CITATION: Silver S, Lo J-F, Gupta A. 1999. Silver cations as an antimicrobial agent: clinical uses and bacterial resistance.
APUA Newsletter 17(3): 1-3.


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Silver cations as an antimicrobial agent: clinical uses and bacterial resistance
Simon Silver, Jeng-Fan Lo and Amit Gupta
Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois, USA


Silver cations (Ag+) are important, often misunderstood compounds that play a significant role as effective and legitimate antimicrobial agents, used particularly in the treatment of burns. Their spectrum of uses is broad and generally unfamiliar, ranging from beneficial clinical applications, to commercial- and folk-practices that are of questionable value but of little harm, to "snake oil" products and frauds found through the internet and in health food stores.

In an effort to better understand and anticipate the uses of these compounds, we recently studied plasmid-mediated resistance to silver.1 Advances in molecular genetics have allowed us to use epidemiological tools to establish the range and diversity of resistance systems. Past difficulties in measuring Ag+ resistance have been overcome.2 The importance of the halide concentration and initial cell number in such measurements has been shown, and after the sequence of the first silver resistance gene cluster was complete,1 we found closely homologous genetic determinants surprisingly abundant in hospital collections of enteric bacteria, both from silver-exposed and not-knowingly silver-exposed sources (A. Gupta et al., in prep.).

Clinical Uses of Silver Products
Silver cations are microcidal at low concentrations, and are without serious side effects for humans. Argyria (irreversible discoloration of the skin resulting from subepithelial silver deposits) is rare and mostly of cosmetic concern. The widest and best-known medicinal use of silver preparations is as preferred antimicrobial agents for the treatment of serious burns.3 Silver sulfadiazine cream that contains 1% silver sulfadiazine plus 0.2% chlorhexidine digluconate is the mostly widely used product, marketed as Silvazine in the United States for human and veterinary use. Flamazine is the same product in other countries, largely in the United Kingdom, Canada and continental Europe. Ag-coated nylon is increasingly being used to cover burn wounds and traumatic injuries to humans4 and large animals.5 Silver sulfadiazine-coated methacrylate sheet material that provides a stable base for sustained release of Ag+ over days is also being investigated.6 These silver-containing fabrics are easier to apply and remove from large burns than is the residue of a cream. Sometimes a low voltage DC current is applied across a sheet to accelerate release of Ag+ from the cloth.4,7 Additional clinical uses include aseptic coverings for plastic surgery, traumatic wounds, leg ulcers, skin grafts, incisions, abrasions and minor cuts. Plastic indwelling catheters coated with silver compounds8 are being developed to retard the formation of biofilms and stem the incidence of nosocomial infection. The use of Ag-coated nylon threads in electroretinograms has allowed the detection of tissue damage without fear of infection.9 Silver salts have traditionally been administered to the eyes of newborn infants to prevent neonatal eye infections. Dental amalgam, so-called "silver fillings," contain about 35% Ag(0) and 50% Hg(0), but we do not know if sufficient Ag+ is released to have an antimicrobial effect. It is known, however, that the release of Hg2+ from dental amalgams selects for metal-resistant bacteria.10

Bacterial Resistance and Genetics
Bacterial resistance to silver sulfadiazine, with its sometimes tragic consequences, has been periodically reported. An Ag+-resistant Salmonella strain killed three patients and required the closing of the burn ward at Massachusetts General Hospital (MGH).11 Although silver sulfadiazine-resistant bacteria have occasionally been observed in burn ward infections, and while chromosomal mutations of clinical strains to Ag+ resistance may also cause a problem in infection,12 resistance rates have not been followed.

The plasmid-determined gene cluster for silver resistance from the MGH Salmonella11 contained a total of nine genes, seven of which we have named with the two less recognized open reading frames still called ORFs: in order silP(ORF105)silAB(ORF96)C silRS silE.1 The system encodes a periplasmic Ag+-binding protein (SilE) plus two membrane Ag+ efflux pumps (SilCBA and SilP). The central six genes (silA through silS) produce products that are homologous to an unstudied gene cluster on the Escherichia coli genome (currently called ybdE, ylcD, ylcC, ylcB, ylcA and ybcZ, in order) and less closely to other metal resistance systems. In Southern blotting DNA/DNA hybridization analysis of clinical isolates with homologous DNA, the central six genes appear to always be present together, but homologs of the outer two genes, silP and silE, are occasionally missing (A. Gupta et al., in prep.). The six genes, silPORF105ABORF96silC, are co-transcribed in a very long mRNA.1 The regulatory gene pair silRS is co-transcribed separately, and silE is transcribed by itself as a third mRNA.1

Mechanism of Resistance
The functions of silver-resistance gene products can be recognized by homology to other gene products that have been studied. SilP is a membrane P-type ATPase that pumps Ag+ from the cell1,13,14 and is most similar to Cu+ and Cd2+ efflux ATPases. SilCBA (probably together with the ORF96 product) form a second Ag+ efflux pump driven by the membrane potential and not ATP. This pump consists of three proteins, one in the inner membrane (SilA), another in the outer membrane (SilC), and the third bridging the periplasmic space (SilB). Three-protein membrane potential-driven cation/proton exchangers were initially recognized in our laboratory with a bacterial Cd2+/Zn2+/Co2+ system.14

This silver resistance system is the first time we have seen three different mechanisms in a single toxic metal cation resistance determinant. It appears to be transcriptionally controlled by the products of two genes, SilS (a histidine-containing membrane auto-kinase "sensor") and SilR (a cytoplasmic DNA-binding activator "responder" that contains an aspartate residue that is trans-phosphorylated from SilS). SilRS is homologous in sequence to members of the large family of two-component sensor/responder transcriptional regulators that respond to extracellular signals.1,14

SilE is a small periplasmic Ag+-binding protein that binds Ag+ ions specifically at the cell surface, presenting the first line of resistance against Ag+ toxicity. The SilE protein has been purified to homogeneity and extensively studied by J-F Lo et al. (in prep.). The SilE protein contains ten histidine residues that bind five Ag+ cations1 (J-F Lo et al., in prep.). In contrast to other metal-binding proteins, SilE has no cysteine residues. Binding of Ag+ to the SilE protein brings about an unusually large change in protein folding, from essentially disordered, to a predominantly alpha helical structure. At this early stage, we do not know whether silE, which confers some Ag+ resistance by itself, will ever be found alone or how the various sil gene products interact for full resistance.

Non-Clinical Uses of Silver
Our primary concern remains Ag+ usage in the clinic and the selection for Ag+ resistance. The wide spread, often unchecked application of silver products as biocides is adding to the problem. Silver-containing products are used in hospital and hotel water distribution systems to control infectious agents (e.g., Legionella). Silver has been used to sterilize recycled water aboard the MIR space station and on the NASA space shuttle.15 Home-water purification units sold in the US supermarkets contain silverized activated carbon filters and ion-exchange resins (Fig. 1). Silver is a health additive in traditional Chinese and Indian Ayurvedic medicine.16 In Mexico, supermarkets sell Microdyn, colloidal silver in gelatin, to disinfect salad vegetables and drinking water (Fig. 2). Johnson Matthey Chemicals (UK) uses an inorganic composite with immobilized slow-release silver as a preservative in cosmetics and toiletries.17 In Japan, a new compound is mixed into plastics for lasting antimicrobial protection of telephone receivers, calculators, toilet seats, and children's toys.18 Metallic silver-copper containing ceramic disks, marketed as "Clean Power Plus," are sold as an alternative to laundry detergents.19 Silver addition to fabrics (similar to clinical use of Ag-nylon) is proposed to reduce buildup of microbial populations and therefore offensive smells in camping gear and clothing. While folk remedies and "snake oil" preparations are not the same, they are coupled here as representative of applications with suspect benefit.9 Over-the-counter Ag+ health food supplements are probably not effective20 and are frequently mislabeled.21 The non-clinical uses of silver appear endless, with one possible, detrimental side-effect being the lessening of its usefulness as an antimicrobial agent.

What is Needed
The identification of the genes for silver resistance, and the determination of closely related genes in bacteria from environmental and clinical environments, and from diverse geographical locations (A. Gupta et al., in prep.) should eliminate recent skepticism about the existence of silver-resistant bacteria. Now that the means for identifying silver resistance determinants in Enterobacteriaceae is available, similar efforts should be made with respect to other common pathogens, particularly those associated with large burns (i.e., pseudomonads and staphylococci). The wide and rather uncontrolled use of silver products may result in increased resistance, analogous to the emergence of antibiotic- and other biocide-resistant bacteria. Undermining the benefits of these compounds would be unfortunate to the clinical and hygienic uses that depend on the microcidal properties of silver.

References

  1. Gupta A, Matsui K, Lo JF, Silver S. 1999. Nature Medicine 5:183-188.
  2. Gupta A, Maynes M, Silver S. 1998. Applied Environmental Microbiol 64: 5042-5045.
  3. Rosenkranz HS, Carr HS. 1972. Antimicrob Agents Chemother 2: 367-372; Monafo WW, West MA. 1990. Drugs 40:364-373; Fox CL Jr, Rao TN, Azmeth R, Gandhi SS, Modak S. 1990. J Burn Care Rehabilitation 11:112-117.
  4. Deitch EA, Marino AA, Malakanok V, Albright JA. 1987. J Trauma 27: 301-304.
  5. Adams, AP, Santschi EM, Mellencamp MA. 1999. Veterinary Surgery 28: 219-225.
  6. Miller L, Hansbrough J, Slater H, Goldfarb IW , Kealey P, Saffle J, Kravitz M, Silverstein P. 1990. J Burn Care Rehabilitation 11:35-41.
  7. Modak S, Fox P, Stanford J, Sampath L, Fox CL Jr. 1986. J Burn Care Rehabilitation 7: 422-425.
  8. Gabriel MM, Mayo MS, May LL,Simmons RB, Ahearn DG. 1996. Current Microbiol 33:1-5.
  9. The Silver Institute. Washington, DC, USA.
  10. Lorscheider FL, Vimy MJ, Summers AO. 1995. FASEB J 9:504-508, 1499-1500.
  11. McHugh SL, Moellering RC, Hopkins CC, Swartz MN. 1975. Lancet i: 235-240.
  12. Li XZ, Nikaido H, Williams KE. 1997. J Bacteriol 179:6127-6132.
  13. Silver S, Gupta A, Matsui K, Lo J-F. 1999. Metal-Based Drugs 6 (in press).
  14. Silver S, Phung LT. 1996 Annual Review Microbiol 50: 753-789.
  15. Adachi K (editor). Colloidal Silver. Educate-Yourself. Costa Mesa, CA, USA.
  16. Reach for Life Enterprises. Fresno, CA, USA.
  17. Johnson Matthey. London, England, UK.
  18. Amenitop, silica gel microspheres containing a silver-thiosulfate complex. Washington Post, February 5, 1993.
  19. Mass Appeal Marketing. Torrance, CA, USA.
  20. Fung MC, Weintraub M, Bowen DL. 1995. JAMA 274:1196-1197.
  21. US Food and Drug Administration. 1996. Over-the-counter drug products containing colloidal silver ingredients or silver salts. Federal Register, October 15, 61(200): 53685-53688; US Food and Drug Administration. 1994. FDA Health Fraud Bulletin #19, Colloidal Silver, October 7.
 

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