Enhancing the Antibiotic Antibacterial Effect by Sub Lethal Tellurite Concentrations: Tellurite and Cefotaxime Act Synergistically in Escherichia coli

The emergence of antibiotic-resistant pathogenic bacteria during the last decades has become a public health concern worldwide. Aiming to explore new alternatives to treat antibiotic-resistant bacteria and given that the tellurium oxyanion tellurite is highly toxic for most microorganisms, we evaluated the ability of sub lethal tellurite concentrations to strengthen the effect of several antibiotics. Tellurite, at nM or µM concentrations, increased importantly the toxicity of defined antibacterials. This was observed with both Gram negative and Gram positive bacteria, irrespective of the antibiotic or tellurite tolerance of the particular microorganism. The tellurite-mediated antibiotic-potentiating effect occurs in laboratory and clinical, uropathogenic Escherichia coli, especially with antibiotics disturbing the cell wall (ampicillin, cefotaxime) or protein synthesis (tetracycline, chloramphenicol, gentamicin). In particular, the effect of tellurite on the activity of the clinically-relevant, third-generation cephalosporin (cefotaxime), was evaluated. Cell viability assays showed that tellurite and cefotaxime act synergistically against E. coli. In conclusion, using tellurite like an adjuvant could be of great help to cope with several multi-resistant pathogens.


Introduction
The constant emergence of clinically-relevant pathogens exhibiting high levels of antibiotic resistance is nowadays a worldwide health problem that poses new challenges to the scientific community. Such scenario is even more worrying given the ability of some pathogens to use antibiotics as the sole carbon source [1].
During the last 50 years, the pharmaceutical industry has introduced only one new antibiotic into the market. However and even if new compounds with antibiotic ability are discovered, the emergence of resistant strains is only a matter of time. This is the main reason to look for new treatments, and in this context the use of compounds strengthening the antibiotic effect is a choice that worth to be evaluated [2]. Recently, it has been reported that some antibiotics can act in a synergistic manner when used in conjunction with genetically-modified bacteriophages or organometallic compounds, among others [3,4,5].
In 2007, Kohanski et al. [6] showed that a common mechanism underlying the toxicity of bactericidal antibiotics involves the generation of the highly reactive oxygen species (ROS), hydroxyl radical. Overall, this observation evidences that the general mechanism(s) underlying antibiotic-toxicity are not fully understood to date. On the other hand, in 1932 Fleming reported the antibacterial properties of tellurite (TeO 3 22 ) and penicillin [7] and since then TeO 3 22 has been used routinely to isolate telluriteresistant strains as Escherichia coli O157, Proteus spp., and other bacteria [8].
During the last years our group has been interested in studying the underlying molecular mechanism(s) of tellurite toxicity. It has been shown that part of it results from ROS generation [9], damage to metabolic enzymes [10,11], glutathione depletion [12] or lipid peroxidation [13]. In this context, based on the high toxicity exhibited by TeO 3 22 against bacteria, its numerous cell targets [14] and its apparent low noxiousness to eukaryotic cells [15], we hypothesized that TeO 3 22 could increase significantly the antimicrobial effect of antibiotics.
In this work we report that sub lethal tellurite concentrations increase the effect of ampicillin, tetracycline, chloramphenicol or cefotaxime against E. coli and Pseudomonas aeruginosa. A similar, but reduced effect was observed with the highly tellurite-and antibiotic-resistant Staphylococcus aureus. Especially interesting was the effect with cefotaxime, a widely-used, third-generation cephalosporin, which was found to act synergistically with tellurite against E. coli.

Results
The ability of non-lethal tellurite concentrations to increase the antibacterial effect was assessed by determining growth inhibition zones. Antibiotics targeting different cellular processes were tested in the absence or presence of sub lethal tellurite concentrations.
Bacterial species displaying distinct susceptibility to antibiotics and TeO 3 22 were evaluated to determine if the potentiating effect was also observed with tellurite-or antibiotic-resistant bacteria. Tellurite-mediated antibiotic-potentiating effects were observed with AMP, CHL, TET, GEN and CTX when E. coli (highly sensitive to tellurite, MIC 4 mM) was grown in tellurite-amended LB plates. Approximately a 3-fold increase in growth inhibition zones was observed with TET, CHL and CTX (Fig. 1A). In turn, when P. aeruginosa was exposed to different antibiotics in the presence of 4 mM TeO 3 22 (MIC/80), a significant potentiatingeffect was observed only with CHL and GEN (Fig. 1B). Similar results were obtained with S. aureus grown in 200 mM tellurite (MIC/20)-containing plates (Fig. 1C).
Differences in growth inhibition areas observed among these bacterial species are most probably due to their different susceptibility to tellurite and antibiotics. P. aeruginosa exhibited smaller inhibition zones than E. coli, which may reflect antibiotic resistance genes that are absent in E. coli K12-derived laboratory strains [16]. In our hands and depending on the particular antibiotic, MIC values for these antibacterials decreased 25-75% in the presence of sublethal tellurite concentrations.
Particularly interesting was the effect in E. coli exposed to CTX, where the most significant inhibition zone increase was observed in the presence of tellurite (Fig. 1A). CTX, a third-generation cephalosporin, is routinely used to treat infections caused by Gram-negative and Gram-positive pathogens and also as prophylactic strategy [17]. Given the effect observed in sensitivity to CTX and its clinical relevance, the tellurite-dependent potentiation on CTX effect was further explored.
The minimal concentration of tellurite displaying CTX potentiation was determined. A dose-dependent effect was observed when tellurite concentrations ranging from 1/10 up to 1/1,000 of E. coli MIC were evaluated (Fig. 2). Although the maximal effect was observed at 400 nM, half of this concentration was used since the potentiating effect was still significant and because this concentration seems not to affect eukaryotic cells [15,18,19].
The cefotaxime MIC for E. coli was diminished 4 fold (0.13 to 0.03 mg/ml) when grown in the presence of tellurite. Surprisingly, the CTX MIC for the antibiotic-resistant bacteria P. aeruginosa decreased .30 fold (300 to 9.3 mg/ml) in the presence of 4 mM tellurite. Since the CTX MIC is the same for pathogenic [20] and laboratory E. coli strains, these results could be important in terms of future applications of tellurite-mediated cefotaxime potentiation. In this context and aiming to assess if the telluritepotentiating antibiotic effect was also observed with pathogenic bacteria, clinical isolates were exposed to both antibacterials. Growth inhibition zones resulting from antibiotic exposure in the presence or absence of 200 or 400 nM tellurite were determined for 20 clinical coliform isolates from patients suffering urinary infection. A dose-dependent, tellurite-potentiating effect was observed with all tested antibiotics. Interestingly, the most robust effect was again observed with CTX, which was over 2 fold than that observed with other antibiotics as STR, AMK, KAN and TOB (Table 1).
To characterize the type of antimicrobial effect after exposing bacteria simultaneously to tellurite and CTX, cell viability determinations were carried out using different antibiotic concentrations in the presence of the tellurium oxyanion (Fig. 3). Growth and cell viability were not severely affected when E. coli was exposed to 200 nM tellurite. In fact, normal growth and viability was restored after 3 h exposure (Fig. 3, squares).
While growth was not affected when cells were exposed concurrently to 0.065 mg/ml CTX (MIC 0.13 mg/ml) and tellurite (200 nM), the number of viable cells was strongly decreased as determined by CFU counting (Fig. 3A). A similar result was obtained upon exposing to the antibiotic alone, suggesting that the effect observed with both CTX+tellurite depends mainly on antibiotic-mediated damage.
When cells were grown in the presence of 0.13 mg/ml cefotaxime, growth and viability recovered only after 7 h treatment. The observed potentiating-effect at 3 or 7 h exposure cannot be explained as the sum of tellurite-and CTX-independent  (Fig. 3B). This indicates a tellurite/cefotaxime-mediated synergistic effect in E. coli.
Finally, when the potentiating effect was assessed in cells exposed to lethal CTX concentrations (0.5 mg/ml), the synergy was represented by a difference of ,5 Dlog 10 units after 20 h and growth or cell viability was not recovered at all (Fig. 3C).

Discussion
Bacterial multi-resistance to different antibiotics has become a severe problem worldwide. To face this situation, the scientific community and pharmaceutical industry have made important efforts to discover new compounds exhibiting antibacterial properties. However, in the last 40 years these efforts have resulted in the discovery of only 2 new antibiotics, the oxazolidinone linezolid and the lipopeptide daptomycin [21,22].
The conventional treatment of bacterial infections currently lies in administering antibiotics alone or in combination [23], or using last-generation antibiotics as the case of the multi-resistant Enterobacteriaceae with carbapenems [24]. In spite of this, strains resistant to these new antibacterials emerge continuously, making the situation critical.
Horizontal gene transfer is the principal mode of acquiring new information by bacteria thus allowing them to cope with new antibacterial agents. In this context, the idea of using 2 different antibiotics to treat bacterial infections seems reasonable but there is still a risk of acquiring resistance determinants. To avoid multiresistance emergence, the use of compounds exhibiting multitarget toxicity is an interesting and novel alternative, since getting a mutation or acquiring genetic determinants against these new compounds is minimal. In this context, using molecules as tellurite to potentiate the antibacterial effect seems to be a fine approach. Although information regarding TeO 3 22 toxicity for eukaryotic cells is scarce to date, it has been shown that 50 mM tellurite (.125-fold the maximal dose used in this work) seems not to affect the viability of eukaryotic cells [15]. In fact, in different cell lines death occurs at ,160-1,600 mM tellurite, as compared to the E. coli 4 mM killing-dose. Despite the important effect in survival observed in neurons [19] and erythrocytes exposed to 100-500 mM tellurite [25], no significant effects have been reported when lower concentrations were used [26]. Indeed, a therapeutic use of tellurite as a red cell antisickling agent has been proposed [24]. Although rats receiving 8 mM tellurite daily doses did not reveal toxic effects over a year, tellurite-treated animals showed increased mortality after 19 months [27,28]. Despite these considerations, it results obvious that the real effect of tellurite on eukaryotic cells has not been well established to date.
Considering the use of tellurite as strategy to kill bacteria without affecting eukaryotic host cells, it was determined that sub lethal TeO 3 22 concentrations increase the susceptibility of different bacterial species to various antibiotics in either LB or Müeller-Hinton media (not shown). The fact that increased growth inhibition zones were observed with most tested antibiotics in the presence of TeO 3 22 (Fig. 1) suggests that this condition may not be related to the antibiotic's specific target. This allows hypothesizing a common mechanism underlying the observed potentiating effect. As shown by Kohanski et al. for bactericidal antibiotics [6] and by our group for tellurite [9], these compounds promote oxidative stress which could in part explain their toxicity. Experiments to address this issue are under way in our laboratory.
Tellurite-mediated potentiation of TET, GEN and CHL is probably consequence of a combined effect upon protein synthesis (mediated by the antibiotic) and tellurite-induced protein oxidation. In this context, E. coli protein misfolding/mistranslation or oxidation has been observed upon exposure to some aminoglycosides [29] or tellurite [9], respectively.
Major changes in growth inhibition zones observed with Gram negative bacteria facing simultaneously tellurite and antibiotics are probably consequence of their high tellurite susceptibility as compared to that exhibited by Gram positive microorganisms [14]. Differences in growth inhibition areas between E. coli and P. aeruginosa may be explained because of the high antibioticresistance levels exhibited by the last bacterium. In spite of this, its susceptibility to antibiotics can be increased in the presence of low tellurite concentrations (Fig. 1B). On the other hand, a less robust effect was observed when S. aureus was exposed to tellurite and antibiotics, probably because the high resistance to both toxicants exhibited by this Gram positive rod.
A synergistic effect, evidenced by a difference of .2 log units in cell viability, was observed when characterizing the magnitude and the type of tellurite-mediated CTX-potentiating effect (Figs. 3B and C). This was also the case with growth curves, where an important decrease in OD 600 was observed when exposing to both antimicrobials (Fig. 3). Although viability was rather unaltered, increased turbidity was observed when E. coli was exposed to  0.13 mg/ml CTX (Fig. 3B), a result that might be explained by cell filamentation upon exposition to b-lactam agents as cefotaxime [30]. Cell viability was recovered only 7 h after exposing to a lethal cefotaxime concentration (0.5 mg/ml) (Fig. 3C), a result that may reflect a decreased antibiotic bioavailability because of covalent linkage formation with bacterial penicillin binding proteins (PBPs), as has been described for other b-lactam antibiotics [31].
Since hydroxyl radical and superoxide formation occurs during E. coli exposure to tellurite or bactericidal antibiotics, respectively [6,9], the observed tellurite/cefotaxime synergistic effect would be most probably due to an oxidative stress outbreak. This idea is reinforced even with sub lethal antibiotic concentrations, where enhanced DNA damage and mutation rate are observed [32]. Experiments to address this issue are currently being carried out in our laboratory.
The idea of using TeO 3 22 lies on its extremely high toxicity to bacteria as compared to other metals or non metals as chromium, lead, or manganese [14]. In addition to establishing an oxidative stress status (9), the existence of multiple tellurite cell targets (13, 14) makes the emergence of strains resistant to the antibioticpotentiating strategy is almost negligible.
Our findings strongly suggest that the use of tellurite (or similar antimicrobials) as an antibiotic-potentiating adjuvant is a novel and feasible strategy to face the antibacterial multi-resistance problem. It is also particularly promising given that the antibacterial-potentiating effect was observed with antibioticresistant clinical isolates.
Finally, unveiling the molecular mechanism of the antibioticpotentiating effect described in this work should contribute to the development of new molecules or compounds to be applied in new therapies to treat infections caused by antibiotic-resistant bacteria.
Growth inhibition zones were determined for each antibiotic in the absence or presence of 200 or 400 nM tellurite for all 20 isolates and averaged. Results were expressed as the mean of 3 independent trials.

Ethics statement
An individual, informed, written consent was obtained from each participant allowing the samples to be used in this study. Procedures for handling clinical samples were carried out in as recommended in Biosafety Laboratory Manual (3 rd Ed., WHO, 2005). Microbiological procedures as well as carrying out this study were specifically approved by the Ethics Committee of Facultad de Ciencias, Universidad San Luis Gonzaga, Ica, Perú.
Viability assays E. coli grown to OD 600 ,0.3 was exposed to tellurite (0.05 mg/ ml) alone or in combination with CTX (0.065, 0.13 or 0.5 mg/ml). At different time intervals samples were serially diluted (1:10) in PBS buffer, pH 7.0. Ten ml of each dilution were seeded on LB plates to determine the number of colony-forming units (CFU). The CFU/ml was determined using the formula: [(#colonies)*(dilution factor)]/(volume plated in ml) as previously described [6].
Growth curves E. coli grown to OD 600 ,0.4 was incubated in the presence of CTX or tellurite (see above for concentrations) at 37uC with shaking and absorbance was recorded at 600 nm.