Unprecedented Silver Resistance in Clinically Isolated Enterobacteriaceae: Major Implications for Burn and Wound Management

Abstract

Increased utilization of inorganic silver as an adjunctive to many medical devices has raised concerns of emergent silver resistance in clinical bacteria. Although the molecular basis for silver resistance has been previously characterized, to date, significant phenotypic expression of these genes in clinical settings is yet to be observed. Here, we identified the first strains of clinical bacteria expressing silver resistance at a level that could significantly impact wound care and the use of silver-based dressings. Screening of 859 clinical isolates confirmed 31 harbored at least 1 silver resistance gene. Despite the presence of these genes, MIC testing revealed most of the bacteria displayed little or no increase in resistance to ionic silver (200 to 300 μM Ag(+)). However, 2 isolates (Klebsiella pneumonia and Enterobacter cloacae) were capable of robust growth at exceedingly high silver concentrations, with MIC values reaching 5,500 μM Ag(+). DNA sequencing of these two strains revealed the presence of genes homologous to known genetic determinants of heavy metal resistance. Darkening of the bacteria's pigment was observed after exposure to high silver concentrations. Scanning electron microscopy images showed the presence of silver nanoparticles embedded in the extracellular polymeric substance of both isolates. This finding suggested that the isolates may neutralize ionic silver via reduction to elemental silver. Antimicrobial testing revealed both organisms to be completely resistant to many commercially available silver-impregnated burn and wound dressings. Taken together, these findings provide the first evidence of clinical bacteria capable of expressing silver resistance at levels that could significantly impact wound management.

FIG 1

SRKP grown on LB agar supplemented with 3.0 mM Ag⁺ over a period of 3 days. Conditions were as follows (from left to right): 0.0 mM Ag⁺ at 24 h, 3.0 mM Ag⁺ at 24 h, 3.0 mM Ag⁺ at 48 h, and 3.0 mM Ag⁺ at 72 h. Colonies increased in size and developed a dark, metallic appearance over the period of incubation.

FIG 2

SEM images of SRKP and SSKP grown in the presence or absence of Ag⁺. Conditions were as follows: (A) SRKP grown on 3.0 mM Ag⁺; (B) SRKP grown on 0 μM Ag⁺; (C) SRKP grown on 100 μM Ag⁺; (D) SSKP grown on 100 μM Ag⁺; (E) SSKP grown on 0 μM Ag⁺. Note the presence of silver nanoparticles when SRKP was grown in high concentrations of silver. Subsequent EDS analysis confirmed the presence of silver localized on the cell surface.

FIG 3

Bar graph illustrating the percent reduction of challenge organisms after 24 h of dynamic contact with commercially available silver-impregnated wound dressings. Isolates SSKP and SSEC were reduced by 100% and were excluded from the figure for clarity. Error bars represent standard deviations. Asterisks (with absence of bars) indicate that there was no reduction in CFU per milliliter. Values are means of two independent experiments (n = 2).

FIG 4

Bar graph illustrating CZOI measurements of challenge organisms after 24 h of static contact with commercially available silver-impregnated wound dressings. Error bars represent standard deviations. Asterisks indicate no measurable CZOI. Values are means of two independent experiments (n = 2).

FIG 5

SDS-PAGE results with outer membrane preparations from K. pneumoniae and E. cloacae strains used in this study. Isolates were grown in LB supplemented with 100 μM Ag⁺. All silver-resistant and silver-sensitive isolates underexpressed or lacked the OmpF homologue, which was detected only from control cultures.