Download Bactericidal activity of the organo

J Antimicrob Chemother 2012; 67: 2165 – 2172
doi:10.1093/jac/dks185 Advance Access publication 24 May 2012
Bactericidal activity of the organo-tellurium compound AS101 against
Enterobacter cloacae
Miriam Daniel-Hoffmann1, Benjamin Sredni1,2† and Yeshayahu Nitzan1*†
1
Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel; 2Safdié Institute for AIDS
and Immunology Research, Bar-Ilan University, Ramat-Gan 52900, Israel
*Corresponding author. Tel: +972-3-5318592; Fax: +972-3-7384058; E-mail: [email protected]
†Both authors contributed equally to this study.
Received 29 January 2012; returned 27 February 2012; revised 2 April 2012; accepted 17 April 2012
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.
Keywords: antimicrobial activity, motility and biofilm formation inhibition, ultrastructural changes
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 b-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.14 E. 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
# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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Daniel-Hoffmann et al.
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 378C 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 48C until used.
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
378C 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 mL drops of AS101 containing final amounts of
0.094, 0.1875, 0.375, 0.75 and 1.5 mg/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 378C and diameters of the inhibition zones were measured in millimetres.
O
CH2
+
NH4
CH2
O
Figure 1. Chemical structure of AS101.
2166
The MIC of AS101 was determined by the broth dilution method.19 Cultures of E. cloacae were grown aerobically overnight on BHA dishes at
378C, 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 378C. 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 378C. 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 mL 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 mM 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
378C, after which swimming or swarming was measured. Twitching
was measured after 24– 48 h of incubation at 378C.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 378C 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
—
Cl3Te
MIC and MBC determinations
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.
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Antimicrobial activity of organo-tellurium against E. cloacae
X-ray microanalysis (XRMA)
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
30
Diameter of inhibition zone (mm)
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
25
20
15
10
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
48C. A 100 mL sample was suspended on a glass grid covered with
1-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.
5
0
0
0.094
0.1875
0.375
0.75
1.5
AS101 (µg)
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.
Table 1. MIC and MBC tests of AS101 on E. cloacae
AS101 concentration (mg/L)
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.
BHA
NA
Growth
Viability
0.00
1.17
2.35
4.70
9.40
18.75
37.5
75.0
+
+
+
+
+
+
+
+
2
2
2
2
2
2
2
2
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 mg (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).
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.
Results
Antibacterial activity of AS101
Effect of AS101 on motility and biofilm formation
The antibacterial activity of the tellurium compound AS101 was
indicated by inhibition of the growth of the nine E. cloacae isolates
Three motility parameters of E. cloacae were tested: swimming,
swarming and twitching. Each parameter was measured in a
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Daniel-Hoffmann et al.
2.1
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
OD at 660 nm
1.8
1.5
1.2
Treatment with AS101 (mg/L)
0.9
Test
0.6
0.3
0
0
30
60
90
120 150 180 210 250
Time (min)
AS101 at log phase
Viable bacteria per mL
1010
108
106
104
AS101
Control
102
10
0
0.5
1
1.5
2
Time (h)
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.
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.
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
2168
0.58
1.17
2.35
4.7
9.4
90+1.0
7+0.3
5+0.3
+
ND
ND
ND
+
ND
ND
ND
+
ND
ND
ND
+
1.0
0.0*
0.0*
2
1.0
0.0*
0.0*
2
ND, not done.
Experiments were repeated three times independently.
*Results indicated are statistically significant (P≤ 0.01).
AS101 at lag phase
Control
Swimming (mm)
Swarming (mm)
Twitching (mm)
Biofilm formation
0
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.
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.
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
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Antimicrobial activity of organo-tellurium against E. cloacae
0.36
Swelling (OD at 400 nm)
0.34
0.32
0.30
Stachyose
AS101
0.28
Arabinose
AS101(No porin)
AS101(BSA)
0.26
1
2
3
4
5
6
Time (s)
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.
Electron microscopy examination
Table 3. XRMA of E. cloacae following AS101 treatment
Percentage atomic weight in the
samples
Treatment
None (control)
AS101 (9.4 mg/L)
Na
Mg
P
0.36
0.54
1.63
2.82
1.72
1.08
Table 4. Elemental analysis by ICPMS of E. cloacae before and after
treatment with 9.4 mg/L AS101
Element concentration (mg/L)
Element
P
Na
K
S
control
after treatment with AS101
2.05+0.23
4.94+0.18
0.68+0.08
0.48+0.06
1.42+0.11*
5.79+0.20*
0.21+0.10*
0.35+0.05*
Each experiment was repeated three times independently.
*Results indicated are statistically significant (P≤ 0.01) by the t-test.
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.
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.
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
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Daniel-Hoffmann et al.
(a)
(b)
(c)
(d)
(e)
(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).
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),
2170
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
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Antimicrobial activity of organo-tellurium against E. cloacae
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.
This study was conducted as part of the requirements for a PhD by
M. D.-H.
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.
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