REDUCTION IN THE BACTERIAL LOAD

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Session 267 PresentaGon 2300 REDUCTION IN THE BACTERIAL LOAD ON THE SKIN IN A CLINICAL SETTING David W. Stroman Co-authors: K. Mintun, A. Epstein, C. Brimer, C. Patel, J. Branch, K. Najafi

The Skin Microbiome From Grice and Segre Nat Rev Micro 2011 9(4): 244-253.

RaGonale of Study Design Hypothesis: In view of the mechanism of ac0on of HOCl and its rapid biocidal ac0vity in solu0on, the removal of bacteria on the surface of the skin would be measurable a=er a single applica0on Primary ObjecGve: To quan0ta0vely determine the bacterial load on the surface of the skin just below the lower eyelid before and 20 minutes a=er a single applica0on of Avenova.

Aspects of Design and Demographic Data To be eligible, subjects had lid inflammagon and for whom daily lid hygiene would be recommended Microbiological specimens were collected OU Before and 20 minutes a^er first HOCl applicagon Demographic data 36 subjects 71 of 72 ocular specimens were processed Female - 22; Male - 14 Average age was 63; range 19-88 yrs

Recovered Bacterial Species Staphylococcus: S. aureus, S. capi0s, S. caprae, S. epidermidis, S. haemoly0cus, S. hominis, S. lugdunensis, S. pasteuri, S. simulans, S. warneri Corynebacterium: C. accolens, C. bovis, C. confusum, C. macginleyi, C. propinquum, C. tuberculostearicum Bacillus: B. circulans, B. pumilus Enterococcus: E. faecalis Micrococcus: M. luteus Rothia: R dentocariosa Streptococcus: S. mi0s Propionibacterium: P. acnes, P. avidum, P. granulosum Prevotella: P. oris Enterobacter: E. aerogenes Moraxella: M. osloensis Neiserria: N. flavescens Pantoea: P. agglomerans Pseudomonas: P. aeruginosa, P. oryzihabitans

Bacterial Diversity on the Skin below the Eyelid Bacterial Groups Number of Species Number of Strains Time = 0 Time = 20 min Time = 0 Time = 20 min Gram-posiGve 22 12 117 35 Anaerobes 4 2 39 23 Gram-negaGve 5 1 6 1

Diversity of Recovered Bacteria (before treatment) Gram-posiGve species Anaerobe species Gram-negaGve species

Diversity of Recovered Bacteria (a^er treatment) Gram-posiGve species Anaerobe species Gram-negaGve species

Bacterial Load on the Skin below the Eyelid Bacterial species* Number of strains CFUs Time = 0 Time = 20 min Time = 0 Time = 20 min Staphylococci 98 24 2,362,786 10,094 (0.43%) S. epidermidis 60 18 1,830,774 9,898 (0.54%) P. acnes* 37 23 12,582 896 (7.1%) *Strain level analysis was not performed

ReducGon of Staphylococcal Load in 20 minutes 10000000 1000000 100000 10000 99.6% 99.5% 1000 100 Time = 0 Time = 20 10 1 All Staphylococci S. epidermidis

SuscepGbility Profile of S. epidermidis AnCbioCc SuscepCble/Resistant Isolates Percent Resistant Isolates Time = 0 Time = 20 min Time = 0 Time = 20 min Oxacillin (Methicillin) >2 43/17 13/5 28% 28% Penicillin >1 33/27 8/10 45% 56% Tobramycin >1 44/16 12/6 27% 33% Clindamycin >1 54/6 15/3 10% 10% Erythromycin >1 32/28 9/9 47% 50% Ciprofloxacin >1 44/16 12/6 27% 33% Tetracycline >2 55/5 15/3 8% 17% Trimethoprim >4 43/13 14/4 22% 22% Sulfamethoxazole >64 7/53 3/15 88% 83%

Summary of Primary Findings The diversity of microbial species is not changed significantly a^er treatment with HOCl Staphylococcal isolates were 60% of the total isolates recovered prior to treatment S. epidermidis isolates were 36% of the total isolates recovered prior to treatment P. acnes isolates were 22% of the total isolates recovered prior to treatment Total staphylococci recovered were reduced by 99.6% 20 minutes a^er treatment with HOCl Total S. epidermidis recovered were reduced by 99.5% SuscepGbility profile of S. epidermidis isolates revealed that one HOCl treatment reduced the the bacterial load of isolates with resistant phenotypes equally well as those with suscepgble phenotypes

Now, for the rest of the story...

Bacterial Groups Species/Isolates Emerging Post-HOCl Original Species Species Number of Unique Isolates Time = 0 Time = 20 min Time = 20 min Time = 0 Time = 20 min New Isolates Time = 20 min 22 12 12 117 35 54 Anaerobes 4 1 2 39 23 6 GramposiGve GramnegaGve 5 0 1 6 0 1 Bacterial species Number of Unique Isolates New Isolates Time = 0 Time = 20 min Time = 20 min Staphylococci 98 24 44 S. epidermidis 60 18 31 P. acnes* 37 23 6 *Strain level analysis not performed

SuscepGbility Profile of new S. epidermidis Isolates AnCbioCc % Resistant strains % Resistant strains Time = 0 Time = 20 min At 20 min Oxacillin (Methicillin) 28% 28% 23% (7/31) Penicillin 45% 56% 35% (11/31) Tobramycin 27% 33% 29% (9/31) Clindamycin 10% 10% 16% (5/31) Erythromycin 47% 50% 45% (14/31) Ciprofloxacin 27% 33% 16% (5/31) Tetracycline 8% 17% 10% 3/31) Trimethoprim 22% 22% 10% (3/31) Sulfamethoxazole 88% 83% 77% (24/31)

CONCLUSIONS The diversity of microbial species is not changed significantly a^er treatment with HOCl Total S. epidermidis recovered were reduced by 99.5% 20 minutes a^er treatment with HOCl HOCl treatment reduced the bacterial load of isolates suscepgble and/or resistant phenotypes to various angbiogcs equally well Skin near lid margin will be recolonized a^er HOCl treatment rapidly by isolates from surrounding skin. This data supports the need for mulgple treatments with HOCl per day for opgmal benefits The resumpgon of the clinical study will assess diversity and the bacterial load a^er two weeks of HOCl applicagons