Abstract

Objectives

The antibacterial effect of the organo-tellurium compound AS101 on the Gram-negative bacterium Enterobacter cloacae is shown in this study for the first time.

Methods

The antimicrobial effect of the drug was shown by inhibition of growth, by inhibition of biofilm formation and by its ability to penetrate the bacterial cell and to cause damage and ultrastructural changes.

Results

AS101 was found to be a bactericidal drug with MICs and MBCs of 9.4 mg/L. It inhibits bacterial growth and causes a six orders of magnitude decrease in viability in a protein-rich medium, but not in a protein-poorer medium, unless 2-mercaptoethanol is added. Subinhibitory concentrations inhibit motility and biofilm formation. AS101 enters the bacterium through its porins and causes bacterial damage to Na+/K+ pumps and leakage of potassium, phosphorous and sulphur. Ultrastructural changes within the bacterial cell and on its surface demonstrate an incomplete surface with a concavity in the centre that looks like a hole from which aggregates are liberated as well as cell lysis.

Conclusions

AS101 has antibacterial activity, which may be useful against E. cloacae and other species of Enterobacteriaceae as a substitute for current antibiotics that have become ineffective due to increasing bacterial resistance.

Introduction

The non-toxic immunomodulator ammonium trichloro(dioxyethylene-O,O′)tellurate (AS101) is a low molecular weight (312 Da) synthetic organo-tellurium compound.1 AS101 possesses immunomodulating properties2–5 and has been shown to exert beneficial effects in several preclinical and clinical studies. It has also been shown to possess antibacterial ability in the caecal ligation and puncture (CLP) model by increasing the survival rate of septic mice6 and by reducing the mortality and number of wounds in a goldfish model infected with Aeromonas salmonicida bacteria and subjected to stress.7 The above models suggested an indirect effect of AS101 on Gram-negative bacteria. This laboratory has recently shown a direct antimicrobial effect of AS101 on extended-spectrum β-lactamase (ESBL) and non-ESBL-producing strains of Klebsiella pneumoniae.8

Enterobacter cloacae comprises part of the normal flora of the gastrointestinal tract of 40%–80% of the population and is widely distributed in the environment.9,10 Like most members of the Enterobacteriaceae family, these microorganisms are important nosocomial pathogens capable of causing opportunistic infections among hospitalized or debilitated9,11–14 and immunosuppressed patients.14E. cloacae has also emerged as an important pathogen that causes bacteremia9,13–16 and high-risk infections in neonatal intensive care units.11–13,17 Due to the increase in antibiotic-non-susceptible strains, the effect of the immunomodulator AS101 was also investigated in E. cloacae. In the present study, the antimicrobial potential of AS101 on E. cloacae, inhibition of biofilm formation and its ability to penetrate the E. cloacae cell as well as damage caused by this compound were examined.

Materials and methods

Bacterial strains

Nine clinical strains of E. cloacae were studied in this research. E. cloacae strains were isolated at the Clinical Microbiology Laboratory from pus (four strains) and from blood cultures (five strains) and were kindly provided by Dr Z. Lazarovich, head of the Clinical Microbiology Laboratory at Assaf Harofe Medical Center, Zerifin, Israel. The strains were tested for their susceptibility to antibiotics by the standardized agar diffusion technique on Mueller–Hinton agar (Difco, Detroit, MI, USA). Antibiotic discs were purchased from Becton-Dickinson and their content in micrograms per disc as well as their symbol are indicated in parentheses. All nine strains were non-susceptible to ampicillin (AM 10), cefalotin (CF 30), cefoxitin (FOX 30), erythromycin (E 15) and trimethoprim/sulfamethoxazole (SXT 25). They were susceptible to tetracycline (TE 30), polymyxin B (PB 300) and the aminoglycosides tobramycin (NN 10) and amikacin (AN 30). These strains were also susceptible to the quinolones ciprofloxacin (CIP 5) and ofloxacin (OFX 5).

Bacterial growth and treatment conditions

All nine strains were grown overnight at 37°C under aerobic conditions on brain heart agar (BHA). Cultures were transferred into brain heart infusion (BHI) liquid medium to obtain an initial optical density (OD) of 0.1 at 660 nm (about 1.2 × 108 cfu/mL). Cultures were allowed to grow further with agitation at 180 rpm. In most experiments, treatment with AS101 began when the cultures reached an OD of 0.4 at 660 nm (about 5 × 108 cfu/mL). In some experiments the drug was added to the bacterial cultures at the beginning of the incubation when the OD was 0.1 at 660 nm. Bacterial growth was determined at 660 nm with a Biochrom LKB spectrometer (Novaspec, Cambridge, UK). Viable bacteria were monitored and calculated by counting the number of cfu after appropriate dilution on agar plates. Bacterial cultures grown under the same conditions but without the drug served as controls.

Organo-tellurium compound AS101

AS101 (Figure 1) was supplied by Professor M. Albeck from the Department of Chemistry at Bar-Ilan University, Ramat-Gan, Israel, as a solution of 150 mg/L in PBS at pH 7.4, and was maintained at 4°C until used.

Figure 1.

Chemical structure of AS101.

Figure 1.

Chemical structure of AS101.

Agar diffusion test

The agar diffusion test was performed using the Kirby-Bauer diffusion method.18 Cultures of E. cloacae were grown aerobically overnight at 37°C on BHA dishes, then suspended in BHI until an OD of 0.1 at 660 nm (about 1.2 × 108 cfu/mL) was obtained and were allowed to grow with agitation of 180 rpm to obtain exponential growth up to an OD of 0.4 at 660 nm. The cells were then diluted 1 : 100 and spread uniformly on agar plates containing the indicated medium. For testing the antibacterial activity, 10 μL drops of AS101 containing final amounts of 0.094, 0.1875, 0.375, 0.75 and 1.5 μg/drop were placed on the surface of BHA plates or in comparison tests on nutrient agar (NA). The plates were then incubated aerobically for 24 h at 37°C and diameters of the inhibition zones were measured in millimetres.

MIC and MBC determinations

The MIC of AS101 was determined by the broth dilution method.19 Cultures of E. cloacae were grown aerobically overnight on BHA dishes at 37°C, then suspended into BHI to obtain an OD of 0.1 at 660 nm and agitated at 180 rpm to obtain exponential growth cultures (about 5 × 108 cfu/mL). Tubes containing BHI medium and 2-fold dilutions of AS101 from a concentration of 75 mg/L to 2.3 mg/L were inoculated with ∼105 bacteria and incubated aerobically for 24 h at 37°C. The MIC was determined as the lowest concentration of AS101 that prevented bacterial growth. The MBC was determined by sampling all the tubes without visual turbidity from the MIC test. Aliquots of 0.1 mL were spread on BHA plates. The MBC was considered to be the lowest concentration of AS101 that totally inhibited the growth of any cfu within 24 h of incubation at 37°C. A culture containing PBS at the same volume as the drug and inoculated with the same inoculum of E. cloacae cells was used as a positive control.

Testing the motility and the ability of E. cloacae to form a biofilm

Motility assays were performed as described by Marr et al.20 and Overhage et al.21 with some minor modifications. Overnight cultures of E. cloacae were grown on Luria-Bertani (LB) agar plates (Difco). Single colonies of E. cloacae were inoculated into the special swimming, swarming or twitching agar plates as 1–2 μL aliquots. Swimming motility was evaluated on BM2 + glucose plates. The latter medium contained 62 mM potassium phosphate buffer at pH 7 in addition to 7 mM (NH4)2SO4, 2 mM MgSO4, 10 μM FeSO4 and 0.4 % (w/v) glucose. Agar (Becton-Dickinson), 0.3% (w/v), was added to this medium. Swarming was examined on modified BM2 + glucose plates, where 0.5 % casamino acids substituted for the 7 mM (NH4)2SO4. The modified medium contained 0.5% (w/v) agar (Becton-Dickinson). Twitching was evaluated on LB plates (Difco) that contained 1 % agar. For treatment with AS101, the drug was added to the medium before solidification in all three types of plates at final concentrations of 4.7 or 9.4 mg/L. Plates without the drug served as controls. Plates were incubated for 20 h at 37°C, after which swimming or swarming was measured. Twitching was measured after 24–48 h of incubation at 37°C.22 All experiments were repeated three times.

Biofilm formation was detected as described in recent work,23,24 with some modifications. Overnight cultures were diluted 1 : 100 into fresh BHI medium in borosilicated glass tubes (75 × 12 mm) that contained 2-fold decreasing concentrations of AS101 from 9.4 mg/L (MIC) to 0.55 mg/L. A culture without AS101 served as a control. All cultures were incubated at 37°C for 15 h. Static biofilm was detected by discarding the medium and rinsing the tubes twice with double-distilled water. Tubes were loaded with 4 mL of 1% Crystal Violet for 15 min. The dye was washed out with double-distilled water and the appearance of a biofilm was demonstrated by the existence of a violet ring in the tubes.

Reconstitution of pore-forming proteins in liposomes and swelling assay

Proteins from the outer membranes of E. cloacae were separated according to the method described by Tada and Yamaguchi.25 The pore-forming proteins (porins) were purified by the method described by Wexler and Getty.26 The purified porin molecules were reconstituted in liposomes that were prepared according to the procedures of Nikaido and Rosenberg27 and Wexler et al.28 The liposome swelling assay was performed as described by Nikaido and Rosenberg27 and was also described by our laboratory.29–31 Liposome swelling was detected by a Cary 200 Scan Spectrometer combined with a computer program.

X-ray microanalysis (XRMA)

XRMA combined with scanning electron microscopy (SEM) was used for elemental analysis of individual cells whose content was fixed by deep freezing immediately after treatment. The method has been described for bacterial cultures previously by Daniel-Hoffmann et al.,8 Malik et al.32 and Nitzan et al.33 All X-ray spectra were taken with the same count rate.34

Inductively coupled plasma mass spectrometry (ICPMS) detection

Analysis by ICPMS, which is analytical atomic spectrometry, was performed in aqueous homogeneous medium. E. cloacae cultures at the logarithmic phase were treated for 10 min with 9.4 mg/L AS101 or with PBS as control and were centrifuged for 5 min at 5000 g. The supernatants were discarded and the pellets were treated with 1 M HNO3 for 15 min and washed twice. The chemical elements were analysed by ICPMS (Ultima-2; Ji Jobin Yvon Horiba, Longjumeau, France) and their concentrations calculated. Calibration standards at 0.1, 1.0, and 10.0 ppm were used for each set of analyses. The mean and standard deviation data were based on three replicates per sample. Several of the samples were re-diluted and analysed as duplicates to ensure reproducibility.35

Transmission electron microscopy (TEM)

Bacterial cells at the log phase were treated with 9.4 mg/L AS101 for 0.5 h and processed for electron microscopy analysis. Cell pellets were first washed in 0.05 M cacodylate buffer (pH 7.2) and then fixed for 2 h in 0.05 M cacodylate 1.5% (w/v) glutaraldehyde (pH 7.2). The same buffer was then used for overnight washing of the sample, followed by 2 h of fixation with 2% (w/v) osmium tetroxide and dehydration with ethanol. Finally, the samples were embedded in Durcupan, and thin sections were prepared with an LKB Ultratome Nova. The sections were double-stained with uranyl acetate and lead citrate.36 Specimens were examined with a Philips CM-100 transmission electron microscope.

SEM

Bacterial cells at the log phase were treated with 9.4 mg/L AS101 for 20 min and processed for electron microscopy analysis. Cell pellets were first washed three times in phosphate buffer without Ca2+ and Mg2+ and then fixed for 1 h in 2.6% glutaraldehyde (pH 7.2) at room temperature. After fixation, samples were allowed to settle for 24 h at 4°C. A 100 μL sample was suspended on a glass grid covered with ɛ-poly-L-lysine for 1 h, followed by 1 h of fixation with 2% (w/v) osmium tetroxide, and dehydration with ethanol and Freon. Finally, the samples were coated with carbon and the specimens were examined in a JSM-35C scanning electron microscope.

Statistical analysis

The data reported are the average values from a minimum of three experiments for each strain. Differences between bacterial strains were analysed by Student's t-test, with P < 0.05 considered to be statistically significant.

Results

Antibacterial activity of AS101

The antibacterial activity of the tellurium compound AS101 was indicated by inhibition of the growth of the nine E. cloacae isolates when the inhibition zone of the drug was tested on a rich medium (BHA). No susceptibility to AS101 was shown when bacteria were grown on a poorer medium (NA), except for a slight and insignificant inhibition diameter (8 mm) seen with 1.5 μg (Figure 2). The MIC and MBC of AS101 were found to be 9.4 mg/L AS101 for all nine E. cloacae isolates (Table 1). Since the MIC and MBC values for AS101 were the same, it can be concluded that AS101 acts as a bactericidal agent for E. cloacae. In accordance with the MIC determination, cultures of E. cloacae were treated in BHI with 9.4 mg/L AS101 at the beginning of the lag phase and at the beginning of the log phase. The same growth inhibition was evident when AS101 was added at the lag phase, and no further growth was observed. When AS101 was added at the log phase (Figure 3, top panel), inhibition began immediately after AS101 was added and a decrease in OD was observed. The same results were obtained when cultures (at the lag phase or the log phase) were treated with AS101 in nutrient broth (NB) supplemented with 2-mercaptoethanol (0.012 mM).

Table 1.

MIC and MBC tests of AS101 on E. cloacae

  AS101 concentration (mg/L)
 
0.00 1.17 2.35 4.70 9.40 18.75 37.5 75.0 
Growth − − − − 
Viability − − − − 
  AS101 concentration (mg/L)
 
0.00 1.17 2.35 4.70 9.40 18.75 37.5 75.0 
Growth − − − − 
Viability − − − − 
Figure 2.

Agar diffusion test of E. cloacae treated with different doses of AS101. Diameters of inhibition zones were measured when bacteria were treated with AS101 on BHA or NA.

Figure 2.

Agar diffusion test of E. cloacae treated with different doses of AS101. Diameters of inhibition zones were measured when bacteria were treated with AS101 on BHA or NA.

Figure 3.

Top panel: Growth curves of E. cloacae treated with AS101 (9.4 mg/L) at the lag phase or at the log phase. Cultures without addition of AS101 served as a control. Arrows indicate addition of AS101. Bottom panel: Viability curves of E. cloacae following treatment with AS101 (9.4 mg/L) at the log phase. Cultures without addition of AS101 served as a control.

Figure 3.

Top panel: Growth curves of E. cloacae treated with AS101 (9.4 mg/L) at the lag phase or at the log phase. Cultures without addition of AS101 served as a control. Arrows indicate addition of AS101. Bottom panel: Viability curves of E. cloacae following treatment with AS101 (9.4 mg/L) at the log phase. Cultures without addition of AS101 served as a control.

The killing curve of E. cloacae treated in BHI with 9.4 mg/L AS101 clearly showed that the viability decreased by more than two orders of magnitude during the first 30 min of exposure to AS101 (Figure 3, bottom panel). The number of bacteria continued to decrease until the bacterial culture was totally diminished (more than eight orders of magnitude decrease) within 2 h from the beginning of treatment.

Effect of AS101 on motility and biofilm formation

Three motility parameters of E. cloacae were tested: swimming, swarming and twitching. Each parameter was measured in a special sort of plate and special agar concentration (see Materials and Methods section). Swimming was influenced mostly by the MIC (9.4 mg/L) and the 2-fold diluted (4.7 mg/L) concentrations of AS101 compared with the untreated control (Table 2). Swarming and twitching were also totally inhibited by the above concentrations of AS101.

Table 2.

Motility assays (swimming, swarming and twitching) and biofilm formation ability of E. cloacae before and after treatment with the MIC (9.4 mg/L) and decreasing subinhibitory concentrations of AS101

Test Treatment with AS101 (mg/L)
 
0.58 1.17 2.35 4.7 9.4 
Swimming (mm) 90 ± 1.0 ND ND ND 1.0 1.0 
Swarming (mm) 7 ± 0.3 ND ND ND 0.0* 0.0* 
Twitching (mm) 5 ± 0.3 ND ND ND 0.0* 0.0* 
Biofilm formation ± − − 
Test Treatment with AS101 (mg/L)
 
0.58 1.17 2.35 4.7 9.4 
Swimming (mm) 90 ± 1.0 ND ND ND 1.0 1.0 
Swarming (mm) 7 ± 0.3 ND ND ND 0.0* 0.0* 
Twitching (mm) 5 ± 0.3 ND ND ND 0.0* 0.0* 
Biofilm formation ± − − 

ND, not done.

Experiments were repeated three times independently.

*Results indicated are statistically significant (P ≤ 0.01).

Static biofilm formation was inhibited at AS101 concentrations of 9.4 and 4.7 mg/L (Table 2). At a concentration that is 4-fold lower than the MIC (2.35 mg/L), biofilm formation was very weak. At even lower AS101 concentrations (1.17 and 0.58 mg/L), biofilm formation was very significant and was equal to that of the positive control (without AS101).

Penetration of AS101 into the bacterial cell

The question of whether the compound penetrates the cell was examined in order to gain insight into the mechanism of action of AS101 on E. cloacae bacterial cells. Penetration of AS101 was demonstrated using a liposome swelling assay. In this assay, porin molecules were inserted into the surface of the liposome. The porin used for this assay was the one expressed on the outer membrane of an E. cloacae strain. It was purified and was shown to be a trimeric porin with a MW of 120 kDa. The results shown in Figure 4 demonstrate that AS101 (MW 312 Da) exhibits an ability to penetrate the liposomes through the specific E. cloacae porin. Arabinose (MW 150 Da) had a much higher penetration rate than AS101, since it has a lower molecular weight. On the other hand, stachyose (MW 666 Da) could not enter these liposomes as a result of its high molecular weight. AS101 could not penetrate liposomes that did not have any porins incorporated into their surface. The same was true when the protein BSA was incorporated instead of a porin, i.e. no AS101 penetrated the liposomes. These results suggest that AS101 enters the E. cloacae bacterial cell through a porin and not through the phospholipid envelope of the outer membrane and acts intracellularly on the viability of this bacterium.

Figure 4.

Liposome swelling assay for AS101 using porins purified from the outer membrane of E. cloacae that were reconstituted into liposomes. The sugar stachyose served as a negative control and the sugar arabinose served as a positive control. BSA reconstituted into liposomes and unreconstituted liposomes served as controls.

Figure 4.

Liposome swelling assay for AS101 using porins purified from the outer membrane of E. cloacae that were reconstituted into liposomes. The sugar stachyose served as a negative control and the sugar arabinose served as a positive control. BSA reconstituted into liposomes and unreconstituted liposomes served as controls.

XRMA and ICPMS detection

Ionic fluxes during the activity of AS101 can be determined by XRMA-SEM. As seen above, AS101 entered through the porin and penetrated the periplasm and the cytoplasmic membrane. This kind of interference can change the transportability of some elements into the cell. The XRMA results (Table 3) indicate that E. cloacae treated with 9.4 mg/L AS101 exhibited an increase in the influx of Na and Cl, which may indicate damage to the Na+/K+ pumps located in the cytoplasmic membrane. An increase in Mg was observed, indicating damage to the stability of the cell membrane. On the other hand, a decrease in the level of P was observed, indicating loss of ATP or even damage to DNA.

Table 3.

XRMA of E. cloacae following AS101 treatment

Treatment Percentage atomic weight in the samples
 
Na Mg 
None (control) 0.36 1.63 1.72 
AS101 (9.4 mg/L) 0.54 2.82 1.08 
Treatment Percentage atomic weight in the samples
 
Na Mg 
None (control) 0.36 1.63 1.72 
AS101 (9.4 mg/L) 0.54 2.82 1.08 

The ICPMS detection method was used to confirm the former results. ICPMS is an analytical atomic spectrometry method that can identify most elements (except H, N, O, C and F) and quantify their concentration in ppm units (mg/L). E. cloacae cultures were treated with 9.4 mg/L AS101 for 10 min and then treated with HNO3, and elemental concentrations were measured using the ICPMS detection instrument. Table 4 demonstrates an influx of Na and an efflux of K, P and even S, where the latter indicates protein loss due to AS101. Untreated E. cloacae cells demonstrated higher amounts of K, P and S, while the amounts of sodium were lower than in the treated bacterial cells.

Table 4.

Elemental analysis by ICPMS of E. cloacae before and after treatment with 9.4 mg/L AS101

Element Element concentration (mg/L)
 
control after treatment with AS101 
2.05 ± 0.23 1.42 ± 0.11* 
Na 4.94 ± 0.18 5.79 ± 0.20* 
0.68 ± 0.08 0.21 ± 0.10* 
0.48 ± 0.06 0.35 ± 0.05* 
Element Element concentration (mg/L)
 
control after treatment with AS101 
2.05 ± 0.23 1.42 ± 0.11* 
Na 4.94 ± 0.18 5.79 ± 0.20* 
0.68 ± 0.08 0.21 ± 0.10* 
0.48 ± 0.06 0.35 ± 0.05* 

Each experiment was repeated three times independently.

*Results indicated are statistically significant (P ≤ 0.01) by the t-test.

Electron microscopy examination

The results indicated that the possible action of the tellurium compound AS101 on E. cloacae cells would eventually lead to a cytotoxic destructive process that could be demonstrated by TEM. An isolate of E. cloacae was grown to the logarithmic phase on BH medium and treated with 9.4 mg/L AS101 for 30 min. Figure 5(a) shows cell lysis following AS101 treatment compared with the control, which was not treated with AS101 (Figure 5b). It seems that treatment with AS101 not only damaged the surface of the cells, but also caused acute damage inside the cell that eventually led to cell death.

Figure 5.

TEM of an E. cloacae culture treated at the mid-log phase with 9.4 mg/L AS101 (a). The same culture without AS101 served as a control (b). SEM of an E. cloacae culture treated with 9 mg/L AS101 (c). The same culture without AS101 served as a control (d). SEM of E. cloacae cells treated with 9 mg/L AS101 where aggregates are liberated from the cavity (e). Enlargement of a cell where a phage is coming out of the cavity (f).

Figure 5.

TEM of an E. cloacae culture treated at the mid-log phase with 9.4 mg/L AS101 (a). The same culture without AS101 served as a control (b). SEM of an E. cloacae culture treated with 9 mg/L AS101 (c). The same culture without AS101 served as a control (d). SEM of E. cloacae cells treated with 9 mg/L AS101 where aggregates are liberated from the cavity (e). Enlargement of a cell where a phage is coming out of the cavity (f).

The morphology of E. cloacae cells treated with AS101 was observed using SEM, analysed under the same conditions as for TEM. The cell wall of the bacteria treated with the tellurium compound was damaged (Figure 5c). A concavity that looks like a hole was observed in the centre of treated cells, which implies cell wall perforation. This concavity was not observed in the untreated cells (Figure 5d). In some cells, aggregates were liberated from the concavity and it seemed that the content of the bacterial cell spilled out (Figure 5e). Enlargement of such cells indicated the shape of an unidentified phage coming out of the concavity in the cell wall (Figure 5f).

Discussion

It was previously shown that AS101 has antimicrobial potential against K. pneumoniae.8 In the current study, presentation of the spectrum of the antibacterial effect of AS101 was expanded to another species of the Enterobacteriaceae family. This time the drug was examined against E. cloacae. Infectious diseases caused by Enterobacteriaceae are currently treated with aminoglycoside antibiotics such as gentamicin or tobramycin. However, these antibiotics can be toxic,37,38 especially for patients with renal failure. Furthermore, long-term treatment with these antibiotics may lead to the development of resistance. In contradistinction, AS101 has been shown to be non-toxic in various cell cultures as well as in in vivo models39 using the concentrations mentioned in this research. AS101 was also used in phase II clinical trials with cancer patients with no toxicity40 and is currently being used in phase II clinical trials for psoriasis and atopic dermatitis. It thus appears that AS101 may provide a potential substitute or supplement to antibiotic treatments.

The potential activity of AS101 against E. cloacae was characterized by several methods for the first time. Treatment of E. cloacae isolates with AS101 caused bacterial growth inhibition on agar, which was medium dependent. In this study, AS101 inhibited E. cloacae growth, especially in a rich medium (BHI), and did not act in NB unless 2-mercaptoethanol was added with the drug. The same phenomenon was found with K. pneumoniae.8 It seems that the reason is not only because BHI contains a higher protein concentration than NB medium (15-fold),41 but also because it contains a high level of thiols. This is an outcome of the fact that Te (IV) compounds readily interact with thiols to form Te(thiol)4 compounds.42 All of these findings led us to suggest that AS101 may require a carrier to cross the bacterial cell barriers and that it uses free thiols as a carrier. These results indicate that thiols play an important role in the mechanism of action of AS101 and support its clinical potential of AS101.

The MICs and MBCs of AS101 in the protein-rich medium were found to be 9.4 mg/L for all nine isolates tested. This drug concentration immediately inhibited growth of the bacteria when they were treated at the lag phase. When bacteria are treated at an early stage of the logarithmic phase, growth is stopped and a decrease in OD is seen within 30 min from the beginning of the treatment. Viability also decreased immediately, and total eradication of the culture was obtained in less than 2 h (a decrease of more than seven orders of magnitude).

The results obtained in this research indicate that AS101 can penetrate the cells of E. cloacae through the outer membrane. The major role of the outer membrane of Gram-negative bacteria is to serve as a permeability barrier to prevent the entry of noxious compounds and, at the same time, allow the influx of nutrient molecules27,43 through pore-forming proteins (porins). The entrance of AS101 through porins that were purified from E. cloacae and incorporated into liposomes, imitating the outer membrane, led us to suggest a possible mechanism whereby AS101 penetrates the E. cloacae cell. The pore of the E. cloacae porin is large enough (in diameter) to allow a molecule the size of AS101 (MW 312 Da) to penetrate through it. An alternative possibility, that AS101 might enter the outer membrane directly, was ruled out since AS101 did not penetrate into liposomes not incorporated with the porin or into liposomes in which BSA was incorporated instead of the porin. These results can be extrapolated to other members of the Enterobacteriaceae family and may be true for other Gram-negative bacteria.

One of the concerns of infections in hospitals is the ability of bacteria to form a biofilm on surfaces, equipment and inside catheters.44–46 The formation of a biofilm inside a catheter can be life threatening when the bacterial mass breaks off from the inner wall of the catheter and is flushed by the infusion fluid directly into the patient's bloodstream, thus causing sepsis.44 An important process in the development of a biofilm is the motility of bacteria on surfaces.47 In the present study it was unequivocally demonstrated that the motility of E. cloacae is inhibited following treatment with AS101. All three characteristic forms of motility, swimming via flagella,47 swarming via flagella and pili46 and twitching via pili46 were impaired following treatment with AS101. The impairment is apparent not only at the MIC (9.4 mg/L) of the drug, but also at a 2-fold subinhibitory concentration (4.7 mg/L) of the drug against E. cloacae. It was also found that E. cloacae bacteria did not succeed in forming a biofilm, even at a subinhibitory concentration of half (4.7 mg/L) of the MIC (9.4 mg/L), and at one quarter (2.35 mg/L) of the MIC the biofilm was very weak. The ability of AS101 to inhibit the virulence factors for motility and attachment to surfaces of cells or artificial devices (biofilm formation) is very important, since it can be very beneficial to the clinician. This finding also supports the above results that AS101 molecules penetrate through porins and act intracellularly on inner-cell components that are responsible for the production of flagella and pili.

Further proof for the phenomenon that AS101 molecules act inside the cell was provided by XRMA and ICPMS. Both methods indicated that upon penetration into the bacterial cell, AS101 damages the cytoplasmic membrane as well as the pumps connected to it. It seems that AS101 can induce damage to Na+/K+ pumps, which causes leakage of ions through the damaged membrane. The influx of Na+ into the cell and leakage of P or even S clearly indicate leakage of ATP, nucleotides and even proteins from cells. Cells cannot multiply under such conditions and their viability is decreased.48 The XRMA results with E. cloacae are similar to results demonstrated for K. pneumoniae with the same drug.8 The TEM and SEM images support the findings obtained by XRMA and ICPMS. The empty cells seen by TEM are probably a result of leakage of the cell contents. In some cells, aggregates in the chromosomal area are seen, which may indicate damage to the bacterial chromosome. These aggregates have been shown in our former work with AS101-treated K. pneumoniae.8 A similar picture of aggregates in the chromosomal area was obtained in another work of this laboratory.48 These aggregates were assumed to be repair centres for double-stranded DNA by RecA due to activation of the SOS response.49 SEM demonstrated a hole at the centre of treated cells, which implies cell wall perforation. This was also seen in AS101-treated K. pneumoniae.8 Some AS101-treated E. cloacae cells were seen to liberate small bodies from this hole. These small bodies that leak from the cell could be endotoxin fragments, which are known to be liberated during antibiotic treatment of infections caused by Gram-negative microorganisms.50 Another possibility is that AS101 treatment causes a stress response that leads to the induction of prophages within the bacterial cell and the progeny are spilled out through the hole. This is supported by our observation of what appears to be a phage particle emerging from a damaged bacterial cell. No experimental proof was obtained to date for this phenomenon, probably because of the lack of a proper propagating bacterial strain that will show plaques on agar plates or lysis in a liquid medium.

The antimicrobial potential of AS101 is exhibited in this study on E. cloacae, another member of the Enterobacteriaceae family. This family is known to contain many pathogenic species that cause severe infections in hospitalized patients and are now being tested with AS101. AS101 may provide a potential substitute or supplement to current antibiotic treatment that has been demonstrating increasing resistance.

Funding

This work was partly supported by the Dr Tovi Comet-Walerstein Cancer Research Chair, the Dave and Florence Muskovitz Chair in Cancer Research and the Frieda Stollman Cancer Memorial Fund. This research was also supported in part by the Rappaport Foundation for Microbiology, Bar-Ilan University, Israel (to Y. N.).

Transparency declarations

None to declare.

Acknowledgements

We wish to thank Mrs Rachel Dror for her excellent technical assistance and helpful comments, Dr Izabella Pechatnikov for helping with the liposome swelling assay, and Mr Ofir Avidan for his help in preparing samples for TEM, and for helpful discussions and assistance.

Some of the material included in this manuscript for the bacterium E. cloacae was previously published in the following abstract: Hoffman-Daniel M, Albeck M, Sredni B, Nitzan Y. The bactericidal activity of the tellurium compounds AS 101 on Enterobacter cloacae. Annual Meeting of the Israel Society for Microbiology, Rehovot, Israel, 2008. P-26. p. 76.

This study was conducted as part of the requirements for a PhD by M. D.-H.

References

1
Albeck
M
Tamari
T
Sredni
B
Synthesis and properties of ammonium trichloro(dioxyethylene-O-O) tellurate (AS-101)—a new immunomodulating compound
Synthesis Stuttgart
 , 
1989
, vol. 
8
 (pg. 
635
-
6
)
2
Sredni
B
Caspi
RR
Klein
A
, et al.  . 
A new immunomodulating compound (AS-101) with potential therapeutic application
Nature
 , 
1987
, vol. 
330
 (pg. 
173
-
6
)
3
Sredni
B
Caspi
RR
Lustig
S
, et al.  . 
The biological activity and immunotherapeutic properties of AS-101, a synthetic organotellurium compound
Nat Immun Cell Growth Regul
 , 
1988
, vol. 
7
 (pg. 
163
-
8
)
4
Sredni
B
Rosenthal-Galili
Z
Michlin
H
, et al.  . 
Restoration of murine cytomegalovirus (MCMV) induced myelosuppression by AS101
Immunol Lett
 , 
1994
, vol. 
43
 (pg. 
159
-
65
)
5
Rosenblatt-Bin
H
Kalechman
Y
Vonsover
A
, et al.  . 
The immunomodulator AS101 restores T(H1) type of response suppressed by Babesia rodhaini in BALB/c mice
Cell Immunol
 , 
1998
, vol. 
184
 (pg. 
12
-
25
)
6
Kalechman
Y
Gafter
U
Gal
R
, et al.  . 
Anti-IL-10 therapeutic strategy using the immunomodulator AS101 in protecting mice from sepsis-induced death: dependence on timing of immunomodulating intervention
J Immunol
 , 
2002
, vol. 
169
 (pg. 
384
-
92
)
7
Okun
E
Albeck
M
Avtalion
RR
, et al.  . 
The immunomodulator AS101 protects against Aeromonas salmonicida infections in stressed goldfish by regulating an IL-10-cytokine
2003
Appendix III: published article, reprinted from the International Cytokine Society Annual Meeting
Dublin, Ireland
(pg. 
157
-
61
)
8
Daniel-Hoffmann
M
Albeck
M
Sredni
B
, et al.  . 
A potential antimicrobial treatment against ESBL-producing Klebsiella pneumoniae using the tellurium compound AS101
Arch Microbiol
 , 
2009
, vol. 
191
 (pg. 
631
-
8
)
9
Gaston
MA
Enterobacter—an emerging nosocomial pathogen
J Hosp Infect
 , 
1988
, vol. 
11
 (pg. 
197
-
208
)
10
Rozenberg-Arska
M
Visser
MR
Cohen
J
Powderly
WG
Enterobacteriaceae
Infectious Diseases
 , 
2004
London
Mosby
(pg. 
2189
-
99
)
11
Dalben
M
Varkulja
G
Basso
M
, et al.  . 
Investigation of an outbreak of Enterobacter cloacae in a neonatal unit and review of the literature
J Hosp Infect
 , 
2008
, vol. 
70
 (pg. 
7
-
14
)
12
Krzymińska
S
Mokracka
J
Koczura
R
, et al.  . 
Cytotoxic activity of Enterobacter cloacae human isolates
FEMS Immunol Med Microbiol
 , 
2009
, vol. 
56
 (pg. 
248
-
52
)
13
Chen
HN
Lee
ML
Yu
WK
, et al.  . 
Late-onset Enterobacter cloacae sepsis in very-low-birth-weight neonates: experience in a medical center
Pediatr Neonatol
 , 
2009
, vol. 
50
 (pg. 
3
-
7
)
14
Watson
JT
Jones
RC
Siston
AM
, et al.  . 
Outbreak of catheter-associated Klebsiella oxytoca and Enterobacter cloacae bloodstream infections in an oncology chemotherapy center
Arch Intern Med
 , 
2005
, vol. 
165
 (pg. 
2639
-
43
)
15
Bodey
GP
Elting
LS
Rodriguez
S
Bacteremia caused by Enterobacter—15 years of experience in a cancer hospital
Rev Infect Dis
 , 
1991
, vol. 
13
 (pg. 
550
-
8
)
16
Falkiner
FR
Enterobacter in hospital
J Hosp Infect
 , 
1992
, vol. 
20
 (pg. 
137
-
40
)
17
Castro
B
Montesinos
I
Fuster-Jorge
P
, et al.  . 
Epidemiology of Enterobacteriaceae causing bloodstream infections in neonatal intensive care unit patients
Enferm Infec Microbiol Clin
 , 
2010
, vol. 
28
 (pg. 
227
-
32
)
18
Bauer
AM
Kirby
WMM
Sherris
JC
, et al.  . 
Antibiotic susceptibility testing by a standardized simple disk method
Am J Clin Pathol
 , 
1966
, vol. 
45
 (pg. 
493
-
6
)
19
Gavan
TL
Town
MA
A microdilution method for antibiotic susceptibility testing—an evaluation
Am J Clin Pathol
 , 
1970
, vol. 
53
 (pg. 
880
-
5
)
20
Marr
AK
Overhage
J
Bains
M
, et al.  . 
The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility
Microbiology
 , 
2007
, vol. 
153
 (pg. 
474
-
82
)
21
Overhage
J
Campisano
A
Bains
M
, et al.  . 
Human host defense peptide LL-37 prevents bacterial biofilm formation
Infect Immun
 , 
2008
, vol. 
76
 (pg. 
4176
-
82
)
22
Kohler
T
Curty
LK
Barja
F
, et al.  . 
Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili
J Bacteriol
 , 
2000
, vol. 
182
 (pg. 
5990
-
6
)
23
Jackson
DW
Suzuki
K
Oakford
L
, et al.  . 
Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli
J Bacteriol
 , 
2002
, vol. 
184
 (pg. 
290
-
301
)
24
Reshamwala
SMS
Noronha
SB
Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis
Arch Microbiol
 , 
2011
, vol. 
193
 (pg. 
711
-
22
)
25
Tada
Y
Yamaguchi
J
A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium
FEMS Microbiol Lett
 , 
1990
, vol. 
58
 (pg. 
115
-
8
)
26
Wexler
HM
Getty
C
The isolation and characterization of a porin protein from Bacteroides fragilis
Anaerobe
 , 
1996
, vol. 
2
 (pg. 
305
-
12
)
27
Nikaido
H
Rosenberg
EY
Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins
J Bacteriol
 , 
1983
, vol. 
153
 (pg. 
241
-
52
)
28
Wexler
HM
Getty
C
Fisher
G
The isolation and characterization of a major outer-membrane protein from Bacteroides distasonis
J Med Microbiol
 , 
1992
, vol. 
37
 (pg. 
165
-
75
)
29
Elazar
M
Halfon
D
Pechatnikov
I
, et al.  . 
Porin isolated from the outer membrane of Erwinia amylovora and its encoding gene
Curr Microbiol
 , 
2007
, vol. 
54
 (pg. 
155
-
61
)
30
Gribun
A
Katcoff
DJ
Hershkovits
G
, et al.  . 
Cloning and characterization of the gene encoding for OMP-PD porin: the major Photobacterium damsela outer membrane protein
Curr Microbiol
 , 
2004
, vol. 
48
 (pg. 
167
-
74
)
31
Magalashvili
L
Pechatnikov
I
Wexler
HM
, et al.  . 
Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure
Anaerobe
 , 
2007
, vol. 
13
 (pg. 
74
-
82
)
32
Malik
Z
Babushkin
T
Sher
S
, et al.  . 
Collapse of K+ and ionic balance during photodynamic inactivation of leukemic cells, erythrocytes and Staphylococcus aureus
Int J Biochem
 , 
1993
, vol. 
25
 (pg. 
1399
-
406
)
33
Nitzan
Y
Ashkenazi
H
Photoinactivation of Deinococcus radiodurans: an unusual Gram-positive microorganism
Photochem Photobiol
 , 
1999
, vol. 
69
 (pg. 
505
-
10
)
34
Zierold
K
Schäfer
D
Preparation of cultured and isolated cells for X-ray microanalysis
Scanning Microsc
 , 
1988
, vol. 
2
 (pg. 
1775
-
90
)
35
Patra
CR
Moneim
SSA
Wang
E
, et al.  . 
In vivo toxicity studies of europium hydroxide nanorods in mice
Toxicol Appl Pharmacol
 , 
2009
, vol. 
240
 (pg. 
88
-
98
)
36
Jap
BK
Walian
PJ
Biophysics of the structure and function of porins
Q Rev Biophys
 , 
1990
, vol. 
23
 (pg. 
367
-
403
)
37
Thomas
T
Galiani
D
Brod
RD
Gentamicin and other antibiotic toxicity
Ophthalmol Clin North Am
 , 
2001
, vol. 
14
 (pg. 
611
-
24
)
38
Bath
AP
Walsh
RM
Bance
ML
, et al.  . 
Ototoxicity of topical gentamicin preparations
Laryngoscope
 , 
1999
, vol. 
109
 (pg. 
1088
-
93
)
39
Nyska
A
Waner
T
Pirak
M
, et al.  . 
Toxicity study in rats of a tellurium based immunomodulating drug, AS-101: a potential drug for AIDS and cancer patients
Arch Toxicol
 , 
1989
, vol. 
63
 (pg. 
386
-
93
)
40
Sredni
B
Albeck
M
Tichler
T
, et al.  . 
Bone marrow-sparing and prevention of alopecia by AS101 in non-small-cell lung cancer patients treated with carboplatin and etoposide
J Clin Oncol
 , 
1995
, vol. 
13
 (pg. 
2342
-
53
)
41
Nitzan
Y
Balzam-Sudakevitz
A
Ashkenazi
H
Eradication of Acinetobacter baumannii by photosensitized agents in vitro
J Photochem Photobiol B
 , 
1998
, vol. 
42
 (pg. 
211
-
8
)
42
Albeck
A
Weitman
H
Sredni
B
, et al.  . 
Tellurium compounds: selective inhibition of cysteine proteases and model reaction with thiols
Inorg Chem
 , 
1998
, vol. 
37
 (pg. 
1704
-
12
)
43
Nikaido
H
Molecular basis of bacterial outer membrane permeability revisited
Microbiol Mol Biol Rev
 , 
2003
, vol. 
67
 (pg. 
593
-
656
)
44
Hall-Stoodley
L
Costerton
JW
Stoodley
P
Bacterial biofilms: from the natural environment to infectious diseases
Nat Rev Microbiol
 , 
2004
, vol. 
2
 (pg. 
95
-
108
)
45
Bryers
JD
Medical biofilms
Biotechnol Bioeng
 , 
2008
, vol. 
100
 (pg. 
1
-
18
)
46
Schroll
C
Barken
KB
Krogfelt
KA
, et al.  . 
Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation
BMC Microbiol
 , 
2010
, vol. 
10
 (pg. 
179
-
89
)
47
O'Toole
GA
Kotler
R
Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development
Mol Microbiol
 , 
1998
, vol. 
30
 (pg. 
295
-
304
)
48
Ashkenazi
H
Pechatnikov
I
Nitzan
Y
Low-intensity photosensitization may enhance RecA production
Curr Microbiol
 , 
2006
, vol. 
52
 (pg. 
317
-
23
)
49
Cox
MM
Regulation of bacterial RecA protein function
Crit Rev Biochem Mol Biol
 , 
2007
, vol. 
42
 (pg. 
41
-
63
)
50
Prins
JM
van Deventer
SJH
Kuijper
EJ
, et al.  . 
Clinical relevance of antibiotic-induced endotoxin release
Antimicrob Agents Chemother
 , 
1994
, vol. 
38
 (pg. 
1211
-
8
)

Author notes

Both authors contributed equally to this study.