01/28/2016 16:40 FAX 8013925192 MOTHER EARTH MINERALS 14)001 1/28/16 To: Jim Dooley From; Mother Earth Minerals (Five Pages) MIC and MBC Evaluation of Pure Products Antimicrobial Product against Selected Bacteria: Staphylococcus aureus E.coli
- 01/28/ 2016 16 :4 0 FAX 8013925192 MOTHER EARTH MINERALS @ 003 Rese a rc h Cor pora t ion 3437 SW 24 Avenue fox: 352-378-6483 Gir.mdllc, Ft. ;.2607 E-rmiil: rnfo -Oabr:r.rum l'el: 352.-372,-0436 Web: www.abcr.com A FOOD TESTING LABORATORY SlNCE 1967 PROJECT REPORT RESEARCH MICROBIOLOGY DEPARTMENT DAIE: August 29, 2006 PREPARED FOR: CLIENT CONTACT: OBJECTIVE: EXPERIMENTAL APPROACH: A!ESTPRODUCT AND.MATERIALS MIC and MBC Evaluation of Pure Products Antimicrobial Product against Selected Bacteria. To dcterm.i.ne the MIC (minimal inhibitory concentration) and MBC (miuimal bactericidal concentration (MBC) for Pure Products antinlicrobi.nl product against E. coli and Staphylococcus aureus using standard NCCLS/CLSl roethods. The client provided sufficient Pure Products antimicrobial product to use in the study. B. TEST MICROOROANISMS Generic Escherichia coli (ATCC #11229) and methicillin resistant Staphylococcus aureus (ATCC #700698) were used.in the MIC and MBC evaluations. Generic E. coli is a common Gram-negative bacterial indicator of human/animal feces in food and water. S. aurew is 11 common Gram-positive bacterial pathogen associated with clinical (e.g_, skin and nosocomial infections). Antibiotic resistant strains of S. aureus are of particular clinical concern. C. MIC AND MBC PROCEDURES MIC (minimal inlubitory concentration) assays for the antimicrobial were conducted separately against each selected microorganism (see section B) using the broth macro-dilution procedure for antibiotic susceptibility testing per NCCLS/CLSl methods (''National Committee for Clinical Laboratory Sta.ndatds"; now.referred to as "Clinical and Laboratory Standards Institute"). The inoculum for each microorganism was prepared and the MlC assay conducted. according to respective culture methods de5cribcd by NCCLS (M7-A5, 2000). The E. coli and S. aureus cultures were purified and cultured ming pre-poured Tryptic Soy agar (TSA) plates (incubated.for 20-24 h. at 35 C). Bacterial suspensions were each prepared from the respective ISA plates and diluted per NCCLS method (to a 0.5 McFarland standard
01/28/2016 16:40 FAX 8013925192 MOTHER EARTH MINERALS @002 AquaVivos (dba Pure Products) Its Effectiveness Against E.Coli Recent tests were conducted to determine the effectiveness of AquaVivos (dba Pure Products) against Escherichia coli (E. coli) bacteria. The tests were conducted over a 24 hour period by ABC Research Corporation. a food testing laboratory, in Gainesville, FL. Table 2. Minimal Bactericidal Concentration (l\'lbc) Results for E. coli. 1\ntimkrobinl lbctl.rii Counts CFU.iml l.oqrn C FU/ml Initial fooculum 590.000.c;'ii r I,, J :6 Sun'h or Coun 1 <10 <1.HO l.01!1, RNI ucti,,n >4.77!,. Rt d uct'iun >99.998 J :12 Sun h or Conni <10 <l.00 Loit1111 Rtduction :>4.17 It> R\'duction >99.1)98 1:24 Sunfror Cl)unt..: 10 <1.00 lo-.. Rt dt1cf i11n >4.77 % Rl'd uction ;:-..99.998 bl8 S!:!n'lt or Coml1 <10 <1.00 l.0 1, Rl cl ucfom >4.77 % Rtdudion >99.998 1:% Sllnin,r Co1m1 t.200 3.0S I,f'lg 11 ]l1. duco,,n 2.69 "!., Rc. duction 99.8 Noti;-s: l ) (\, 11':\l:- xpn.:s o.:j ::i ('f l.i \\, km)" fr,rm in&! un.it/ml). 2) l.\l Rl.!d llt.:l inn (lo!:,! 0.:\1U1H f initinl in\'ul um) - iii' count,.,f ulk t survin.1r:-.)..1} Th MBC!i...:..,ll kalll n -h)g \'J' 99. l)'ji, rcducli\,.ll ) w.is al 1 1:4 dilutk,n "'t' t.h an1i1nk1'<,h i,.i. The dilution rate of 1:48 (1 part AquaVivos to 48 parts water) provided the most efficient results, reducing the E. colibacteria 99.998%.
01/28/2016 16:41 FAX 8013925192 fother EARTH MINERALS 14]004 Page 2 of 4 in sterile 0.85% saline) to obtain respective cell suspensions to inoc'ltlate the rnacrodihrtion series. The final inoculum auspensions were enumerated by making appropriate serial dilutions in Butterfield's phosphate buffer (BPB) and spread plating 0.1 ml of each dilution onto pre-poured TSA plates. The coi:respomling MIC assays were conducted using Mueller Hinton broth (MHB) to prepare the macro-dilution series of the antimicrobial product (i.e., from 1 :6 through 1;24576) in M.HB. The (MIC) dilution tubes were incubated for 24 h at 35QF. The MIC is the lowest concentration of antimicrobiaj agent that completely inhibits growth of the bacteria. Minimal bactericidal concentration (MBC) assays were determined concw:rently against each microorganism per standard methods (i.e., Manual of Clinical Microbiology, 2003). After incubation of the MHB tubes and detcnnination of the MIC, the surviving bacteria in each tube with concentrations at and above the MIC was enumerated in duplicate using spread plating technique and pre-poured TSA plates. Using the initial.inoculwn counts and the viable counts in respective MHB tubes, t..1..e lethality of each res,pective tube was determined. The MBC is the lowest concentration of antimicrobial which yields at least 99.9% reduction (i.e., 3 log-units) during the MIC incubation (i.e., 24 h at 35 C). RESULTS: The minimal inhibitory concentration (MIC) results for. the Pure Product antimic.robia1 against E. coli and Staphylococcus aureus are presented in Table 1. The MIC value for the antimicrobial against E. coli was at the 1:48 dilution (i.e.) 2.08% concentration of active agent) and that for the S. aureus at the l :24 dilution (i.e., 4.17% concentration of active ageot). The minimal bactcrici.dru concentration (MBC).results for the Pure Product antimic.rnbial against E. coli and Staphylococcus aureus are presented in Tables 2 and 3, respectively. Note th.at these bactericidal (reduction) results apply to a 24 h period in a growth medi'un1per the; standard NCCLS/CLSI methods. There was a greater than 4.77 logwunit (>99.998%) reduction of E. coli at the antimicrobial 's l :48 dilution and a 3.08 log10-unit reduction (99.8%) at the 1:96 dilution. For S. atireus, there was a 2.11 log 1o-unit (99.2%) reduction at the l ;6 dilution, a 2.13 Jog 1o-unit (99.3%) reduction at the 1:12 dilution, and a l.13 1og 1o-unit reduction (92.6%) at the 1:24 dilution. In summary, these results indicated that the PUi\'i Pr:oduct antimicrobial was more effective in the inhibition and reduction of E. coli (a Gram-negative bacterium) than of S. aureus (a Grampositive bacterium). James E. (Ken) Kennedy, Ph.D. Vice President., Research Microbiology ABC Research Corp.
01/28/2016 16:42 FAX 8013925192 MOTHER EARTH MINERALS @5 Page 3 of 4 Table 1. Minimal Inhibitory Concentration (MIC) Results for E. coli and S. aureus Anthnlcrobial E. coli (growth) $. IJW'eU.'J (l!rowth) 1:6 - - 1:12-1:24 - -lmicl 1:48 - lmic) + l:96 + + 1:192. + + 1:384 + + 1:768 + + 1;1536 + + l:3075 + + 1:6144 + + 1,12288 + + 1:24576 + + Note: 1) The MIC is the lowest concentrntion of antimicrobial agent that completely inhibits growth of the bucteria. 2) Toe MlC was at the 1:48 dilution of the a,o.timicrobiri for E. coli and the I :24 dilution of the a.ntimkrobial for!;'. aureus.
01/28/2016 16:42 FAX 8013925192 MOTHER EARTH MINERALS @6 Page 4 of 4 Table 2. Minimal Bactericidal Concentration (MBC) Results for E. coli. Antimicrobial - - Bacterial Counts CFU/ml Log 10 CFU/ml Initial Inocuh1m 590,000 5.77 1:6 Sul"'iVor Cowit <10 <l.00 Log10 R.edudlon >4.77 % Reduction >99.998 1:12 Survivor Co,Jnl <10 <l.00 Lo210 Reduction >4.77 % Red111:tton >99.998 1:24 Survivor Coun1 <10 <l.00 Lo210 Reduction >4,77 % Reduction :>99.998 1:48 Sunolvor Cou111 <10 <1.00 Log10 Reduction >4.77 8/u Reduction :>99.998 1;96 SW'Vlvor Couni 1.,200 3.08 Loirn Reduction 2.69 % Reductio11 99,8 Notes: 1) Couots expressed as CFU (colony forming units/ml). 2) Log Reduction = (log count of initial {noculum) - (log count of subject survivors). 3) Th1,; MBC (i.e., at least a 3-log or 99.9% reduction) was at a 1:48 dilutio.n of th<: antimicrobial. Tab)e 3. Minimal Bactericidal Concentr11tion (MBC) 'Results for $. aureus. Antimict'obtal Baci:eria.l Counts CFU/ml Login CFU/anl Initial Inoeuhm1 270,000 5.43 1;6 Survivor Counc 2.100 3.32 Log 18 Reduction 2.11 o/u Rllduclf o11 99.Z t:u Survivor Couni 2,000 3.30 LOf!tu Reduction 2.13 % Reduction 99.3 1:14 Survi'\lor Count 20,000 4.30 Login Reduction 1.13 % Reduction 92.6 Notes: 1) Counts expressed as CFU (colony fonning units/ml). 2) Log Reduction = (log count of initial jnoculum) - (log count of 6Ubject survivo:ri;). 3) The MBC (i.e., at least a 3 log or 99.!)%.reductioo) was not achieved at any dilution of the ant.in:ucrobial.