Fitoterapia 129 (2018) 237–240

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Treating urinary tract infections due to MDR E. coli with Isothiocyanates – a T

phytotherapeutic alternative to antibiotics?
⁎
Nico T. Muttersa, , Anja Mampela, Rebecca Kropidlowskia, Klaus Biehlera, Frank Güntherb,
Ioana Băluc, Veronika Malekc, Uwe Franka
a
Institute for Infection Prevention and Hospital Epidemiology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany
b
Institute of Medical Microbiology and Hospital Hygiene, University of Marburg, Marburg, Germany
c
Heidelberg University Hospital, Centre for Infectious Diseases, Heidelberg, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Multidrug-resistant (MDR) bacteria are increasingly causing urinary tract infections (UTI), which
Isothiocyanates has been linked to frequent use of antibiotics. Alternative treatment regimens are urgently needed and natural
Urinary tract infection isothiocyanates (ITC) may represent one. ITCs are natural plant products found in nasturtium (Tropaeoli majoris
Multidrug resistance herba) and horseradish (Armoraciae rusticanae radix).
Uropathogenic E. coli
Purpose: The objectives were to (1) assess the antimicrobial effects of nature-identical ITCs for UTI treatment
caused by uropathogenic E. coli (UPEC), (2) to evaluate a potential influence of antimicrobial resistance on ITC
susceptibility, and (3) to test whether ITCs affect UPEC penetration into human uroepithelial cells.
Methods: We tested 217 clinical UPEC isolates, 54.5% of which were classified as MDR, for susceptibility against
ITCs. ITC susceptibility testing was performed by broth dilution using a mixture of three synthetic ITCs.
Internalization was tested using human T-24 bladder carcinoma cells in an internalization assay co-incubated
with UPEC (n = 5) and ITCs.
Results: The mean minimal inhibitory concentration (MIC) 90 was 0.17 mg/ml, showing very high susceptibility
against ITCs. Interestingly, MDR E. coli were significantly less susceptible than non-MDR strains (p = .01).
Internalization of UPEC was decreased by 31.9% in the mean when treated with ITCs. Overall, ITCs exerted a
strong antimicrobial activity against clinical UPEC isolates and reduced internalization into uroepithelial cells.
Conclusion: ITCs might present a promising treatment alternative for UTIs, expressing both high antimicrobial
activity as well as blocking the pathogenic process of human cell penetration by UPEC. Clinical studies, however,
are needed to confirm activity of ITCs in UTIs in vivo.

1. Introduction spent on the management of UTIs annually [5].

Urinary tract infections (UTIs) are common bacterial infections in
out - as well as inpatient care and the second most common reason to
prescribe antibiotics [1], often prematurely. Almost every third woman
will be diagnosed with a UTI, requiring antibiotic therapy before the
age of 25 [2]. Uropathogenic Escherichia coli (UPEC), the most fre-
quently isolated species in UTIs [3], are able to survive antibiotic
treatment through internalization and cause relapses. An Italian study
estimated that the costs associated with a single episode of UTI amount
to 239 Euros [4]. Taking the high frequency of UTIs into account, the
economic impact is considerable. An estimated one billion US dollars is
Besides economic factors, the increasing spread of antibiotic re-
sistance is of growing concern [6]. Large epidemiological studies
showed high resistance rates among pathogens causing UTIs [3, 7]. The
Antimicrobial Resistance Epidemiological Survey on Cystitis, for in-
stance, rated > 10% of their E. coli isolates as multidrug-resistant
(MDR) [3]. Hence, finding alternative treatment and relapse prevention
strategies is of utmost importance [8].
Nature-identical isothiocyanates (ITCs), set free by enzymic hydro-
lysis of glucosinolates found in Brassicaceae vegetables, are considered
such an alternative [9, 10] as they might exhibit antimicrobial activity
[11, 12]. ITCs derive from nasturtium (Tropaeoli majoris herba) and

Abbreviations: CFU, colony-forming unit; ESBL, Extended-Spectrum-Betalactamase; ITC, natural isothiocyanates; MDR, Multidrug-resistant; MIC, minimal in-
hibitory concentration; UPEC, uropathogenic E. coli; UTI, urinary tract infections
⁎
Corresponding author.
E-mail address: nico.mutters@uniklinik-freiburg.de (N.T. Mutters).

https://doi.org/10.1016/j.fitote.2018.07.012
Received 21 June 2018; Received in revised form 16 July 2018; Accepted 18 July 2018
Available online 19 July 2018
0367-326X/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
N.T. Mutters et al.
horseradish (Armoraciae rusticanae radix) and are therefore found in
nature [11, 12]. The objective of this study was to assess the clinical
potential of nature-identical ITCs for the treatment of UTIs. We assessed
its antimicrobial efficacy and its capability to reduce internalization of
UPEC. Therefore, we tested the ITC susceptibility of E. coli isolated from
clinical patients with signs and symptoms of UTI. Furthermore, we
compared MDR and non-MDR strains to detect possible differences in
their susceptibility to ITC. We then assessed the internalization beha-
vior of five different E. coli strains into cultivated human uroepithelial
cells with and without ITC present.
2. Material and methods
2.1. Antimicrobial susceptibility testing
We isolated 217 E. coli strains from UTI patients of the Freiburg
University Hospital. UTI with E. coli was defined according to German
laboratory standards (M. Hauch et al. Microbiological-infectious disease
diagnostic quality standards [MiQ]). Briefly, E. coli UTI was considered
by isolation in midstream urine of: (1) pure culture of E. coli in
10^3–10^4 CFU or (2) isolation of E. coli in > 10^4 CFU or (3) isolation
of E. coli pure culture in puncture urine with any CFU. Additionally, we
included five reference strains (E. coli ATCC 11,229, 11,775, 12,155,
25,922, 9,637) for standard antimicrobial susceptibility testing using
the VITEK2 (bioMérieux, France) system. Interpretation of results was
done according to EUCAST clinical breakpoints. Strains were classified
as MDR or non-MDR as previously described [13]. Briefly, strains were
classified as MDR when they were ESBL positive (expressing penicillin
and cephalosporin resistance) and quinolone resistant.
2.2. ITC susceptibility testing
We used allyl - ITC (Sigma – Aldrich with a purity of 95% and Merck
KGaA with a purity of at least 94%), benzyl - ITC (Sigma – Aldrich with
a purity of 98% and Fluka Analytical with a purity of at least 97%) and
2 - phenyl – ethyl - ITC (Fluka Analytical with a purity of at least 95%).
A mixture of 38% (v / v) allyl - ITC, 50% benzyl - ITC and 12% 2 –
phenyl – ethyl - ITC was obtained to reflect the proportions of active
agents in a phytotherapeutic drug (Angocin® Anti-Infekt N, Repha
GmbH) available in Germany [14]. To dilute this lipophylic mixture,
8% (v / v) polysorbate 80 (Merck Schuchardt OHG) served as a solvent.
One ml of a stock solution, containing 2% (v / v) of the pure ITC
mixture, was used in a serial dilution. Subsequently, 0.5 ml double
concentrated Mueller Hinton broth (Merck KGaA), containing 10 ^ 6
colony - forming units per ml (CFU / ml), was added to each tube.
Resulting ITC concentrations ranged from 0.02 to 10.80 mg / ml.
To rule out contamination, a mixture of polysorbate and Mueller
Hinton broth was used as a negative control. Additionally, the poly-
sorbate was mixed in a one – to - one ratio with the bacterial solution to
prove growth - enhancing test conditions. The quality of the bacterial
solution used was also tested. It was, therefore, diluted to a con-
centration of 10 ^ 4 CFU / ml. A sample of 20 μl was then plated on
Mueller Hinton agar (Merck KGaA). All samples were incubated at 36 °C
for 24 h. The lowest ITC concentration with no visible bacterial growth
indicated the minimal inhibitory concentration (MIC).
2.3. UPEC internalization
2.3.1. Cell lines and bacteria
Human T-24 bladder carcinoma cells (Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, DSMZ No. ACC376) were
used as human test cells. T-24 bladder carcinoma cells were chosen
because they represent a well-established infection model [15] and
results therefore are highly reproducible and reliable. We included four
E. coli isolates from UTI patients of the Freiburg University Hospital and
one reference strain (ATCC 11775). Bacteria were inoculated on
238
Fitoterapia 129 (2018) 237–240
Columbia 5% sheep blood agar plate (Heipha Dr. Müller GmbH, Ger-
many) and incubated under aerobic conditions at 36 °C for 24 h.
2.3.2. Internalization tests
T-24 cells were grown in 96-well cell culture plates including 100 μl
of McCoy 5A medium. Subsequently, 50 μl of bacterial suspension
(1 × 10^5 colony forming units [CFU]/ml) and 50 μl of the ITC solution
or 50 μl of McCoy 5A medium as control was added and incubated at
37 °C and 5% CO2. Incubation times were set to 0, 1, 2 and 3 h. All
measurements were done eightfold. Subsequently, cell monolayers
were washed twice with 200 μl PBS and incubated with 20 μl antibiotic
suspension containing 15 μg/ml Gentamicine and 5 μg/ml Imipenem,
solved in McCoy's 5 A medium, for 1 h at 36 °C and 5% CO2 to kill all
bacteria not internalized in human cells. After removal of the anti-
biotics by multiple washing with McCoy 5A medium, 50 μl Trypsin was
added to detach the cell monolayer and 50 μl of 2% Triton solution was
added. The remaining cell solution was plated onto Mueller Hinton agar
plates and incubated for 12 h at 36 °C; CFUs were counted to assess the
remaining, thus internalized UPEC.
2.4. Statistical analysis
Statistical analysis was performed using Statistical Package for the
Social Sciences (SPSS 20.0, Chicago, USA). Descriptive statistics were
used to explore data. The distribution of data was tested with Shapiro-
Wilk test. Variables were then analyzed using non-parametric tests. P-
values of < 0.05 were considered significant. All tests were two-tailed.
3. Results
3.1. Susceptibility testings
The majority of all tested strains 54.5% (121/222) were classified as
MDR E. coli. The MICs for ITC obtained by broth dilution ranged
from < 0.02 to 0.67 mg ITC/ml. The mean overall MIC 90 shows that a
concentration of 0.17 mg ITC ml was necessary to inhibit visible bac-
terial growth in 90% (200 / 217) of all tested strains. Comparing non-
MDR to MDR strains, the latter showed significantly higher MIC values
(0.16 ± 0.07 mg/ml versus 0.18 ± 0.08 mg/ml, p = .01) (Fig. 1), al-
though the difference in absolute numbers was rather small.
Fig. 1 shows the relative frequency of different MIC values in MDR
and non MDR-strains.
3.2. UPEC internalization
Internalization of UPEC could be reduced by 10 to 67% when
treated with ITCs over all incubation periods (Table 1), except for one
strain (3279) that actually showed increased internalization when in-
cubated for 1 or 2 h (+30% and + 10.4% respectively).
After 3 h of incubation also this strain showed reduced inter-
nalization (13%). Overall, mean reduction of internalization for all
strains was highest after 3 h of incubation (41.1%) compared to 1 or 2 h
(16.7% and 38%, respectively) of incubation.
4. Discussion
Our data demonstrate a strong antimicrobial activity of ITCs against
UPEC represented by very low MIC 90s. Additionally, ITCs are able to
inhibit internalization of UPEC into human uroepithelial cells especially
after longer periods of incubation. However, this effect is far less con-
sistent than direct antimicrobial activity and appears to differ between
strains. Possibly, strains also have varying ability to penetrate human
cells since this mechanism depends on many different interactions be-
tween surface molecules and results from different genetically de-
termined virulence factors. Unfortunately, we did not evaluate those
differing genetic features of our tested strains. However, clinical data
N.T. Mutters et al. Fitoterapia 129 (2018) 237–240

Fig. 1. ITC susceptibility of MDR and non-MDR strains; MIC distribution.

Table 1 our MIC 90 would not be reached in vivo.

Internalized E. coli colony-forming units (CFU) with and without ITC incuba- Before dismissing the clinical potential of ITC one should take the
tion. following points into consideration. First, when measuring ITC only, a
Strain CFU with CFU without % reduction incubation times, possible additional ITC release from mercapturic acids [20, 21] is not
ITC ITC h addressed. This might explain the substantial deviation from the results
of previous studies measuring mercapturic acids. Additionally, Martón
ATCC 11775 0 0 0
et al. struggled with high intra - and inter - personal variability. They,
0.8 1.7 −52,9 1
1.8 3.9 −53,8 2 therefore, saw the need for larger studies [20]. In general, most in vivo
9.8 19.2 −49,0 3 studies concerning ITC conversion rates included healthy participants
3532 0 0 0 exclusively [18–20]. However, physiopathological changes due to UTI
0.4 0.6 −33,3 1 possibly promote ITC release [20, 21] and have not been taken into
2.7 4.7 −42,6 2
account yet.
10.8 23.3 −53,6 3
3553 0 0 0 Until now, the same antimicrobial efficacy of ITC was assumed for
0.9 1 −10,0 1 MDR and non-MDR bacteria. As a possible explanation, Conrad et al.
4.3 13.1 −67,2 2 discussed specific cellular targets differing from those of standard an-
32.8 61.3 −46,5 3
tibiotics in 2007 and 2013. Our data, however, suggests a slightly re-
3279 0 0 0
2.6 2 30,0 1
duced ITC susceptibility of MDR uropathogenic E. coli. This might be
5.3 4.8 10,4 2 due to higher expression/activity of unspecific efflux pumps, which are
20.8 23.9 −13,0 3 frequently found in MDR strains. However, the differences in MICs,
3453 0 0 0 although statistical significant are not large and clinical impact might
1.9 2.3 −17,4 1
be negligible.
3.8 6 −36,7 2
8.4 14.9 −43,6 3 In conclusion, the mixture of three synthetic ITC showed potent
antibacterial activity against clinical isolates of uropathogenic E. coli, as
well as impeding their migration into uroepithelial cells, which may
concerning the use of ITC in the treatment of UTIs are scarce. Goos et al. prevent UTI relapses. In vivo studies need to evaluate a possible clinical
tested a phytotherapeutic agent containing glucosinolates in cases of use of preparations containing ITC precursors.
upper respiratory and urinary tract infections in children and adults.
However, neither ITC excretion nor bacteriuria were quantified [16,
17]. Funding
Our in vitro experiments confirmed that ITCs inhibit the migration of
UPEC into uroepithelial cells to some extent. Due to different me- This study was financially supported by Repha Gmbh, Langenhagen,
chanisms of action - direct inhibition of bacterial growth and reduction Germany Grant number Cuno-No. 503026. Repha GmbH was neither
of internalization - ITCs may play a clinical role in UTI treatment. involved in conducting the experiments nor the interpretation of the
However, it is yet unknown if sufficient ITC concentrations can be results nor the writing of this manuscript.
reached in vivo after oral ITC administration. Conversion rates pre-
viously published for orally administered glucosinolates, however,
range widely. In early studies, mercapturic acids found in urine after Conflict of interest
the ingestion of glucosinolates were taken as proof of high ITC excretion
rates. For allyl - ITC the conversion rate came to 15% after ingesting The authors declare no conflict of interest.
cooked cabbage, 37% after eating raw cabbage and 68% after mustard
consumption [18]. For phenyl – ethyl – ITC, percentages ranged from a
minimum of 30% after eating raw cress [19] to almost 90% after Acknowledgements
drinking cress juice [18]. A recent pilot study measuring free ITC only
suggests conversion rates of < 6% [20]. According to Martón et al.´s Partial results were presented at the 24th ECCMID in Barcelona,
data, one ml urine would contain 0.13 mg of the ITC mixture. Thereby, Spain.

239
N.T. Mutters et al.
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