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This study compares Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to the selenium and tellurium oxyanions selenite (SeO32−), tellurate (TeO42−), and tellurite (TeO32−). P. aeruginosa planktonic and biofilm cultures reduced the selenium and tellurium oxyanions to orange and black end-products (respectively) and were equally tolerant to killing by these metalloid compounds. S. aureus planktonic cell cultures processed these metalloid oxyanions in a similar way, but the corresponding biofilm cultures did not. S. aureus biofilms were approximately two and five times more susceptible to killing by tellurate and tellurite (respectively) than the corresponding planktonic cultures. Our data indicate that the means of reducing metalloid oxyanions may differ between the physiology displayed in biofilm and planktonic cultures of the same bacterial strain.
The physiological distinctiveness of biofilm bacteria relative to the solitary, planktonic bacterial population is controversial. Recognized as the predominant form of bacteria in nature, the biofilm is a surface adherent, irregularly structured community of microorganisms encased in an extracellular polymeric matrix . Architecturally, the bacterial biofilm consists of mushroom shaped microcolonies interspersed with an amorphous network of water filled channels . Diffusion of nutrients and oxygen, entrained through the channels from the bulk phase, result in chemical gradients that restrict the growth of bacteria in the biofilm . Correlative to structure, metabolic activity within the biofilm is thus heterogeneous, and generally decreases with depth . Biofilms present with a well-known 10–100-fold increase in tolerance to antibiotics relative to the planktonic form [3–7], and a demonstrable tolerance to the heavy metals Cu2+, Zn2+ and Pb2+. It is unknown whether biofilms are less susceptible to antimicrobials because of structure dependent metabolic heterogeneity or because of the existence of a ‘persister’ cell phenotype.
The group VIA elements selenium and tellurium are naturally present in the environment in low abundance . Typically, these metalloids are disseminated into specific ecological niches from the effluent of mining, photographic, and electronics industries as well as from the manufacturing of rubber [9,10]. Microbiologists commonly employ potassium tellurite (K2TeO3) as a selective agent for the isolation of enterohaemorrhagic Escherichia coli. The precise mechanism of tellurite toxicity is to date unknown, and there have been at least six genetically unique resistance determinants characterized in Gram-negative bacteria .
In this study we comparatively examine the metalloid oxyanions selenite (SeO32−), tellurate (TeO42−), and tellurite (TeO32−) for toxicity against aerobically grown biofilm and planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus. We report that biofilm and planktonic cultures of P. aeruginosa reduce metalloid oxyanions similarly and are similarly sensitive to the toxicity of these compounds. In contrast, S. aureus biofilms and planktonic cells reduce metalloid oxyanions in different ways, and the apparent loss of this biochemical mechanism of metalloid reduction in biofilm cultures coincides with hypersensitivity to tellurium oxyanions. This suggests that the S. aureus biofilm displays distinct physiology relative to the planktonic form.
2Materials and methods
2.1Bacterial strains and growth media
Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 were stored at −70 °C in 8% DMSO (dimethylsulfoxide) in high-salt Luria–Bertani medium (pH 7.1) enriched with 0.001% vitamin B1 (LB + B1). Assays for metalloid oxyanion toxicity were performed using LB + B1 medium, and subcultures, minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) assays were performed on plates prepared with LB + B1 and 1.5% (w/v) granulated agar.
Biofilms were formed in the MBEC™ high throughput (HTP) device (MBEC Bioproducts Inc., http://www.mbec.ca), and preparation of the inoculum and growth of the biofilms was performed according to the manufacturer's instructions. Briefly, the MBEC™ HTP device consists of a shallow trough that houses a lid with 96 plastic pegs. The peg lid may be fit over a standard 96-well microtitre plate. The trough has shallow channels that allow flow of an inoculated suspension over the pegs when the device is placed on a rocker. This method facilitates the formation of 96 statistically equivalent biofilms on the surface of the pegs that can then be exposed to antimicrobial challenge for MBEC determination [3,12]. The planktonic population of each well in the challenge plate is seeded by bacteria shed from the surface of the biofilm peg. These sloughed bacterial cells serve as the inoculum for MIC and MBC determinations. In these experiments, all incubations were performed at 35 °C and 95% relative humidity. Biofilms were disrupted from individual pegs broken from the lid, or from all pegs at once, by sonication on high with an Aquasonic sonicator (model 250HT, VWR Scientific) as previously described [3,12]. For a further description of the methods, see .
2.3Stock solutions of metalloid compounds
Selenite (H2SeO3, BDH Inc.), tellurite (K2TeO3, Sigma) and tellurate (K2TeO4, Johnson Mathey Electronics) were made up to concentrations of 40, 10 and 1 mg/ml (approximately 315, 57.0 and 5.2 mM, respectively) in double distilled water. Stock solutions were syringe filtered and stored in sterile glass vials at 20 °C until use. Working solutions of selenite, tellurite and tellurate were prepared from these stocks in LB + B1 media at concentrations of 8192, 2048 and 256 μg/ml (approximately 64.5, 11.7 and 1.3 mM), respectively. Serial two fold dilutions were made in the wells of a sterile 96-well microtiter plate (the challenge plate) leaving the first well of each row as a sterility control and the second as a growth control (i.e. no metalloid oxyanion).
In order to differentiate between the bactericidal and bacteriostatic properties of these metalloid oxyanions, it was necessary to employ a neutralizing agent to eliminate carry-over of metalloid toxicity to the recovery media. Tellurite and selenite are known to mediate thiol oxidation in vivo . Glutathione is used by the bacterial cell as a reduction-oxidation buffer to reductively eliminate various inorganic toxins, and is thus the basis for its use as a neutralizing agent in this experiment [14,15]. A stock solution of 0.25 M reduced glutathione (Sigma) was prepared in double distilled water, syringe filtered, and stored at −20 °C until use. A final concentration of 5 mM glutathione was used in the LB + B1 recovery medium for MBEC determination (the recovery plates). The concentration of neutralizing agent (5 mM) was in molar excess of the MIC for tellurium oxyanions. For planktonic cultures, glutathione was prepared at 5 times the desired neutralizing concentration (i.e. 25 mM) in 0.9% saline. Ten microliters aliquot of this solution was then added to the wells of a sterile 96-well microtiter plate (the neutralizing plate) to which 40 μl from the cultures in the challenge plate were added. The final concentration of neutralizing agent used to treat the planktonic cultures was thus equal to that used to treat biofilm cultures.
2.5Metalloid oxyanion susceptibility testing
Metalloid susceptibility testing using the MBEC™-HTP assay was performed by the method of Harrison et al. , which is concisely summarized here. Biofilms formed on the lid of the MBEC™ device were rinsed once with 0.9% saline and then transferred to the challenge plates. The challenge plates were incubated for 24 h at 35 °C and 95% relative humidity. After 24 h exposure, the peg lid was removed and rinsed twice with 0.9% saline, and the biofilm disrupted by sonication into recovery medium. Minimum inhibitory concentrations (MICs) were determined by reading the turbidity of the challenge plate at 650 nm on a 96-well microtiter plate reader.
Neutralization plates were prepared from the planktonic cultures as described above. MBCs and MBECs were qualitatively determined by spotting 25 μl from each well of the recovery and neutralization plates onto LB + B1 agar, and incubating for 48 h at 37 °C. Pictures of the planktonic and biofilm cultures were taken after the toxicity assays were completed (i.e. following slightly greater than 24 h of exposure).
2.6Viable cell counts
Viable cell counts were obtained for biofilms by breaking off four pegs from the peg lid and suspending them in 200 μl of 0.9% saline in a 96-well plate. The microtitre plates were sonicated as described above. The disrupted biofilm cultures were serially diluted 10-fold, plated onto LB + B1 agar, and incubated for 24 h at 37 °C.
3.1Biofilm formation on the surface of the MBEC™ device
Pseudomonas aeruginosa and S. aureus biofilms were grown to a mean density of approximately 6.0×106 cfu/peg in 10 and 24 h of incubation, respectively. For every susceptibility assay, four pegs were broken off the peg lid, sonicated, and plate counts determined to ensure that the appropriate number of cells had formed in the biofilm. One-way analysis of variance (ANOVA) was used to demonstrate that these biofilms were statistically equivalent (data not shown). Scanning electron microscopy (SEM) has been used previously by our research group to examine biofilm formation on the pegs of the MBEC™ device. SEM photographs indicate that S. aureus and P. aeruginosa form a bacterial layer encased in extracellular polymeric substance, and do not simply adhere to the pegs as solitary, planktonic cells.
3.2Relative tolerance of biofilm and planktonic cells to metalloid oxyanions
We assayed bacterial susceptibility to metalloid oxyanions in three ways: inhibition of planktonic growth (MIC), killing of planktonic bacteria (MBC) and killing of biofilm bacteria (MBEC). Metalloid oxyanion MIC, MBC and MBEC values for P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 are summarized in Tables 1 and 2, respectively. The mean and standard deviation are reported for all MIC, MBC and MBEC values. Each assay was performed eight times (four times in each of two independent experiments).
Table 1. Relative levels of resistance of P. aeruginosa ATCC 27853 biofilms and planktonic cultures to metalloid oxyanions (all values are in mM)
12 ± 4
14 ± 4
13 ± 4
0.7 ± 0.1
2.8 ± 2.5
2.5 ± 2.3
Table 2. Relative levels of resistance of S. aureus ATCC 29213 biofilms and planktonic cultures to metalloid oxyanions (all values are in mM)
aThere was no observed killing of S. aureus planktonic cells at the maximum concentration of tellurate employed in this study.
12 ± 4
14 ± 4
13 ± 4
1.0 ± 0.4
1.3 ± 0.7
6 ± 1
1.3 ± 0.7
In the case of selenite, there were no differences between inhibitory and bactericidal concentrations observed for either P. aeruginosa or S. aureus (i.e. MIC=MBC=MBEC). For P. aeruginosa, this was also the case for tellurate, though it must be noted that there was no observed inhibition of growth at the maximum concentration of tellurate employed (1.3 mM). This maximal concentration was limited by the low solubility of K2TeO4 in LB + B1 medium. P. aeruginosa biofilm and planktonic cultures were equally susceptible to killing by tellurite, and the bactericidal concentrations were approximately four times greater than the inhibitory concentration of planktonic growth (i.e. MIC < MBC=MBEC). Bactericidal concentrations of tellurite for S. aureus biofilm and planktonic cultures were approximately two and eight times greater than the inhibitory concentration of planktonic growth, respectively. Notably, S. aureus biofilms were approximately five times more susceptible to killing by tellurite than the corresponding planktonic cultures (i.e. MIC < MBEC < MBC). S. aureus biofilms were killed at the threshold of the highest concentration of tellurate used for these assays (1.3 mM), whereas planktonic cells were not. This implies, given the sensitivity of the assay system, that S. aureus biofilms were at least two times more susceptible to killing by tellurate than corresponding planktonic cultures.
3.3Reduction of the metalloid oxyanions
In water or in LB + B1 broth, selenite (SeO32−), tellurate (TeO42−), and tellurite (TeO32−) are colorless. After 24 h of exposure, S. aureus planktonic cultures reduced tellurate (Fig. 1, panel A), tellurite (Fig. 1, panel B), and selenite (Fig. 1, panel C) to their elemental forms, resulting in grey–black and orange colored cultures, respectively. The corresponding S. aureus biofilm cultures, grown on the surface of the pegs, did not accumulate highly colored end-products characteristic of metalloid reduction (panels D, E and F, respectively). As a comparison, biofilm and planktonic cultures of P. aeruginosa ATCC 27853 reduced tellurate (Fig. 1, panels G and J), tellurite (Fig. 1, panels H and K) and selenite (Fig. 1, panels I and L) to their elemental forms, resulting in grey to black and orange colored cultures, respectively.
Many Gram-positive human bacterial pathogens are known to be naturally resistant to tellurite, including Corynebacterium diphtheriae, Enterococcus faecalis, Streptococcus pneumoniae as well as S. aureus[17,18]. The reduction of selenite  and tellurite  to form highly colored end-products has been previously described for E. coli. The reduction of tellurate (TeO42−) and tellurite (TeO32−) to elemental tellurium (Te0) results in the deposition of colloidal Te0 particles within the cell, resulting in a characteristic grey to black color [20,21]. Reduction of selenite (SeO32−) to insoluble elemental selenium (Se0) and its subsequent association with the bacterial cell wall or membrane results in orange–red colored bacterial cultures . Tellurite reduction is thought to be mediated in part by the terminal cytochrome oxidase of the electron transport chain  and through intermediary reactions with glutathione . A similar mechanism of reduction likely occurs here for P. aeruginosa and S. aureus. In this experiment, we observed an apparent loss of metalloid reduction in S. aureus biofilm cultures and a hypersensitivity to tellurium oxyanion toxicity. Although by no means directly correlative or conclusive, this does support the hypothesis that reduction itself may be a mechanism for cellular defense against metalloid toxicity.
An important consideration in studies of tellurite resistance is the role of the electron transport chain in response to aerobic or anaerobic growth conditions. In P. aeruginosa, electron transport activity of the respiratory chain has been directly correlated with reduction of K2TeO3. Another well-studied example is the Gram-negative, facultative phototroph Rhodobacter capsulatus. R. capsulatus cultures grown anaerobically in the light are highly resistant to TeO32−, whereas cultures grown aerobically in the dark are highly sensitive to this metalloid oxyanion (D. Zannoni, personal communication). In this example, anoxia and increased light flux result in reduced soluble c-type cytochrome content in parallel with reduced cytrochrome c oxidase activity . Concomitantly, NADH-dependent respiration is catalyzed by a pathway with a terminal quinol oxidase . Pertinent to this study, biofilms represent metabolically heterogeneous microbial populations with regions of both high and low oxygen tensions. Biofilm bacteria in proximity to the air–liquid interface grow aerobically, whereas those cells nearest the substratum grow anaerobically . The observation that S. aureus biofilms are hypersensitive to tellurium oxyanions suggests that biofilm susceptibility to tellurate and tellurite may not be explained solely in terms of oxygen limitation.
The physiological uniqueness of biofilm cells is controversial. Biofilm formation is regarded as a stepwise process that is developmentally regulated by quorum-sensing . In an early study by Whiteley et al. , gene array data indicated remarkable similarity between stationary phase planktonic cells and biofilms, and that as little as 1% (∼50 genes) of the P. aeruginosa genome was expressed differentially between the two modes of growth. Subsequent transcriptomics in P. aeruginosa have shown that between 353  and 616  genes are regulated by quorum-sensing, although no quorum-sensing regulated genes were identified in the initial study by Whiteley et al. . A recent study has indicated that as much as 10% of the E. coli genome is differentially expressed during biofilm growth . Rather than a concordant model of biofilm specific gene expression, microarray data have left the distinction between biofilm and planktonic cell physiology uncertain.
One of the advantages of the MBEC™ device is that the wells of the challenge plate are seeded by bacteria sloughed from the surface of the biofilm. In this experiment, the challenge plate was a 96-well microtitre plate containing serial dilutions of metalloid oxyanions, and the planktonic bacteria lost from the biofilm served as the inoculum for MIC and MBC determinations. This situation may be reflective of naturally extant environment systems and as a model of infection, where a biofilm forms a recalcitrant nucleus that sheds bacterial cells into the surrounding milleu. Our observation – that S. aureus planktonic cells reduce metalloid oxyanions whereas biofilms of the same microorganism do not – suggests that the gene expression pattern of planktonic cells was distinct from biofilm cells, and that a switch in gene expression occurred between biofilm and planktonic modes of growth. The observation that S. aureus planktonic cells were more tolerant than biofilm bacteria to tellurium oxyanions also suggests a difference in expression.
Collectively, our data suggest that biofilm and planktonic bacteria correspond to distinct physiological states of the same microorganism, and further, that bacterial metalloid reduction may differ by attachment of the microorganism to a surface. This study indicates a potential for metalloid compounds to be employed as S. aureus biofilm antimicrobials, and for the potential exploitation of non-pathogenic P. aeruginosa biofilms in bioremediation of metalloid contaminated environmental niches.
This work was supported by Natural Science and Engineering Research Council of Canada (NSERC) grants to RJT and HC.