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Applied and Environmental Microbiology, December 1998, p. 5042-5045, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Halides on Plasmid-Mediated Silver
Resistance in Escherichia coli
Amit
Gupta,*
Maria
Maynes,
and
Simon
Silver
Department of Microbiology and Immunology,
University of Illinois, Chicago, Illinois 60612-7344
Received 14 September 1998/Accepted 5 October 1998
 |
ABSTRACT |
Silver resistance of sensitive Escherichia coli J53 and
resistance plasmid-containing J53(pMG101) was affected by halides in
the growth medium. The effects of halides on Ag+ resistance
were measured with AgNO3 and silver sulfadiazine, both on
agar and in liquid. Low concentrations of chloride made the differences
in MICs between sensitive and resistant strains larger. High
concentrations of halides increased the sensitivities of both strains
to Ag+.
 |
TEXT |
Silver salts and compounds are used
widely as environmental biocides and as clinical antimicrobial agents
(2, 7, 10, 13, 17). Ag+-resistant bacteria have
been isolated (2, 8, 18), and resistance has sometimes been
plasmid linked (5, 12, 19). However, most reports have been
preliminary (e.g., references 5, 8, and
16) and the existence of silver-resistant bacteria in clinics has been questioned (10, 17), largely because of the compounding effects of environmental factors on toxicity and resistance (2, 3). Silver-binding components such as
microbial biomass, proteins, lignin, and chloride are such factors.
Among silver salts, AgNO3 is water soluble,
Ag2SO4 and Ag-acetate are sparingly soluble,
and AgCl, AgBr, AgI, and Ag2S are basically insoluble
(4).
The purpose of this report is to set out easy-to-use conditions for
measuring silver sensitivity and resistance in familiar and widely used
media, Luria-Bertani (LB) agar and broth (14), so as to
facilitate wider identification of silver resistance in nature.
Metal resistances including silver resistance are found in environments
exposed to compounds that might provide for selection (3, 7,
17). Environmental toxicity of heavy metals to microorganisms is
affected by both biotic and abiotic factors (3, 13,
16). These factors, for example, alter the bioavailability of metal ions (1, 3, 15). For Hg2+, and as
we will show for Ag+ below, addition of
Cl
increases toxicity to bacteria (6),
presumedly by increasing membrane permeability (11). The
amount of bioavailable Hg2+ decreased when Cl
levels exceeded 1 mM (1). This reduction was suggested to be
due to an increase in the proportion of negatively charged mercury or Hg(II) complexes
(HgCl3
/HgCl42
[1]). Similarly, in early and preliminary studies, the
difference between Ag+-sensitive and -resistant cells was
most clear in the presence of Cl
, while in the absence of
Cl
, cells with or without the resistance plasmid were
both sensitive to Ag+ (16).
The genes for Ag+ resistance have been identified recently
(9). The cluster of seven genes (and two open reading frames of unknown function) is organized into three divergently
transcribed units. The gene products are tentatively identified
by homologies with other available sequences, but
frequently these are also for proteins that have not been
directly isolated. SilE is a periplasmic metal-binding protein
that has been purified and measured for Ag+-binding
properties (9). SilS and SilR are a presumed
two-component membrane sensor and transcriptional responder, and SilCBA
and SilP are a presumed Ag+ efflux chemiosmotic
cation-proton antiporter and a P-type ATPase, respectively.
The effects of halide ions on Ag+ resistance of
Escherichia coli K-12 strains J53 (Ag+
sensitive) and J53(pMG101) (Ag+ resistant) (9,
12) were measured on LB agar (14) streaked with
log-phase cultures at a density of 200 Klett turbidity units (which
corresponds to 2 g [wet weight] of cells per liter) (see Fig. 1
and 2). Since Ag+ is bactericidal rather than
bacteriostatic, as are some other toxic heavy metals, the numbers of
cells streaked and their growth phase have great influence on growth
appearing on the agar plate (reference 16 and data
not shown). NaCl alone, without Ag+, was toxic at high
concentrations: at 40 g of NaCl per liter, the resistant
J53(pMG101) cells showed reduced growth whereas the sensitive strain
J53 reproducibly showed unimpaired growth (data not shown). A similar
difference in NaCl sensitivity was found in liquid media (see below).
We do not know whether this enhanced sensitivity to NaCl is associated
with the silver resistance determinant or another region on the 180-kb
(9) plasmid. In LB agar with added Ag+ and no
added NaCl, E. coli J53 grew with up to 100 µM
Ag+ and J53(pMG101) grew at 600 µM Ag+ (Fig.
1A). E. coli J53 grew
with up to 50 µM Ag+ with 10 g of NaCl per liter
(171 mM [Fig. 1B]), but there was no growth at 50 µM
Ag+ with higher NaCl levels (Fig. 1C and D). Growth of
E. coli J53(pMG101) was barely reduced at 600 µM
Ag+ and 10 g of NaCl per liter (Fig. 1B), but in
20 g of NaCl per liter there was no growth at 200 µM
Ag+ and a marked reduction in growth at 100 µM
Ag+ (Fig. 1C). In 30 g of NaCl per liter, slight
growth of E. coli J53(pMG101) was observed only at 50 µM Ag+ (Fig. 1D). These results on agar media (Fig.
1) were quantitatively reproducible from experiment to experiment
and were consistent with those obtained in liquid media (see below). It
seemed likely that the effects of NaCl on silver toxicity result from
the low solubility of AgCl (solubility product, 1.56 × 10
10 mol/liter [20]) and the formation
of water-soluble complex anions, AgCl2
and AgCl32
(4), at higher
chloride levels. Similar chemistry occurs with the other halides,
bromide and iodide (solubility products of AgBr and AgI,
7.7 × 10
13 and 1.5 × 10
16
mol/liter, respectively [20]), and formation of
anionic complexes (AgX2
and
AgX32
, where X is Cl,
Br, or I) with relative stabilities of I
> Br
> Cl
(4). In addition to the
anionic silver complexes, cationic species
(Ag2X+ and
Ag3X2+) are formed in aqueous
solutions (4). The water-soluble ionic Ag-halide complexes
might have increased access to the cell membrane, resulting in
increased bioavailability of Ag+. At low halide levels,
silver is excluded by precipitating as AgX, but at higher
concentrations there is an increase in the proportion of accessible
anionic complexes, resulting in decreased Ag+ resistance.

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FIG. 1.
Growth of E. coli J53 (sensitive, left)
and J53(pMG101) (resistant, right) on LB agar with NaCl and
AgNO3 after 20 h at 37°C. LB was supplemented with
AgNO3 and no added NaCl (A), AgNO3 and 10 g of NaCl per liter (B), AgNO3 and 20 g of NaCl per
liter (C), and AgNO3 and 30 g of NaCl per liter (D).
The numbers below the panels represent the AgNO3
concentration added to each petri dish.
|
|
Growth experiments were conducted with NaBr- and NaI-supplemented LB
agar and AgNO3 (Fig. 2). The
Ag+ sensitivity of E. coli J53 increased
with the addition of low levels (2.5 g/liter) of NaBr (Fig. 2A),
although NaBr alone was not toxic up to 30 g/liter (data not shown). A
high level (30 g/liter) of NaBr reduced the Ag+ resistance
of J53(pMG101) substantially (Fig. 2A), as was found with 30 g of
NaCl per liter (Fig. 1D). The addition of 5 g of NaI per liter (35 mM) reduced the Ag+ sensitivity of E. coli
J53 (Fig. 2B), allowing growth in 0.6 mM Ag+, presumably by
removing the Ag+ as precipitated AgI. Higher
I
levels (10 or 20 g/liter [Fig. 2B]) increased the
sensitivity of the resistant strain, presumably by solubilizing
Ag+ in a more "bioavailable" anionic complex.

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FIG. 2.
Growth of E. coli J53 and J53(pMG101) on
LB agar plates supplemented with NaBr and AgNO3 (A) or NaI
and AgNO3 (B). The numbers on the sides of the panels
represent the NaBr or NaI concentration, and the numbers below the
panels represent the AgNO3 concentration added to each
petri dish.
|
|
Since Ag-sulfadiazine is the primary silver compound in clinical
use (7, 13), it is appropriate to measure differences between AgNO3 and Ag-sulfadiazine. Sulfadiazine in this
compound is thought to function not as an antibiotic, but rather to
complex silver in a water-insoluble form that is "slow released"
for bioactivity (2, 13). The blackening on the skin from
Ag+ reduction with AgNO3 is not seen with
Ag-sulfadiazine. In the absence of added NaCl, Ag-sulfadiazine was
slightly more toxic than AgNO3 (compare Fig.
3 with Fig. 1A). Growth of sensitive E. coli J53 was reduced at 100 µM Ag-sulfadiazine,
and resistant J53(pMG101) grew at up to 600 µM Ag-sulfadiazine. The
toxicity of Ag-sulfadiazine to sensitive and resistant cells increased with the addition of NaCl (Fig. 3), as had been the case with AgNO3 (Fig. 1). Comparing the toxicity of AgNO3
with that of Ag-sulfadiazine, the latter was less (roughly
twofold) toxic after the addition of NaCl (compare Fig. 3 with
Fig. 1). For example, growth of resistant J53(pMG101) was reduced at
200 µM Ag-sulfadiazine, compared to 100 µM AgNO3
(with 20 g of NaCl per liter), and growth of resistant J53(pMG101)
occurred at 100 µM Ag-sulfadiazine, compared to 50 µM
AgNO3 (with 30 g of NaCl per liter [Fig. 3]).

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FIG. 3.
Growth of E. coli J53 and J53(pMG101) on
LB agar supplemented with NaCl and Ag-sulfadiazine. The numbers on the
sides of the panels represent the NaCl or Ag-sulfadiazine concentration
added to each petri dish.
|
|
Although growth on agar plates is the more ready means of
distinguishing Ag+-sensitive from Ag+-resistant
E. coli (Fig. 1 to 3), liquid growth experiments showed basically similar results (Fig. 4). The
effects of chloride ions on Ag+ resistance of E. coli J53 and J53(pMG101) were measured in LB broth (14)
containing NaCl and/or AgNO3, inoculated with 2 Klett turbidity units (20 µg [wet weight] of cells per ml) of log-phase cells. Low NaCl levels enhanced Ag+ resistance of
E. coli J53(pMG101) from 400 µM Ag+
to more than 600 µM Ag+ (Fig. 4A, C, and D). Growth of
sensitive E. coli was unaffected at 50 µM
Ag+, but turbidity was reduced at 100 µM Ag+.
With added higher NaCl concentrations, there was reduced growth of
sensitive cells at 50 µM Ag+ (Fig. 4D to F). Addition of
5 or 10 g of NaCl per liter (85 or 171 mM) to LB allowed growth of
E. coli J53(pMG101) in 600 µM Ag+ (Fig.
4C and D), but growth was reduced by Ag+ at higher levels
(20 or 30 g/liter) of NaCl (Fig. 4E and F). In LB liquid cultures, NaCl
(50 or 60 g/liter) by itself, without added silver, was more toxic for
strain J53(pMG101) than for the plasmidless strain J53 (Fig. 4B).

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FIG. 4.
Growth of E. coli J53 ( ) and
J53(pMG101) ( ) in LB liquid broth. LB media were supplemented with
AgNO3 (no added NaCl) (A), NaCl (no added
AgNO3) (B), AgNO3 and 5 g of NaCl per
liter (C), AgNO3 and 10 g of NaCl per liter (D),
AgNO3 and 20 g of NaCl per liter (E), and
AgNO3 and 30 g of NaCl per liter (F). Turbidity was
measured after 20 h of growth at 37°C by a Klett-Summerson
colorimeter with a Kodak 56 green filter.
|
|
These results provide a basis for measurement of Ag+
sensitivity and resistance by bacterial isolates, which is useful
since silver compounds are increasingly being used as microbicidal
agents (2, 10, 13, 17). Environmental silver-binding
components are quite diverse and radically affect the proportion of
silver that is bioavailable. In clinical settings, Cl
and
proteins bind Ag+; in soil and most aqueous environments,
Cl
and organic S and N ligands predominate for Ag(I) binding.
 |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Department of Energy.
We thank A. O. Summers (University of Georgia, Athens) for
E. coli J53(pMG101) and Portia Harris (Xavier
University, New Orleans, La.) for assistance during this work.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Illinois, 835 S. Wolcott
Ave., Room E-703, Chicago, IL 60612-7344. Phone: (312) 996-3363. Fax: (312) 996-6415. E-mail: agupta{at}uic.edu.
Present address: Division of Biology and Biomedical Sciences,
Washington University, St. Louis, MO 63110.
 |
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Applied and Environmental Microbiology, December 1998, p. 5042-5045, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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