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Bactericidal activity of combinations
As high level acquired resistance to conventional antibio- of Silver–Water Dispersion™ with 19
tics is frequent, it seems reasonable to use combinationtherapy in order to achieve bactericidal synergism. Active antibiotics against seven microbial
silver solutions have shown marked activity against proven bacterial-resistant strains. Hence, a range of antibioticswere tested with Silver–Water Dispersion™ solution to determine antagonism, additive and synergistic effects A. de Souza1, D. Mehta1 and R. W. Leavitt2,3,*
against a panel of microbial strains.
1Viridis Biopharma Pvt Ltd, 6/312, Jogani Industrial Complex, Antibiotic discs used in this investigation are standardized V. N. Purav Marg, Chunabhatti, Mumbai 400 022, India discs by Pathoteq Biological Laboratories, India: Amoxicillin 2American Biotech Labs, 80 West Canyon Crest Road, Alpine, (AX, 30 mcg), Carbenicillin (CN, 100 mcg), Cefopera- Utah, 84004, USA3Department of Microbiology/Molecular Biology, 727 WIDB, zone (CP, 75 mcg), Ceftizidime (FG, 30 mcg), Ciproflox- Brigham Young University, Provo, Itah 84602-5199, USA acin (RC, 5 mcg), Clindamycin (CD, 2 mcg), Doxycycline(DX, 30 mcg), Erythromycin (ER, 15 mcg), Gentamycin The recent increase in the incidence of infections due to
(GM, 10 mcg), Kanamycin (KA, 30 mcg), Nalidixic Acid bacterial resistance to antibiotics has been recognized
(NA, 30 mcg), Oxacillin (OC, 1 mcg), Penicillin-G (PG, 10 as an alarming problem, especially in the hospital en-
units), Rifampin (RF, 5 mcg), Streptomycin (SM, 10 mcg), vironment with probability of cross-infection. Silver–
Tetracycline(TE, 5 mcg) Tobramycin (TB, 10 mcg) and Water Dispersion®™ solution as an antibacterial is
claimed to have no bacterial resistance. Nineteen anti-
The antibiotic solutions used in the study are biotics were checked in combination with Silver–Water
Dispersion™ solution against seven microbial organisms
KANAMAC-500 (Kanamycin injection, Macleods Phar- for synergism. First, minimal inhibitory concentrations
maceuticals Ltd, Daman); FORTUM* (Ceftizidime injection, were determined for the individual antibiotics and Sil-
GlaxoSmithKline Pharmaceutical Ltd, India); MIKACIN ver–Water Dispersion™ solution individually. Those
(Amikacin injection, Aristo Laboratories Ltd, Daman).
combinations of individual antibiotics with Silver–
MAGNAMYCIN* (Cefoperazone injection, Astral Pharma- Water Dispersion™ that displayed synergism were
further evaluated through the checkerboard method.
The 32 ppm silver in distilled water (S–W D™) was Synergistic activity of Silver–Water Dispersion™ solu-
obtained from American Biotech Laboratories, Utah, USA.
tion in combination with nineteen antibiotics was tested
The organisms used are: E. coli (MDR) strain from stool against seven bacterial strains, except where an organ-
sample; Ps. aeruginosa (multiple-drug resistant) strain from ism was known to be resistant to the antibiotic. Out of
sputum. These two strains were obtained from P.D. Hin- 96 tests, five were synergistic, 89 additive, and two an-
tagonistic.
duja Hospital (Mumbai); Methicillin-resistant S. aureuswas obtained from Lokmanya Tilak Muncipal Hospital.
Keywords:
Antibiotics, bacterial resistance, Silver–Water Shigella flexneri, Salmonella typhi, S. aureus 6538 P, Ba- cillus subtilis and Candida albicans are in-house labora-tory strains.
Nutrient agar (Hi-Media, Mumbai) used in the antibi- ERIOUS infections, particularly antibiotic-resistant, often result in therapeutic failure when treated with seemingly otic spectrum studies and Silver–Water Dispersion® and appropriate single-drug antibiotic regimens, despite readily antibiotic combination studies contains peptic digest of achievable minimum inhibitory concentrations (MICs).
animal tissue 50.00 g/l; yeast extract 1.5 g/l; beef extract The mutations responsible for antibiotic resistance in bac- 1.5 g/l; sodium chloride 5.00 g/l; agar type I 25 g/l; pH teria do not arise as a result of the ‘need’ of the organism. 7.4 ± 0.2. Nutrient broth (Hi-Media, Mumbai) used in the Futuyma1 has noted that: ‘… The adaptive needs of the macrodilution method (MIC) contains peptic digest of species do not increase the likelihood that an adaptive animal tissue 50.00 g/l; yeast extract 1.5 g/l; beef extract mutation will occur; mutations are not directed towards 1.5 g/l; sodium chloride 5.00 g/l; glucose 5 g/l; pH 7.4 ± 0.2.
the adaptive need of the moment. . . .’ Mutations have causes, Actively growing 16-h-old culture was surface-spread but the species need to adapt isn’t one of them’. Alterna- using sterile cotton swabs onto the nutrient agar surface tives must therefore be sought to overcome infections (Hi-Media). The plates were kept aside for absorption for 15 min. The antibiotic discs were then placed onto the Silver–Water–Dispersion™ solution has been shown as agar surface and the plates were incubated at 37°C for an effective antibiotic against many Methicillin-resistant 24 h. All plates were examined for any zones of inhibition Staphylococcus aureus (MRSA) and multiple drug-resistant around the antibiotic discs that would indicate sensitivity (MDR) strains (Escherichia coli, Pseudomonas aeruginosa).
of the organism. Zone diameters were recorded in milli-metres using a zone reader (Hi-Media) and interpretedaccording to the standard charts provided by the National *For correspondence. (e-mail: [email protected]) Committee for Cultural and Laboratory Standards2.
CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006 MICs of eight bacterial strains with Silver–Water Dispersion® R, Resistant; S, Sensitive; PR, Partially resistant.
Actively growing 16-h-old culture was surface-spread MICs of the Silver–Water Dispersion™ and antibiotic using sterile cotton swabs onto the nutrient agar surface solutions for the seven microbes were determined using (Hi-Media). The plates were laid aside for absorption for the macrodilution broth susceptibility test. A standardized 15 min. Wells were punched into the agar surface using a suspension of approximately 105–106 CFU/ml density 10 mm diameter cork-borer aseptically. Next 100 ml of was obtained by inoculating the culture in nutrient broth Silver–Water Dispersion™ was introduced into the wells. (Hi-Media) and incubating the tubes at 37°C for 4 to 6 h.
The plates were incubated at 37°C for 24 h. Sensitivity was A serial dilution of Silver–Water Dispersion™ was prepared indicated by the zone of inhibition around the well. Zone within a desired range. Similarly, the antibiotic solutions diameter was recorded in millimetres using the zone are also diluted. 100 ml of the standardized culture sus- pension was then inoculated and tubes were incubated at Drug interaction studies were carried out using the agar 37°C for 24 h. MIC was defined as the lowest concentra- well diffusion method. Actively growing 16-h-old culture tion of the inhibiting agent that completely inhibited bac- was surface-spread using sterile cotton swabs onto the terial growth. MIC can be visually examined by checking the nutrient agar surface (Hi-Media). The plates were laid aside for absorption for 15 min. Wells were punched into The study of combined antimicrobial activity of Silver– the agar surface for adding Silver–Water Dispersion™ Water Dispersion™ and antibiotics against seven microbes solution (as above). The antibiotic discs were placed at a was carried out using the checkerboard assay method. In distance which was the average of their zone diameters a final volume of 5 ml in each tube antibiotic solution and obtained individually. The plates were incubated at 37°C Silver–Water Dispersion™ solution diluted from appro- for 24 h. The inhibition pattern obtained was recorded as priate stock solution were added. Standardized culture synergy, additive or antagonistic according to the criteria suspension (105–106 CFU/ml) was prepared by inoculating the organisms in nutrient broth incubated at 37°C for 4–6 h.
CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006 Synergism of Silver–Water Dispersion™ with antibiotics An index of less than 0.5 is considered as evidence ofSynergism; an index of > 2.0 is evidence of antagonism.
A panel of bacterial strains was characterized using the 19 antibiotics under study. Results are shown in Table 1. The yeast C. albicans shows resistance to all the antibioticsunder study, but does show sensitivity to Silver–WaterDispersion™. The two hospital isolates E. coli and Ps.
aeruginosa were found to be largely resistant to the anti-biotics under study, with Ps. aeruginosa showing resis-tance to all antibiotics being studied except for Ceftizidimeand Rifampin. Though MRSA was reported by the hospitalto show multiple resistance, laboratory sub-culturing has probably lowered its resistance. All the eight isolatesstudied showed susceptibility to Silver–Water Dispersion™solution when tested both by the agar cup method as well as by the MIC macrodilution test. Their MIC values are also given in Table 1.
Figure 1.
Checkerboard inhibition result of Ps. aeruginosa with Table 2 shows synergism results of Silver–Water Dis- S–W D™ and Ceftizidime combination.
persion™ in combination with various antibiotics whenevaluated by the disc/cup combination. These tests were 100 ml of this suspension was then inoculated in the tubes not performed when the organism was resistant to the an- and incubated at 37°C for 24 h. The tubes were observed tibiotic. However, it may be noted that no organism was for turbidity to determine inhibition and compared with resistant to Silver–Water Dispersion™. Thus the number of tests completed was 96 out of the possible 133 be- Konman et al.3 described the method for quantifying tween 19 antibiotics and seven organisms. The tests MIC results obtained in terms of Fractional Inhibitory showed that five combinations were synergistic, 89 addi- Concentration (FIC) index, defined as the sum of FIC values of two drugs in combination: FIC index = FIC of drug A + Five synergistic combinations in the agar diffusion method (see Table 2), when checked by macro dilutionshowed only two combinations to be synergistic. We chose the two synergistic and two additive combinations among the five and determined MICs of the antibiotics and Silver–Water Dispersion®, and the checkerboard CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006 MICs of S–W D™, four antibiotics and combinations thereof Figure 2.
MRSA inhibition with Penicillin-G alone (Well A), and in combination with Silver–Water Dispersion® (Well B).
combinations of the four as reported. The FIC index re- behind a host of resistant organisms in the system. These ported in Table 3 is calculated favouring a minimum resistant organisms reappear at a later date straining the value of Silver–Water Dispersion®. When the inhibition immune system. Figure 2 shows Penicillin-G (10 units) is seen from the antibiotic side, the FIC index may be differ- (well A) and Penicillin-G (10 units) in combination with ent. As seen in Figure 1, using an antibiotic of 50 mg/ml Silver–Water Dispersion™ solution (32 ppm) (well B).
and Silver–Water Dispersion® of 3 ppm for the inhibitory Well A inhibition zone is not clear and displays the pres- combination, the FIC index is 1.25 and not 1.4 as reported in ence of resistance to Penicillin-G. Whereas well B having a combination of Penicillin-G and Silver–Water Disper- Table 2 shows that silver is mostly additive to the anti- sion™ demonstrates the powerful clearing ability of Sil- biotic efficacy. In the combinations examined, it is indepen- dent in its bacterial activity except for five out of 96 It is clear that the combination will allow a more complete synergistic combinations. It is also shown to have two clearing of the pathological organism.
combinations which appeared antagonistic. These two 1. Futuyma, D. J., Science on Trial, 1983.
2. Konmen, E., Allen, S. D., Janda, W. M., Schreckenberger, P. C. and To validate the results of Amoxicillin antagonism, Winn, Jr. W. C., Color Atlas and Textbook. (Diagnostic Microbio- 30 mcg dilution of Amoxicillin was prepared in Silver– logy), 1975, 5th edn, pp. 816–844.
Water Dispersion™ (32 ppm) solution. 100 ml of this 3. Bauer, Kirby, Sherris, Turck, Am. J. Clin. Pathol., 1966, 45, 493.
combination was added to a single well, and kept for dif- 4. National Committee for Cultural and Laboratory Standards, Per- formance standards for antimicrobial disc susceptibility tests ed. 4 fusion followed by incubation. No antagonistic effects were noted under these conditions as the zone of inhibitionobserved was 19 mm, comparable to 20 mm with a Silver– Water Dispersion™ and 21 mm with Amoxicillin.
by Meghshree Deshmukh and Ms Zeenal Shah.
Many a times, antibiotics may cause symptoms in patients Received 16 May 2006; accepted 12 June 2006 to temporarily disappear and yet the antibiotics may leave CURRENT SCIENCE, VOL. 91, NO. 7, 10 OCTOBER 2006

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