rapidly than reduction ofStaph. epidermidis(FIGS. 2 and 9). The number of both bacteria decreased sharply after 24 hours of incubation (FIGS. 2 and 9). This appeared to be due to substrate depletion, since addition of arginine bicarbonate to the medium during theStaph. epidermidisincubation only decreased its numbers slightly (FIG. 10). To be noted,Staph. aureus(MRSA) showed no positive selection at all. Almost all of theStaph. aureus(MRSA) bacteria involved had disappeared after 48 to 72 hours (FIG. 10).

In contrast (seeFIGS. 4, 5, 12 and 13), whenStaph. epidermidiswas 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 ofStaph. aureus(MSSA or MRSA), while maintaining growth ofStaph. epidermidis. In other words and needing emphasis is that arginine bicarbonate was able to support the growth ofStaph. epidermidis, while not similarly benefitingStaph. aureus(MSSA or MRSA) at all.

As a Non-limiting Explanation:

(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 (seeFIGS. 7 and 15). This was beneficial for the growth ofStaph. epidermidis, which has proven herein to be a major bacterium for maintenance of a normal skin microflora and for suppressingStaph. 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 (seeFIGS. 7 and 15), which evidently was able to inhibit the growth ofStaph. aureus(MSSA or MRSA) slightly to moderately (seeFIGS. 6 and 14). But, it was not able to strongly inhibitStaph. aureus(MSSA or MRSA), in a medium containing arginine bicarbonate (seeFIGS. 6 and 14vs.4and12). In contrast,Staph. epidermidiswas quickly reduced in this medium (FIGS. 5 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 ofStaph. epidermidis, and its anti-Staph. aureuseffectiveness, resulting in reduction of the growth ofStaph. aureus(MSSA or MRSA).(2) Evidently, as explanation, the pH is not the only factor to affect the survival ofStaph. epidermidisandStaph. 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 ofStaph. epidermidisthat was ultimately obtained was increased. Nonetheless and most importantly, this indicated that arginine bicarbonate can play a significant enhancement role in the growth ofStaph. epidermidisand 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 (seeFIGS. 1, 7, 8 and 15). However, there was moderate reduction during incubation for 72 hours in a medium containing zinc carbonate, and CIL without arginine bicarbonate (seeFIGS. 6 and 14). Accordingly, one can conclude that zinc carbonate is an important ingredient for suppression ofStaph. aureus(MSSA and MRSA) growth, and plays thereof a significant inhibitory role as well.

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.

REFERENCES