Infection

https://doi.org/10.1007/s15010-019-01315-4

ORIGINAL PAPER

In vitro efficacy of phytotherapeutics suggested for prevention

and therapy of urinary tract infections
Julian Marcon1 · Sören Schubert2 · Christian G. Stief1 · Giuseppe Magistro1

Received: 17 February 2019 / Accepted: 30 April 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Purpose To analyse the therapeutic efficacy of various phytotherapeutics and their antimicrobial compounds with regard to
strain specificity and dose dependence.
Methods A representative strain collection of 40 uropathogenic bacteria isolated from complicated and uncomplicated
urinary tract infection was subjected to various virulence assays (bacterial growth, mannose-sensitive agglutination, and
motility) to determine the therapeutic impact of various compounds with antimicrobial activity. We tested proanthocyanidins
(PAC), d-mannose, rosemary extract ­(Canephron®), and isothiocyanates ­(Angocin®).
Results d-mannose efficiently blocked the adhesive properties of all type 1 fimbriae-positive isolates in low concentration
(0.2%), but showed no bacteriostatic effect. PAC also actively blocked agglutination, but the concentration varied consider-
ably among isolates. Escherichia coli required the highest concentration (10%), while Enterobacter cloacae responded to
low concentrations (0.1%). Allyl isothiocyanates not only impaired agglutination in all tested isolates, but also had a dramatic
impact on flagella-mediated motility in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis (p < 0.001). The
administration of rosemary extracts revealed a strong bacteriostatic effect in growth assays. All tested strains were strongly
inhibited by the addition of 10 μg/ml or 1 μg/ml of purified rosemary extractions with the exception of Serratia marcescens.
Morganella morganii responded only to 10 μg/ml.
Conclusion Phytotherapeutics and small-molecular compounds like mannosides have the potential to become an integral
part in a multi-modal treatment concept for the treatment and prevention of urinary tract infections. Their efficiency can be
optimised when strain specificities and therapeutic concentrations are taken into account.

Keywords Urinary tract infections · Phytotherapeutics · Prevention · Therapy · Uropathogenic Escherichia coli (UPEC)

Introduction in clinical practice [2, 3]. Its socio-economic relevance is

mirrored by the staggering financial burden to health care
Antimicrobial resistance (AMR) has emerged as one of the systems worldwide. Annual societal costs of 2.4 billion US
most challenging and threatening problems to public health dollars are caused by 1.3 million emergency room visits and
on a global scale. If we do not change our practice today, we 6.8 million office visits in the US alone [4]. Uropathogenic
might be on the verge of a pre-antibiotic era scenario. Pro- Escherichia coli (UPEC) are the main causative pathogens
jections predict an alarming trend, with AMR as the number isolated from about 80% of uncomplicated UTIs [5]. Glob-
#1 death cause in the near future [1]. This especially holds ally, the resistance rates for the treatment of UPEC are seri-
true for the management of urinary tract infections (UTI), ously high. Resistance rates for common antibiotic drugs,
which are among the most common bacterial infections such as ciprofloxacin, trimethoprim/sulfamethoxazole, and
aminopenicillins, reached 45, 48.2, and 50.4%, respectively
* Giuseppe Magistro [6]. Once, the antibiotic treatment of UTI was considered
Giuseppe.Magistro@med.uni‑muenchen.de an easy and safe matter, but the rising AMR renders the
management more and more challenging.
1
Department of Urology, Ludwig-Maximilians-University, Now, we need to endorse action plans to prevent this
Marchioninistrasse 15, 81377 Munich, Germany
critical development. The four most important corner-
2
Max von Pettenkofer-Institut für Hygiene und Medizinische stones include the design of novel antibiotic agents, the
Mikrobiologie, Munich, Germany

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 J. Marcon et al.

development of alternative antimicrobial strategies, effective aeruginosa. We subjected five isolates of each species to
hygiene and preventive measures, and, finally, the concept different virulence assays.
of antimicrobial stewardship [7–11]. There has been a con- In this study, we tested proanthocyanidins, d-mannose,
siderable developmental void for novel antibiotic agents in rosemary extract, allyl isothiocyanate (AITC), benzyl iso-
the last 3 decades. Consequently, we are in an urgent need thiocyanate (BITC), and phenylethyl isothiocyanate (PITC).
for alternatives. Non-antibiotic approaches for the treatment The composition of the phytomedical compound A ­ ngocin®
or the prevention of UTI comprise a plethora of concepts. consists of 38% AITC, 50% BITC and 12% PITC. Concen-
They are an integral part of current guideline recommenda- trations were applied as indicated. The purified substances
tions now [12]. Hormonal agents, the use of probiotics, food were all purchased from Sigma-Aldrich Chemie GmbH,
supplements to modify the urine composition, the design of Taufkirchen, Germany.
vaccines and immune-stimulatory compounds, intravesical Three relevant virulence mechanisms for the development
instillations, and phytotherapeutics have been evaluated [13, of UTI were assessed. We investigated adhesive properties
14]. mediated by type 1 fimbriae, flagellum-mediated motility,
Especially, the role of phytotherapeutics remains still elu- and bacterial growth. All experimental settings were per-
sive. Although certain underlying modes of action have been formed according to previously published protocols [17, 18].
postulated, results have not been convincing in the clini- Briefly, mannose-sensitive adhesion of bacteria was assayed
cal evaluation. For example, cranberry extracts have a pro- by the ability to agglutinate yeast cells (Saccharomyces cere-
posed efficacy due to its high content of proanthocyanidins visiae) on glass slides [19]. The outcome is evaluated micro-
(PAC). These compounds interfere with the most prevalent scopically by the presence of agglutinated cells or loose cells
and important adhesins of UPEC. Therefore, they appeared (Fig. 1). Motility was analysed using 0.3% Luria–Bertani
to be effective for the prevention of UTI when adminis- soft agar plates. A late logarithmic phase culture (optical
tered on a daily basis. In a first Cochrane review, it was density at 600 nm = 1.0; ­OD600nm) was stabbed into the mid-
concluded that cranberry products can significantly reduce dle of a soft agar plate and incubated at 37 °C. Motility was
the incidence of recurrent UTIs over 12 months (RR 0.65, quantified by measuring the diameter of motile bacteria after
95% CI 0.46–0.90) [15]. However, a more recent Cochrane 8 h of incubation (Fig. 2a). The soft agar plates were sup-
meta-analysis, including more clinical studies, withdrew the plemented with 5 μM AITC, 7 μM BITC, and 8 μM PITC.
former recommendation due to the lack of positive trials Growth curves were assessed in triplicates of 50 ml cultures
[16]. The optimal formulations, dosages, or regimens have in 250 ml Erlenmeyer flasks. Overnight cultures were incu-
not been determined yet. Similarly, the spectrum of patho- bated in 50 ml fresh Luria–Bertani medium to a starting
gens that might respond to treatment has not been identi- ­OD600nm of 0.05 and the optical density was recorded every
fied. Exemplarily, not all uropathogenic bacteria express the 20 min. We used a rich medium to ensure optimal growth
potential targets addressed by proanthocyanidins. conditions and focused on the exponential growth phase,
This lack of information applies to almost all tested phy- which is known to represent the fastest growth kinetics.
totherapeutics. This prompted us to evaluate the efficacy of The selection of assays and inhibitory compounds was
various phytopharmaceuticals in a representative collection the results of an exploratory screening in concert with the
of different uropathogenic species isolated from compli- published literature. Published data are mainly restricted to
cated and uncomplicated UTIs. In this first in vitro study, we single species. In this manuscript, we evaluated the impact
determined the impact of different compounds on relevant on various strains of different origin. Our aim was to high-
virulence features. When phytotherapeutics are considered, light clearly defined effects. Data with an inconsistent out-
we need to first assure that the right targets are hit. These come were not presented. All experiments were performed
results are supposed to give first insights into the potential
spectrum of pathogens appropriate for specific phytothera-
peutic compounds.

Methods

Bacterial strains used in the present work were isolated from

uncomplicated and complicated UTIs. The strain collection
included Escherichia coli, Klebsiella pneumoniae, Proteus
mirabilis, Enterobacter cloacae, Serratia marcescens, Cit- Fig. 1  Mannose-sensitive agglutination mediated by type 1 fimbriae.
robacter freundii, Morganella morganii, and Pseudomonas Microscopically examination of agglutinated cells

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In vitro efficacy of phytotherapeutics suggested for prevention and therapy of urinary tract…

Fig. 2  Flagella-mediated motility. The front of swarming bacteria mediated motility in Escherichia coli, Klebsiella pneumoniae, and
can be measured after spotting bacteria on the centre of a soft agar Proteus mirabilis. Allyl isothiocyanate (AITC), benzyl isothiocyanate
plate. Influence of isothiocyanates on the motility of uropathogenic (BITC), phenylethyl isothiocyanate (PITC), ­Angocin® (38% AITC:
bacteria. Allyl isothiocyanates had the strongest impact on flagella- 50% BITC: 12% PITC). *p < 0.05, **p < 0.01, ***p < 0.001

in duplicates and repeated at least three times. For statistical to inhibit agglutination of E. coli. Purified PAC, as the active
analysis, a paired t test or the Mann–Whitney U test was per- compound in cranberry extracts, was able to interfere with
formed and results were considered statistically significant agglutination only using the 10% solution. Among the iso-
if the p value was lower than 0.05. thiocyanates, only AITC efficiently blocked agglutination at
low concentrations, whereas both BITC and PITC required
higher doses of 10%. The combination of all isothiocyanates,
Results as present in the phytomedical preparation ­Angocin®, dis-
played a strong inhibitory effect for every concentration
Screening for anti‑adhesive compounds applied. Various Enterobacteriaceae express type 1 fim-
briae, but their role for the development of UTIs is unclear.
In our first screening, we investigated the response to vari- We included additional type 1 fimbriae-positive isolates
ous available phytotherapeutics and small molecules like with the ability to agglutinate yeast cells. As observed for
d-mannose in an agglutination assay (Table 1). d-mannose E. coli, d-mannose efficiently blocked agglutination in Kleb-
in various concentrations (0.2%, 2%, 10%) was always able siella pneumoniae, Serratia marcescens, and Enterobacter

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 J. Marcon et al.

Table 1  Inhibition of Escherichia coli Klebsiella pneumo- Serratia marces- Entero-

agglutination mediated by type niae cens bacter
1 fimbriae cloacae

Supplements (concentration, %)
d-mannose

0.2% +++ +++ +++ +++

2% +++ +++ +++ +++
10% +++ +++ +++ +++
Cranberry (PAC)
0.1% --- --- --- +++
1% --- --- +++ +++
10% +++ +++ +++ +++
AITC
0.1% +++ --- --- +++
1% +++ +++ +++ +++
10% +++ +++ +++ +++
BITC
0.1% --- --- --- ---
1% --- --- +++ ---
10% +++ +++ +++ ---
PITC
0.1% --- --- --- ---
1% --- +++ +++ ---
10% +++ +++ +++ ---
AITC:BITC:PITC
­(Angocin®)
0.1% +++ --- --- +++
1% +++ +++ +++ +++
10% +++ +++ +++ +++

In this qualitative assay, the agglutination is determined microscopically

+++ positive inhibition; — no inhibition

cloacae, as well. However, their response to PAC and iso- inhibitory effect can be observed, but this was different for
thiocyanates differed considerably compared to E. coli. With every species.
regard to PAC, Klebsiella pneumoniae reacted similarly to
E. coli. However, Serratia marcescens and Enterobacter Screening for substances interfering with motility
cloacae were responsive to lower concentrations. Regard-
ing the response to isothiocyanates, Enterobacter cloacae The ability to move to a more favourable niche is another
demonstrated inhibition with all dosages of AITC. However, relevant feature associated with bacterial virulence. Some
neither BITC nor PITC showed an inhibitory effect. The uropathogens express the so-called flagella to ascend
mixed preparation of isothiocyanates blocked agglutination within the urinary tract [20]. Flagella-mediated motility
efficiently. Klebsiella pneumoniae and Serratia marcescens can be assayed by spotting bacteria on a soft agar plate and
responded to AITC in higher concentrations. Only the highly by measuring the diameter of moving bacteria (Fig. 2a).
concentrated blocking solution with 10% of BITC was effi- We investigated the effect of isothiocyanates on motility
cient in Klebsiella pneumoniae. For Serratia marcescens, in E. coli, Klebsiella pneumoniae, and Proteus mirabilis
1% of BITC was sufficient. In the case of PITC, the 1% (Fig. 2b). E. coli displayed the strongest impairment by
blocking solution showed an inhibitory effect in Klebsiella 5 μM of purified AITC, with a 90% reduction of motility
pneumoniae and Serratia marcescens. Correspondingly, (p < 0.001). For the 1:10 dilution, this effect was dimin-
the 1% solution of the mixed isothiocyanate compound ished to 33% (p < 0.001). The application of 7 μM BITC
blocked agglutination activity. This first descriptive screen- yielded a strong inhibitory effect, with an 83% decrease of
ing suggests that the overall response to anti-adhesive com- motility (p < 0.001). Supplementation with 8 μM PITC led
pounds depends on the specific species. A dose-dependant to a reduction of 25% (p < 0.05). The mixed preparation of

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In vitro efficacy of phytotherapeutics suggested for prevention and therapy of urinary tract…

isothiocyanates decreased motility by 73% in its undiluted Another important aspect is the introduction of efficient
form and by 62% in a 1:10 dilution (both, p < 0.001). In vaccines and immune-stimulatory agents as a preventive
Klebsiella pneumoniae, AITC was again the strongest inhib- measure. Uro-Vaxom® and U ­ rovac®/StroVac® have been
itor, showing a motility reduction of 79% (p < 0.001). BITC shown to reduce the UTI recurrence rate compared to pla-
impaired motility by 41% (p < 0.01). PITC had no inhibitory cebo in clinical trials and meta-analyses [9, 25]. Newly
­ ngocin®-like preparation impaired motil-
effect at all. The A designed vaccines, such as ExPEC4, are emerging and enter-
ity by 67% (p < 0.001). A similar pattern was also revealed ing the stage of clinical evaluation [26]. The search for alter-
for Proteus mirabilis. AITC and BITC decreased motil- native antimicrobial compounds is attracting more and more
ity by 86% (p < 0.01) and 38% (p < 0.05), respectively. No attention. We strive for equally effective approaches with-
impairment was detected using PITC. Mixed isothiocyanates out the risk of inducing antibiotic resistance mechanisms.
reduced motility by 77% (p < 0.01). Isothiocyanates turned The concept of anti-virulence treatment addresses the most
out to have a strong impact on flagella-mediated motility. important virulence factors of pathogens and turns them into
The pattern was similar in various species, with AITC dis- strongly attenuated bacteria. Adhesion of the pathogen is one
playing the strongest inhibitory effect. of the first essential steps in the pathogenesis of infectious
diseases. In UPEC, up to 13 diverse fimbrial systems can be
Screening for bacteriostatic agents expressed [20]. Fimbriae are complex microbial structures
on the surface with the ability to bind specifically to the host
The next virulence assay evaluated the inhibitory effect on epithelium. Type 1 fimbriae are among the most important
bacterial growth. We tested the influence of d-mannose and adhesins in UPEC. They mediate the first attachment to the
rosemary extracts in E. coli, Proteus mirabilis, Enterobacter urothelium by binding to mannosylated uroplakins. Man-
cloacae, Serratia marcescens, Citrobacter freundii, Morga- nose derivatives are known to block type 1 fimbriae [21].
nella morganii, and Pseudomonas aeruginosa (Fig. 3). We They proved to be promising candidates for the treatment
started to analyse the impact of d-mannose to rule out that and prevention in the murine model of UTI [21, 27, 28].
our observations made for bacterial agglutination were due Experimental data even suggest that the action of manno-
to a possible bacteriostatic effect. As depicted in Fig. 3, sides is not restricted to the urinary tract. They also appear
d-mannose did not affect bacterial growth of the screened to selectively deplete type 1 fimbriae-positive UPEC from
isolates at all. The next step was the investigation of rose- the intestinal microbiota. These experimental results have
mary extracts, as they are proposed to mediate various anti- also been confirmed in clinical trials. Daily administration
microbial effects [22]. The concentrations were 10 μg/ml of 2 g of d-mannose was equally effective as the antibiotic
and 1 μg/ml. With the only exception of Serratia marces- prophylaxis with nitrofurantoin regarding risk reduction of
cens, all included isolates were significantly impaired when recurrent UTIs [29]. Our data support the concept of anti-
the highest concentration of 10 μg/ml was applied. Even virulence treatment using mannosides. No bacteriostatic or
1 μg/ml of rosemary extract was sufficient to inhibit bacterial bactericidal effects were observed in the strains tested in this
growth in most strains, excluding Serratia marcescens and study. However, it was highly effective as a type 1 fimbriae-
Morganella morganii. Again, our results do not speak for a blocking compound. Our results in concert with published
consistent reaction to phytotherapeutics. Our observations data suggest that, by the inactivation of relevant virulence
rather suggest a differentiated response depending on the factors of a pathogen, we are able to efficiently treat and
target and the concentration of the agent. prevent bacterial infections.
Phytotherapeutics might also contain antimicrobial com-
pounds with therapeutic potential. As explained above for
Discussion the use of cranberry products, they are not recommended
due to the lack of positive trials. Our results demonstrate
One of the main questions to be answered is how to tackle that PAC as the active compound is efficiently impairing
the problem of AMR. To overcome this obstacle, we need the adhesive ability of bacteria. However, we clearly show
to act on various levels. The development of novel antibiotic that this effect is strain specificity and dose dependence.
agents is one important mainstay, but it still remains a for- Only type 1 fimbriae-positive strains are responsive. With
midable task. Recently, the novel antibiotic zoliflodacin has regard to UPEC, high concentrations were required for com-
been tested successfully for the oral treatment of urogenital plete inhibition, whereas, in Enterobacter cloacae, low con-
gonorrhoea caused by antibiotic-resistant Neisseria gonor- centrations were sufficient. In clinical trials, the bacterial
rhoeae in a phase 2 trial [23]. Novel antimicrobial combina- spectrum was rarely taken into consideration. With UPEC
tions including ceftolozane/tazobactam, ceftazidime/avibac- as the most common pathogen isolated from UTI, our data
tam, and meropenem/vaborbactam are additional examples suggest that the concentration of PAC reached in the urine
that the efforts made in the last years are bearing fruits [24]. of patients with recurrent UTI might have been insufficient.

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 J. Marcon et al.

Fig. 3  Bacteriostatic effect of rosemary extracts. Impact of D-Mannose and rosemary extracts on the growth kinetics of various uropathogenic
bacteria. ­OD600nm (optical density at 600 nm). *p < 0.05, **p < 0.01, ***p < 0.001

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In vitro efficacy of phytotherapeutics suggested for prevention and therapy of urinary tract…

Further RCTs are warranted to evaluate higher dosages of under our laboratory in vitro settings. We have to admit that
standardised PAC preparations. Phytotherapeutics contain- the pharmacokinetics of the substances used in this study
ing rosemary extracts have recently been analysed in two is still unclear. We still do not know the dosage necessary
RCTs for the treatment of uncomplicated cystitis [30, 31]. to achieve a therapeutic concentration in the urinary tract.
In the study by Wagenlehner et al., herbal treatment was Further studies are warranted to clarify this essential aspect.
non-inferior to the antibiotic therapy with fosfomycin, but Furthermore, our study does not claim to be exhaustive. We
higher rates of pyelonephritis were detected. Furthermore, focused on Gram-negative bacteria only, but are aware that
Sabadash et al. reported that the addition of phytotherapeu- also Gram-positive pathogens like Enterococcus spp. are
tics ­(Canephron®) to fluoroquinolones (ofloxacin) provided relevant uropathogenic bacteria. Accordingly, the phyto-
better relief of bothersome cystitis symptoms compared to therapeutics tested in our analysis are not available in every
fluoroquinolones only. Furthermore, they also reduced the country and more herbal combinations are on the market
rate of recurrent infection. The exact mechanism underlying registered as food supplements. It is important to stress that
this observation is not clear. Our results pinpoint towards no recommendation for clinical practice can be formulated
a bacteriostatic effect of rosemary extracts, which again is based on these in vitro data. We believe that the therapeutic
strain specificity and dose dependence. All tested isolates potential of standardised phytotherapeutics can be optimised
responded to purified rosemary extracts except Serratia when they are applied more specifically. Of course, RCTs
marcescens. For Morganella morganii, a bacteriostatic are necessary to determine the true clinical value of this
effect was also confirmed, but only at higher concentra- approach.
tions. Another interesting herbal combination containing
nasturtium and horseradish indicated clinical efficacy for
the treatment and prevention of recurrent UTIs [32, 33]. The
Conclusions
high concentration of isothiocyanates is considered to play
a relevant role for its therapeutic potential. We investigated
With the challenging problem of AMR still rising, we need
the impact of isothiocyanates on flagella-mediated motility.
to act on various levels to check its progress. Phytotherapeu-
Bacterial motility is another relevant virulence mechanism
tics and small-molecular compounds like mannosides have
for the successful ascension and colonisation of the urinary
the potential to become an integral part in a multi-modal
tract [34–36]. Results of the present study provide evidence
treatment concept for the treatment and prevention of UTI.
for a significant impact on motility. Especially, the addition
of AITC showed the strongest reduction of bacterial motil- Author contributions JM: project development, data collection and
ity in UPEC, Klebsiella pneumoniae and Proteus mirabilis. analysis, and manuscript writing. SS: supervision. CGS: project devel-
The antimicrobial properties of AITC also appear to have opment and supervision. GM: project development, data collection and
analysis, and manuscript writing.
an activity against biofilms, which anticipates a potential
impact on catheter-associated UTIs [37].
The main objective of the current study was to perform Compliance with ethical standards
a systematic analysis of available phytotherapeutics with
Conflict of interest The authors declare that they have no conflict of
antimicrobial properties and their impact on specific viru- interest.
lence and fitness factors in a representative strain collection
of uropathogenic bacteria. It was not the goal to decipher Human and animal rights This article does not contain any studies
with human participants or animals performed by any of the authors.
the exact mechanism underlying the effects observed in our
assays on a molecular level. This study is descriptive in its
nature. Nevertheless, this is the first analysis highlighting the
strain specificity and dose dependence of phytotherapeutics.
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