1 Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4
2 Biofilm Research Group, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4
Correspondence
Howard Ceri
ceri{at}ucalgary.ca
Raymond J. Turner
turnerr{at}ucalgary.ca
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ABSTRACT |
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Supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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Biofilm tolerance to antibiotics and disinfectants is dependent on many factors (reviewed by Lewis, 2001). Metal tolerance in bacterial bioflms is similarly multifactorial (Harrison et al., 2005b
). Factors such as metabolic heterogeneity, due to micronutrient and oxygen restriction (Huang et al., 1998
; Xu et al., 2000
; Stewart, 2002
; Walters et al., 2003
), or changes in physiology due to quorum sensing-regulated gene expression (Beloin et al., 2004
; Stanley & Lazazzera, 2004
) may play a role. Restricted diffusion and/or penetration of charged molecules into the biofilm matrix may also be a contributing factor (Stewart et al., 2001
; Stewart, 2003
). It has also been hypothesized that growth stage-dependent production of specialized survivor cells, termed persisters, may mediate the biofilm tolerance phenomenon (Spoering & Lewis, 2001
; Keren et al., 2004b
; Harrison et al., 2005a
). This latter hypothesis is the focus of the present study.
All genetically homogeneous bacterial populations (tested to date) produce a subpopulation of cells that survive prolonged exposure to high concentrations of bactericidal antibiotics (Bigger, 1944; Moyed & Bertrand, 1983
; Lewis, 2001
; Spoering & Lewis, 2001
; Balaban et al., 2004
; Keren et al., 2004a
; Harrison et al., 2005a
). These persister cells typically occur at a frequency of 106 to 105 in planktonic populations of Escherichia coli (Moyed & Bertrand, 1983
). It has been hypothesized that this proportion may be as high as 102 in biofilms (Spoering & Lewis, 2001
). A hallmark of the persister population is biphasic killing kinetics by antibiotics that are (i) time-dependent (Balaban et al., 2004
; Keren et al., 2004a
) and/or (ii) concentration-dependent (Brooun et al., 2000
; Spoering & Lewis, 2001
). Persisters have been associated with a slow-growth phenotype that was identified by using specialized microfluidic devices and fluorescent microscopy (Balaban et al., 2004
). Strictly speaking, the persistent phenotype is not considered resistant because these cells do not grow at high concentrations of antimicrobials. Rather, persisters simply do not die and thus exhibit multidrug tolerance (Keren et al., 2004b
).
The frequency at which persisters occur in populations of E. coli is regulated by the expression of chromosomal toxinantitoxin genes (Keren et al., 2004b). The best-studied example is the toxinantitoxin pair hipAB. When these genes are deleted from the chromosome, stationary-phase planktonic cells and biofilms of E. coli have decreased tolerance to ciprofloxacin and mitomycin C (Keren et al., 2004b
). Overexpression of hipA is also correlated to joint tolerance of clinically isolated E. coli to
-lactam and fluoroquinolone antibiotics (Falla & Chopra, 1998
). Kim Lewis and colleagues have hypothesized that expression of toxins drives bacteria reversibly into the slow-growing, multidrug-tolerant phenotype by shutting down targets of antibiotics (reviewed by Lewis, 2005
). Expression of the cognate antitoxins causes bacteria to exit this physiological state (Keren et al., 2004b
). Persisters thus represent a recalcitrant nidus that can seed a new population with normal susceptibility once the antibiotic is removed (Bigger, 1944
; Moyed & Bertrand, 1983
; Spoering & Lewis, 2001
; Harrison et al., 2004a
; Keren et al., 2004a
).
Unlike antibiotics, which generally have a single, distinct and highly specific target, metal and metalloid oxyanions may exert toxicity through many mechanisms. Metals may have high-affinity and energetically favourable, site-specific reactions with a target biomolecule. An example is the Painter-type reaction of tellurite ( with glutathione, a reaction that has been observed to occur rapidly in vivo with studies using E. coli (Turner et al., 2001
). Displacement of metals with similar reactivity from proteins may alter biological activity of the target molecule (Schützendübel & Polle, 2002
). Preliminary evidence also suggests that some heavy-metalloid oxyanions (in particular,
) may exert toxicity by destroying the proton-motive force of cell membranes (Lohmeier-Vogel et al., 2004
). Metals may produce hydroxyl and superoxide radicals via reduction or Fenton-type reactions (Stohs & Bagchi, 1995
; Inaoka et al., 1999
; Geslin et al., 2001
). Finally, metals may also exert toxicity through oxidative stress exerted on the free thiol groups of proteins (Stohs & Bagchi, 1995
; Turner et al., 1998
, 1999
).
In this study, we investigated whether bacterial persistence may play a role in the tolerance of E. coli JM109 to the toxicity of metal oxyanions. We chose six water-soluble heavy-metal and -metalloid oxyanions: chromate (), arsenate (
), arsenite (
), selenite (
), tellurate (
) and tellurite (
). With 2 h exposure, we report that biofilms were 1·8310 times more tolerant to metal oxyanions than corresponding planktonic cultures. However, with 24 h exposure, metal oxyanions eradicated biofilm and planktonic cultures at approximately the same concentration in every instance. Subsequently, we evaluated biofilm and planktonic viable cell counts for a range of metal-oxyanion concentrations. We observed that all of the metal oxyanions killed >99 % of biofilm and planktonic-cell populations at concentrations slightly in excess of the planktonic MIC. Notably, we observed no precipitation or colorimetric coordination of oxyanions in exposed biofilms treated with the organic chelator sodium diethyldithiocarbamate (Na2DDTC). This observation would suggest that metal oxyanions may have low affinity for the E. coli biofilm matrix and, consequently, that metal sequestration in the EPS may not be a large contributor to the tolerance of biofilms to metal oxyanions. Finally, we tested E. coli HM22, a strain that bears a gain-of-function allele of hipA that increases the proportion of persisters in stationary-phase populations. Here, we show that multidrug-tolerant persisters from the HipA7 mutant also have a metal-tolerant phenotype. We thus concluded that the short-term survival of <1 % of E. coli JM109 populations fits well with the hypothesis that a small population of persister cells may be responsible for the in vitro tolerance of both planktonic and biofilm cultures to high concentrations of metal oxyanions.
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METHODS |
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Bacterial strains, buffers and media.
E. coli strains JM109, HM21 and HM22 were stored cryogenically at 70 °C in LuriaBertani (LB) medium containing 8 % DMSO. All bacterial cultures were grown in LB medium [pH 7·1; 5 g NaCl, 5 g yeast extract and 10 g tryptone (l double-distilled water)1] enriched with 0·001 % (w/v) vitamin B1 (LB+B1). Subcultures, MBC100 and MBEC determinations and viable cell counts were performed on plates containing LB+B1 medium with 1·5 % (w/v) granulated agar. All growth media used for E. coli strains HM21 and HM22 were supplemented with 75 µg diaminopimelic acid (DAPA) ml1. Serial dilutions were carried out with 0·9 % saline.
Cultivation of biofilms.
Biofilms were grown aerobically in the MBEC-HTP device (MBEC BioProducts) according to the manufacturer's instructions and as described previously (Ceri et al., 1999, 2001
). To summarize briefly, there are two parts to this batch-culture apparatus. The top half of the plastic MBEC-HTP device is a lid with 96 pegs that also fits over a standard 96-well microtitre plate. The bottom half is a fluted trough that guides inoculated growth medium across the pegs when the device is placed on a rocker. The rocker provides the shear force that facilitates biofilm formation. Here, 22 ml inoculum (containing
107 c.f.u. E. coli JM109 ml1 suspended in LB+B1 broth) was transferred into the trough. The device was then placed on a rocking table (Bellco Biotechnology) and incubated at 35 °C for 24 h and 95 % relative humidity at 2·5 rocks min1. Biofilms formed on the lid of the MBEC-HTP device were rinsed once (by placing the lid in a 96-well microtitre plate with 200 µl 0·9 % saline in each well) to remove loosely adherent planktonic bacteria. To verify biofilm formation, four pegs were removed from alternating, parallel rows of the MBEC-HTP device after being rinsed, placed in 200 µl 0·9 % saline and sonicated on an Aquasonic water-table sonicator (VWR International, model 250HT) for 5 min on high, as described previously (Ceri et al., 1999
, 2001
). The disrupted biofilms were diluted serially and plated for viable cell counting.
Antibiotic-susceptibility testing.
Amikacin, ceftrioxone and tobramycin (Sigma Chemical) were prepared at 5120 µg ml1 in double-distilled water, passed through a 0·22 µm syringe filter and stored at 70 °C until use. This stock solution was diluted tenfold in LB+B1 broth, which served as a working solution to prepare challenge media. Serial twofold dilutions of the working solution were made in LB+B1 broth along the length of a 96-well microtitre plate (resulting in a log2 concentration gradient from 512 to 1 µg ml1). The first and last wells of every row were used as sterility and growth controls, respectively. The peg lids (with the rinsed biofilms) were then transferred to the 96-well microtitre plates containing antibiotics. These challenge plates were incubated at 35 °C and 95 % relative humidity for either 2 or 24 h.
Following exposure, the biofilms were removed from the challenge plates and rinsed twice with 0·9 % saline as described above. Biofilm pegs were placed in a 96-well microtitre plate with 200 µl LB+B1 medium in each well and sonicated as described above. Disrupted biofilm cultures from this recovery plate were diluted serially tenfold and plated for viable cell counting. Corresponding planktonic cultures were seeded by bacteria shed from the surface of the mature biofilms (on peg lid of the MBEC-HTP device) into the 96 wells of the antibiotic-challenge plates. Planktonic cells from these cultures were diluted serially tenfold and plated for viable cell counting.
MIC values were obtained by reading OD650 of the challenge plates after 56 h incubation at 35 °C using a THERMOmax microplate reader with SoftMax Pro data-analysis software (Molecular Devices). Agar plates were incubated for 48 h at 35 °C and enumerated to obtain MBC100 and MBEC values. MBEC values were redundantly determined (qualitatively) by reading the OD650 of the recovery plate after 48 h incubation at 35 °C and 95 % relative humidity.
Heavy-metal and -metalloid oxyanion-susceptibility testing.
Stock solutions of copper sulphate (CuSO4.5H2O) (Fisher Scientific), sodium hydrogen arsenate (Na2HAsO4) (Fisher Scientific), potassium dichromate (K2Cr2O7) (J. T. Baker Chemical), selenous acid (H2SeO3) (British Drug Houses) and sodium arsenite (NaAsO2) (Sigma Chemical) were prepared at 40 mg oxyanion ml1 in double-distilled water, passed through a 0·22 µm syringe filter and stored at room temperature until use. Potassium tellurite (K2TeO3) (Sigma Chemical) and potassium tellurate (K2TeO4) (Johnson Mathey Electronics) have low solubility in water. Tellurite and tellurate were prepared similarly except at concentrations of 10 and 1 mg oxyanion ml1, respectively.
Biofilm metal-susceptibility testing was performed according to the method of Harrison et al. (2004a). This method differed from the method used for antibiotic-susceptibility testing (as outlined above) by virtue of an additional neutralizing protocol. Reduced glutathione (GSH; 5 mM) was used as neutralizing agent for all of the oxyanions examined in this study (Harrison et al., 2004a
, b
). In the case of biofilm cultures, 5 mM GSH was added directly to the recovery medium before the disruption of biofilms by sonication. In the case of planktonic cells, 40 µl culture was added to 10 µl 25 mM GSH. The neutralized planktonic cultures were incubated for 10 min at room temperature prior to serial dilution. Serially diluted biofilm and planktonic cultures were plated onto LB+B1 agar and incubated for 48 h at 35 °C. MIC, MBC100 and MBEC values were obtained as described above.
Additional susceptibility testing of exponentially growing planktonic cells.
To validate the MBEC assay used throughout the rest of this study, experiments with exponentially growing E. coli JM109 cells were repeated by using a protocol designed to reflect the CLSI method for antibiotic-susceptibility testing. The inoculum for these tests was prepared by direct colony suspension from streak plates to a 1·0 McFarland standard as described above for biofilm cultivation. This standard was diluted 30-fold in LB+B1 medium and 5 µl aliquots of this 1 in 30 dilution were added to each well of the challenge plate. This corresponded to an initial cell load of approximately 105106 c.f.u. ml1. Challenge and recovery media, neutralizing agents, serial dilution and incubation times were identical to those described above for biofilm testing.
Susceptibility testing of stationary-phase planktonic cells.
To test directly whether known high-persistence (hip) mutants may have a metal-tolerant phenotype, we examined E. coli HM21 (wild-type) and HM22 (hipA7). These strains were grown in batch cultures by inoculating 24 ml LB+B1+DAPA with 1 ml 1·0 McFarland standard. These cultures were grown in sterile 50 ml polypropylene tubes for 16 h at 35 °C on a gyrorotary shaker set to 125 r.p.m. Stationary-phase planktonic cells were harvested by centrifugation (3000 g for 10 min) then suspended in fresh medium containing the appropriate concentration of metal(loid) oxyanions. There was no significant loss of cell viability during centrifugation of either strain (data not shown). Aliquots (20 µl) were removed from the tubes after 6 h exposure, neutralized, diluted serially and plated for viable cell counting as described above. HipA7 has a temperature-sensitive mutation (Keren et al., 2004b) and, during exposure to metals, these cultures were incubated at 30 °C.
Colorimetric coordination of metal cations and oxyanions in biofilms.
Na2DDTC is an analytical reagent used for determining the concentration of some transition metals, as well as arsenic and tellurium, in aqueous solution (Cheng et al., 1982; Turner et al., 1992
). As it pertains to this study, Na2DDTC forms dark brown-, green- and yellow-coloured complexes with Cu2+,
and
, respectively. A visible colour change (mediated by Na2DDTC) of the biofilm pegs in the MBEC-HTP device following metal exposure would indicate that these metal cations and oxyanions had been adsorbed and/or retained by the biofilm matrix (Harrison et al., 2005a
). Biofilms were formed on pegs of the MBEC-HTP device (as described above) and then exposed to Cu2+,
or
for 24 h. Biofilms exposed to these heavy-metal(loid) cations and oxyanions were rinsed twice for 5 min in sterile 0·9 % saline. Subsequently, the peg lid was inserted into a 96-well microtitre plate that contained 200 µl 37·5 mM Na2DDTC in each well. Biofilms were treated with Na2DDTC for approximately 2 min, then photographed by using a Sony 4·0 megapixel Cyber-shot digital camera.
Scanning electron microscopy (SEM).
Pegs were broken from the lid of the MBEC device and rinsed with 0·9 % saline. Biofilms were fixed onto the pegs by incubating at 4 °C overnight in 5 % glutaraldehyde in 0·1 M cacodylate buffer (pH 7·2). The next day, pegs were rinsed with 0·1 M cacodylate buffer, dehydrated with 95 % ethanol and then air-dried for 30 h before mounting. SEM was performed by using a Hitachi model 450 scanning electron microscope as described by Morck et al. (1994).
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RESULTS |
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SEM was used to examine biofilm formation in situ. SEM photomicrographs of E. coli JM109 are presented in Supplementary Fig. S1 (available with the online version of this paper). Consistent with previous reports using this method (Ceri et al., 1999; Olson et al., 2002
; Harrison et al., 2004a
), these pictures show the growth of mature biofilms on the peg surface.
Time-dependent susceptibility of E. coli to antibiotics
As a standard of comparison to metal(loid) oxyanions and as a quality control for our experimental system, we examined the growth inhibition and eradication of E. coli JM109 by three different antibiotics: amikacin, ceftrioxone and tobramycin. The means±SD for observed MIC, MBC100 and MBEC values are summarized in Table 1. Each reported value is based on four to eight independent replicates, and MBEC values obtained from (qualitative) OD650 measurements of recovery plates were also included in this calculation. In no instance did antibiotics eradicate biofilm cultures of E. coli with 2 h exposure. By 24 h, planktonic cultures were eradicated at relatively low concentrations of amikacin (20±8 µg ml1), ceftrioxone (88±48 µg ml1) and tobramycin (36±20 µg ml1). Regardless of exposure time, biofilms could not be eradicated at the highest concentrations of amikacin or ceftrioxone examined (512 µg ml1). Tobramycin eradicated biofilms in a time-dependent manner at very high concentrations (352±192 µg ml1). However, these assays show that E. coli biofilms remained at least 9·851 times more tolerant to antibiotics than planktonic cells (even with prolonged exposure time). This observation is consistent with previously reported results (Ceri et al., 1999
; Walters et al., 2003
; Harrison et al., 2004a
).
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Log killing of E. coli biofilms by heavy-metal(loid) oxyanions
To examine the survival of planktonic and biofilm bacterial populations following exposure to metal(loid) oxyanions, viable cell counts were determined for a range of concentrations following either 2 or 24 h exposure. Mean viable cell counts and log killing of biofilm cultures with respect to concentration of ,
and
are presented in Fig. 3
. The data for
,
and
are presented in Fig. 4
. Every metal oxyanion examined in this study killed a large proportion (2log10 cells or greater) of the biofilm at relatively low concentrations. To continue with the example of tellurite, the majority of both planktonic and biofilm populations were killed at 7 µM
(approx. 12 µg ml1). However, a small fraction of biofilm cells (105 to 103) survived for at least 2 h at concentrations well in excess of 0·1 mM (1632 µg ml1). In addition to the data from Table 2
, a very small number of survivors (below the threshold of detection by viable cell counts) tolerated
at concentrations up to 12 mM (approx. 2048 µg ml1). This surviving fraction was eradicated by 24 h exposure. Only
did not eradicate biofilm or planktonic cultures with 24 h exposure, and only 103 c.f.u. biofilm per peg survived this toxic exposure. High concentrations of
,
,
,
and
eradicated biofilm and planktonic cultures completely with extended exposure times.
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Second (and critical to the seeding of planktonic wells), transfer of the peg lid from the MBEC-HTP device into challenge medium inherently results in the dispersion of a proportion of the E. coli JM109 biofilm. We have noted previously that, for P. aeruginosa ATCC 27853 biofilms, this dispersion event is not statistically significant (J. J. Harrison, R. J. Turner & H. Ceri, unpublished data). However, the proportion of the E. coli JM109 biofilm that is disrupted is larger. In the absence of shear and given sufficient exposure time, the disrupted biofilms resume growing. Here, this works as an advantage. Mean viable cell counts reported in Figs 1, 2 and 3 were based on the number of bacteria recovered from pegs (including growth controls) after transfer and exposure. However, log killing was calculated relative to the initial number of bacteria formed in the biofilm before transfer to the challenge medium (i.e. log killing was based on the growth controls used to calculate the mean biofilm cell density of 3·6±2·9 c.f.u. per peg as described above). We can thus discern that growth of biofilms occurs during exposure to low concentrations of metal(loid) oxyanions. If we compare where the transition between growth and cell death occurs on the log-killing curves in Fig. 3(b, d, f)
and Fig. 4(b, d, f)
, biofilms were not observed to grow at concentrations of metal(loid) oxyanions greater than the planktonic MIC values reported in Tables 1 and 2
. As noted above, the MIC values in Tables 1 and 2
are consistent with literature MIC values and those obtained by using a modified CLSI protocol (Table 2
). This results in the interesting notion that biofilms and planktonic cells may be similarly resistant to growth inhibition by metal(loid) oxyanions.
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Retention and reduction of metal(loid) oxyanions by E. coli biofilms
We have observed previously that biofilms of P. aeruginosa ATCC 27853 adsorb cupric cations (Cu2+) (Harrison et al., 2005a). We thus addressed the question of whether the observed time-dependent killing of E. coli JM109 may be partly due to oxyanion adsorption to the biofilm matrix. As a control, we exposed biofilms of E. coli JM109 to Cu2+ for 24 h, rinsed the biofilms with 0·9 % saline, then treated the exposed biofilms with the chelator Na2DDTC. Correlative with our previous observations, Na2DDTC caused the formation of dark-brown metal chelates in the biofilm (Fig. 5a, d, g
). However, Na2DDTC did not cause the characteristic colorimetric coordination of oxyanions in biofilms exposed to either
(Fig. 5b, e, h
) or
(Fig. 5c, f, i
). Surprisingly, this would suggest that EPS adsorption of oxyanions may not be the primary mechanism of the observed time-dependent tolerance of biofilms to either
or
.
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Survival of the E. coli hipA7 mutant after exposure to metals
To test directly whether persisters are responsible for bacterial tolerance to metal(loid) oxyanions, a hipA7 strain of E. coli was examined. hipA7 is a gain-of-function allele of hipA that increases the proportion of persisters in biofilms and stationary-phase planktonic cultures (Keren et al., 2004b). Stationary-phase E. coli HM21 (wild-type) and HM22 (hipA7) were exposed to bactericidal concentrations of
(1·5 mM),
(1·3 mM) and
(2·2 mM) for 6 h, then enumerated. Relative to the wild-type strain, E. coli HM22 (hipA7) had an 82-fold-, 76-fold- and fivefold-increased proportion of survivors after exposure to these metal(loid) oxyanions, respectively. These data are summarized in Supplementary Fig. S2 (available with the online version of this paper). At the highest concentrations of arsenate, arsenite and selenite tested against E. coli JM109, there was insufficient killing of E. coli HM21 and HM22 at 6 h exposure to observe a hypertolerant phenotype for the hipA7 mutant (data not shown). The starting numbers of cells for E. coli HM21 and HM22 were 5·3±0·6 (x108) and 1·2±0·3 (x108) c.f.u. ml1, respectively. E. coli HM22 entered the stationary phase at lower cell densities than E. coli HM21 (data not shown).
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DISCUSSION |
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E. coli HM22 (hipA7) well-known for its ability to produce a large proportion of persisters recalcitrant to killing by bactericidal antibiotics also produces a subpopulation of metal-tolerant cells that is larger than the population of the isogenic, wild-type strain. Biofilms of E. coli JM109 presented with a transient, time-dependent tolerance to metal oxyanions that was up to two orders of magnitude greater than that of planktonic cells. Long exposures to these highly toxic substances resulted in destruction of planktonic and biofilm cultures at similar concentrations of metal oxyanions. Consistent with the persister-cell hypothesis, this short-term tolerance was mediated by a small fraction of the biofilm population (103 or less). There are two potential explanations for this phenomenon: (i) that persister cells in a biofilm are killed at a reduced rate by metal oxyanions relative to the planktonic persister population, or (ii) that there is a greater population of persisters in a biofilm that are killed at the same rate as planktonic persister cells. In either case, this is different from antibiotic killing of biofilms, where cell death typically reached a plateau characterized by a lack of further antibiotic efficacy (even with prolonged exposure or increased antibiotic concentrations). In the case of tobramycin, which did eradicate biofilms by 24 h exposure, biofilms remained 9·8 times more tolerant to killing than the corresponding planktonic cells. Thus, our data would suggest that metal oxyanions possess a bactericidal action capable of destroying biofilm persisters. To reaffirm this notion, we have observed in a subsequent study that stationary-phase E. coli HM22 may be eradicated by arsenate with 24 h exposure (J. J. Harrison, R. J. Turner & H. Ceri, unpublished data).
The role of E. coli EPS in adsorption of metal oxyanions had previously been unexamined. Here, metal oxyanions may have equilibrated across the biofilm matrix at a slowed rate due to steric and/or ionic hindrance (Stewart, 2003). Intuitively, we can be confident that complete penetration of the EPS matrix eventually occurred if the biofilms were eradicated. The observation that the chelator Na2DDTC does not result in precipitation or coordination of metal oxyanions in exposed biofilms supports the idea that the E. coli JM109 EPS matrix may have low affinity for adsorbing and/or binding metal oxyanions. We noted that this was not the case for the divalent heavy-metal cation Cu2+, which was retained and formed metal precipitates in similarly treated biofilm cultures. Our observations are consistent with reports that the E. coli EPS is polyanionic (reviewed by Sutherland, 2001
). Colanic acid, which is known to be important in E. coli biofilm formation (Potrykus & W
grzyn, 2004
), is the major extracellular polysaccharide of most E. coli strains (Gottesman & Stout, 1991
; Whitfield & Roberts, 1999
). Further evidence for the low affinity of the matrix for metal oxyanions was the observed reduction of tellurite (Fig. 5i
) by biofilm cultures.
The reduction of tellurite by E. coli has been described previously (reviewed by Turner, 2001). Reduction of
to greyblack-coloured, colloidal Te0 is an intracellular reaction potentially mediated by the terminal cytochrome c oxidase of the respiratory electron-transfer chain (Trutko et al., 2000
) and/or through an intermediate Painter reaction of
with glutathione (Turner et al., 2001
). Insoluble Te0 associates closely with cell membranes (Lloyd-Jones et al., 1994
), giving bacterial cultures a characteristic greyblack colour. The observation that E. coli biofilms turn greyblack after exposure to
(Fig. 5i
) is evidence of intracellular Te accumulation and entrapment. This is evidence that the biofilm matrix may not sequester metal oxyanions in the extracellular milieu. A similar case can be made for selenite reduction, another group 6A chalcogen with similar chemistry to tellurite (reviewed by Turner et al., 1998
). In the present study, E. coli biofilm cultures also reduced
to Se0, which resulted in the accumulation of orangered end products on the pegs (data not shown), similar to P. aeruginosa (Harrison et al., 2004b
).
We have recently conducted studies examining the toxicity of metal cations (in both E. coli JM109 and P. aeruginosa ATCC 27853) to further validate the persister-cell phenomenon reported here for metal oxyanions (Harrison et al., 2005a, b
). A characteristic attributed to persisters is the ability of these survivors to give rise to a new population with normal susceptibility (Bigger, 1944
; Moyed & Bertrand, 1983
; Spoering & Lewis, 2001
; Keren et al., 2004a
). We have thus addressed this question for E. coli JM109. Persisters cultivated after exposure to mercury (Hg2+), then to copper (Cu2+) and finally to Cu2+ again yielded a similar proportion of metal persisters in the newly generated populations and produced cultures with normal susceptibility to metals (J. J. Harrison, H. Ceri & R. J. Turner, unpublished data). A similar phenomenon occurred for P. aeruginosa ATCC 27853 (Harrison et al., 2005a
). Collectively, this would indicate that persisters are not mutants within the bacterial culture, but rather specialized survivor cells produced by a genetically homogeneous population.
In E. coli, the proportion of persisters in the bacterial population is in part controlled by the high-persistence (hip) operon (Moyed & Bertrand, 1983; Korch et al., 2003
; Keren et al., 2004b
). The hipA7 mutant produces 10- to 1000-times more persister cells than wild-type E. coli as it approaches stationary phase (Korch et al., 2003
; Keren et al., 2004b
). As the hipA7-mediated high-persistence phenotype was abolished in a relA spoT background, Korch et al. (2003)
postulated that (p)ppGpp synthesis may govern the persistent physiological state. More than 80 homologues of the hipA gene have been identified in Gram-negative and Gram-positive bacteria (Korch et al., 2003
). It is unknown what role the stringent response [which is also governed by (p)ppGpp synthesis] plays in the production of persisters. However, it is known that stringent response-regulated genes play an important role in E. coli biofilm formation (Balzer & McLean, 2002
), entry into stationary phase, bacterial adaptation to nutrient limitation and oxidative stress (Chang et al., 2002
). The persistent response of hip mutants not only reduces cell death due to antibiotic exposure, but also reduces the lethality of heat shock (Scherrer & Moyed, 1988
) and metabolic block (i.e. starvation for DAPA) (Moyed & Bertrand, 1983
). We acknowledge that some of these mechanisms may share overlapping features with each other and the effects of metal toxicity.
Metal oxyanions are known to exert toxicity through oxidative stress (Stohs & Bagchi, 1995; Turner et al., 1998
, 1999
). Additionally, arsenate is toxic by virtue of substitution for inorganic phosphate in glycolytic intermediates, abrogating ATP-producing enzymic steps through spontaneous hydrolysis of arsenophosphates in water (reviewed by Moran et al., 1994
). Although the precise mechanism is unknown, tellurite ultimately functions to uncouple the E. coli transmembrane
pH gradient (Lohmeier-Vogel et al., 2004
). In both cases, ATP levels would be lowered in sensitive cells, mimicking the effects of starvation. Thus, the data in this study and in the literature suggest that persistence may be part of an underlying, highly conserved and generalized mechanism for bacteria to tolerate environmental duress. Within the limits of our current understanding, persisters may be defined phenotypically as the small, slow-growing and physiologically distinct subpopulation of bacterial cells capable of withstanding stress (Balaban et al., 2004
; Keren et al., 2004b
).
Biofilms are infamous for their ability to withstand antimicrobials. Here, we noted that biofilms did not grow at concentrations of metal oxyanions in excess of the planktonic MIC. Based on our data, we may not label bacterial biofilms as resistant. However, biofilms did withstand concentrations of metal oxyanions far in excess of the planktonic MBC100. Biofilms may thus be considered highly tolerant to metal oxyanions. Persisters are known to survive high levels of antibiotics for prolonged exposure times. As it pertains to our model system and E. coli JM109, this is not true for metal oxyanions. In fact, given long exposures, metal oxyanions destroyed biofilm cultures at concentrations similar to those that eradicated planktonic bacteria. In this study, we observed that 1 % or less of the biofilm population survived for short periods of time at concentrations of metal cations in excess of the concentration required to eradicate planktonic bacteria (MBC100). We thus propose that persister cells mediate the time-dependent tolerance of E. coli biofilms to metal oxyanions and, further, that these compounds may be lethal to the slow-growth phenotype possessed by persister cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 November 2004;
revised 26 May 2005;
accepted 22 July 2005.
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