Searching for new tools to counteract the helicobacter pylori resistance: The positive action of resveratrol derivatives

Article abstract of DOI:10.3390/antibiotics9120891

The drug-resistance phenomenon in Helicobacter pylori underlines the need of novel strategies to improve the eradication rate including alternative treatments combining antibiotic and non-antibiotic compounds with synergistic action. In this study, the antibacterial (MIC/MBC) and anti-virulence effects (biofilm reduction and swarming motility inhibition) of resveratrol-RSV and new synthetized RSV-phenol derivatives, with a higher bioavailability, alone and combined with levofloxacin-LVX were evaluated against resistant H. pylori clinical strains. The experiments were confirmed in vivo using the Galleria mellonella model. Among the studied RSV derivatives, RSV-3 and RSV-4 possessed higher antibacterial activity with respect to RSV (MICs from 6.25 to 200 μg/mL and from 3.12 to 200 μg/mL, respectively). RSV, RSV-3, and RSV-4 were able to synergize with LVX restoring its effect in two out of seven clinical resistant strains tested for the study. RSV, RSV-3, and RSV-4, alone and with LVX at sub-MIC and sub-synergistic concentrations, significantly reduced the biofilm formation. Moreover, RSV-3 and RSV-4 reduced the H. pylori swarming motility on soft agar. RSV, RSV-3, and RSV-4 were non-toxic for G. mellonella larvae and displayed a protective effect against H. pylori infection. Overall, RSV–phenol derivatives should be considered interesting candidates for innovative therapeutic schemes to tackle the H. pylori antibiotic resistance.

Full text of DOI:10.3390/antibiotics9120891

antibiotics
Article
Searching for New Tools to Counteract the
Helicobacter pylori Resistance: The Positive Action of
Resveratrol Derivatives
Paola Di Fermo 1,† , Silvia Di Lodovico 1,† , Rosa Amoroso 1 , Barbara De Filippis 1,
Simonetta D’Ercole 2 , Emanuela Di Campli 1, Luigina Cellini 1,* and Mara Di Giulio 1
1
Department of Pharmacy, University “G. d’Annunzio” Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy;
paola.difermo@unich.it (P.D.F.); silvia.dilodovico@unich.it (S.D.L.); rosa.amoroso@unich.it (R.A.);
barbara.defilippis@unich.it (B.D.F.); e.dicampli@unich.it (E.D.C.); mara.digiulio@unich.it (M.D.G.)
Department of Medical Oral and Biotechnological Sciences, University “G. d’Annunzio” Chieti-Pescara,
2
Via dei Vestini 31, 66100 Chieti, Italy; simonetta.dercole@unich.it
*
Correspondence: l.cellini@unich.it; Tel.: +39-0871-355-4560
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 13 November 2020; Accepted: 8 December 2020; Published: 10 December 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The drug-resistance phenomenon in Helicobacter pylori underlines the need of novel
strategies to improve the eradication rate including alternative treatments combining antibiotic and
non-antibiotic compounds with synergistic action. In this study, the antibacterial (MIC/MBC) and
anti-virulence effects (biofilm reduction and swarming motility inhibition) of resveratrol-RSV and
new synthetized RSV-phenol derivatives, with a higher bioavailability, alone and combined with
levofloxacin-LVX were evaluated against resistant H. pylori clinical strains. The experiments were
confirmed in vivo using the Galleria mellonella model. Among the studied RSV derivatives, RSV-3 and
RSV-4 possessed higher antibacterial activity with respect to RSV (MICs from 6.25 to 200
µg/mL
and from 3.12 to 200 g/mL, respectively). RSV, RSV-3, and RSV-4 were able to synergize with LVX
µ
restoring its effect in two out of seven clinical resistant strains tested for the study. RSV, RSV-3,
and RSV-4, alone and with LVX at sub-MIC and sub-synergistic concentrations, significantly reduced
the biofilm formation. Moreover, RSV-3 and RSV-4 reduced the H. pylori swarming motility on
soft agar. RSV, RSV-3, and RSV-4 were non-toxic for G. mellonella larvae and displayed a protective
effect against H. pylori infection. Overall, RSV–phenol derivatives should be considered interesting
candidates for innovative therapeutic schemes to tackle the H. pylori antibiotic resistance.
Keywords: Helicobacter pylori resistance; resveratrol; resveratrol phenol derivatives; antibacterial and
anti-virulence action; Galleria mellonella model
1. Introduction
Helicobacter pylori is a gastroduodenal pathogen that affects more than 60% of the population
worldwide with a higher prevalence in developing countries [1]. This bacterium is able to colonize the
human stomach, thereby inducing inflammation of the gastric mucosa causing chronic or atrophic
gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric
cancer [2,3]. The treatment of H. pylori infection includes different combinations of drugs among
clarithromycin, levofloxacin, amoxicillin, metronidazole, tetracycline, and proton pump inhibitor with
an eradication rate that is sometimes satisfactory. The increase of H. pylori antimicrobial resistance
and the failure of therapeutic schemes underlines the difficulty to treat the H. pylori infection [2].
In particular, in our region (Abruzzo region), the resistance of H. pylori isolates to clarithromycin is
particularly high with values near 73% [4,5].
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www.mdpi.com/journal/antibiotics

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In the last two decades, the alarming antibiotic resistance phenomenon became a key factor in
the treatment failure of H. pylori infection, and it strongly suggests us to improve the eradication
rate performing susceptibility tests as well as to investigate novel strategies to improve the current
therapeutic schemes [4–7].
H. pylori strains, together with genotypic resistance, are also able to express survival strategies
entering the dormant state and forming biofilm [8–12]. These antibiotic tolerant conditions widely
contribute to the treatment failure and recurrence of infection becoming a significant threat to public
health. In fact, H. pylori has been included by WHO in the list of pathogens for which identification of
novel treatment strategies is urgent.
Recent literature displays the synergistic effect of non-antibiotic compounds when combined with
standard therapies with the aim to restore the antimicrobial drug efficacy [13–16]. This hopeful scenario
could be related to the manifold effect on different target sites on the bacterial cell, pharmacokinetic or
physicochemical effects (e.g., improvement of solubility or bioavailability), and/or action on a bacterial
resistance mechanism [17
between antibiotics and non-antibiotic compounds, resulting in a potentiated effect against H. pylori
strains [ 13]. In particular, the Pistacia vera L. oleoresin or bovine lactoferrin capability to synergize,
,18]. Some of our recent studies emphasize the combined synergistic effect
4,
in vitro and in vivo, with levofloxacin (LVX) by reducing its antimicrobial concentration under the
breakpoint, was demonstrated.
Over recent years, resveratrol (RSV) attracted great attention for its multifaceted biological activities
like anti-inflammatory, anti-carcinogenesis, and anti-aging, including antimicrobial activity [19].
Resveratrol is the 3,5,40-trihydroxy-trans-stilbene (Figure 1A), which is a naturally occurring compound,
found, in particular, in grape and in grapeskin, acting like a phytoalexin synthetized in response to
microbial attack [20
Gram-negative microorganisms. Among antimicrobial properties [22
synthesis and ATP hydrolysis in Escherichia coli and Mycobacterium smegmatis [24
,
21]. Resveratrol inhibits the growth of bacteria including both Gram-positive and
23], RSV is able to inhibit ATP
25]. This phenolic
,
,
compound produces, in E. coli cells, DNA fragmentation and upregulation of the SOS stress-response
regulon, together with a morphological transformation in elongated cells due to the suppression of ftsZ
gene expression. Moreover, RSV displays anti-virulence properties such as antibiofilm, antimotility
activities, and it also alters the bacterial exotoxin expression and the quorum sensing system [26–30].
The role of RSV in effecting the oxidative stress and inflammation in H. pylori-infected mucosa has also
been described [23].
Figure 1. Resveratrol (RSV) (A) and its derivatives RSV-1–5 (B).
Although RSV possesses antimicrobial benefits, it is noteworthy that its availability is limited.
In fact, it is only produced on a nanogram scale in plants, and, thus, it is hardly to be obtained in large
quantities from their natural sources. In addition, RSV has been associated with poor bioavailability
(less than 1%) especially due to its extensive metabolism [31
optimization approaches have been developed and RSV was the precursor of more active derivatives,
obtained by chemical modification of a stilbene scaffold [33 34]. This approach fits into a context
,32]. To overcome this limitation, various
,

Antibiotics 2020, 9, 891
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regarding the use of natural products as a chemical lead for development of antibacterial agents [35–38].
In a recent work, we reported the effects of a series of RSV analogs on viability of three pancreatic
cancer cell lines [36]. In order to enlarge the pharmacological activities of these compounds, the interest
was to explore the antimicrobial activity against H. pylori. For this purpose, among the studied RSV
derivatives, we selected the derivatives RSV-1–5 (Figure 1B). They kept the 40-hydroxyl group because
its importance for biological activity has been largely reported [39] while the 3,5-hydroxy group was
replaced with a substituent in the 4-position.
In the first step of this study, we evaluated the antibacterial effect of RSV and new synthetized
derivatives, RSV-1–5, against resistant H. pylori clinical strains. Subsequently, the most antibacterial
derivatives were studied for their antimicrobial and anti-virulence effects also combined with LVX,
which is an antibiotic commonly used in H. pylori therapy. In detail, (i) RSV and the most antibacterial
derivatives were combined with LVX for detecting a potential restorative effect in resistant H. pylori
strains, and (ii) RSV and the most promising antibacterial derivatives were detected for their
anti-virulence activity by evaluating the inhibition of biofilm formation alone or combined with
LVX and through the swarming motility. The detected in vitro antimicrobial data was confirmed
in vivo by using the Galleria mellonella model.
The main aim of this work was to explore the potential capability of these novel compounds to
synergize with LVX by reducing the level of bacterial persistence/tolerance.
2. Results
This study evaluates the antibacterial and anti-virulence properties of RSV and new RSV derivatives
and their capability to potentiate the LVX antibacterial action.
The antibacterial effects of RSV and RSV derivatives (1–5) were evaluated against resistant H. pylori
clinical isolates in terms of MIC and MBC determination (Table 1). As shown, the MIC values of
RSV ranged from 200 to 800
compounds in terms of antibacterial activity being RSV-3 and RSV-4 with MIC values ranging from
6.25 to 200 g/mL and from 3.12 to 200 g/mL, respectively. The detected MBC values were generally
µg/mL, among the tested RSV derivatives, with the best promising
µ
µ
one-step or two-step higher than MIC values for RSV and each RSV derivative.
Table 1. Minimum Inhibitory Concentration (MIC, µg/mL) and Minimum Bactericidal Concentration
(MBC, µg/mL) of RSV and RSV derivatives (RSV-1–5) against resistant H. pylori strains.
Substances
H. pylori
strains
LVX
MIC
RSV
MBC
RSV-1
RSV-2
RSV-3
RSV-4
RSV-5
MBC MIC
MIC
MBC
MIC MBC
MIC
MBC
MIC
MBC MIC MBC
11F/11
2A/12
7A/12
12A/12
5A/13
13A/13
26A/13
ATCC
43629
1.00
0.50–1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
2.00
200
200
200
200
800
200
800
400
200
200
200
>800
400
200
200
>800
100
200
100
200
800
400
>800
400
400
400
400
200
200
200
400
100
50
200
200
400
800
200
100
100
25
25
25
6.25
200
6.25
50
50
25
25
50
400
50
3.12
3.12
50
25
50
50
100
400
50
200
200
200
200
100
100
200
400
200
400
400
100
200
200
25
200
3.12
100
1.00
2.00
>800
100
100
200
0.12
0.12
200
800
100
>800
25
100
6.25
50
12.5
25
100
100
Since RSV-3 and RSV-4 resulted in the most antibacterial compounds with higher antibacterial
activity, they were selected together with the lead compound to detect their synergism with LVX,
their antibiofilm activity combined with LVX, and their action on H. pylori swarming motility.
In Table 2, the best combinations of RSV, RSV-3, or RSV-4 and LVX with the values of FIC Index
(FIC I) for all resistant H. pylori clinical strains are shown. Synergisms were recorded in H. pylori 7A/12
and H. pylori 13A/13 with FIC I from 0.24 to 0.28 for H. pylori 7A/12 and from 0.26 to 0.28 for H. pylori
13A/13. For these two micro-organisms, the LVX MICs, in the presence of RSV, RSV-3, and RSV-4,

Antibiotics 2020, 9, 891
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were reduced from two to eight times by restoring its antimicrobial efficacy. Additive actions were
recorded for H. pylori 11F/11, 2A/12, 12A/12, 5A/13, and 26A/13. The antagonism was not recorded.
Table 2. Best combinations (µg/mL) of RSV, RSV-3 and RSV-4 and LVX with the values of FIC Index
(FIC I) for resistant H. pylori clinical strains.
Best Synergistic Combinations
H. pylori
Strains
LVX+RSV
FIC I
LVX+RSV-3
FIC I
LVX+RSV-4
FIC I
11F/11
2A/12
7A/12
12A/12
5A/13
13A/13
26A/13
1.00+0.50
0.50+0.50
0.25+6.00
1.00+0.50
1.00+24.0
0.25+6.00
2.00+24.0
1.50
1.00
0.28
1.50
1.03
0.28
1.03
1.00+0.09
0.50+0.09
0.25+0.09
1.00+6.25
1.00+0.78
0.25+0.09
2.00+12.5
1.00
1.00
0.25
2.00
1.00
0.26
1.25
1.00+0.01
0.50+0.09
0.12+6.25
1.00+25.0
1.00+0.78
0.25+0.09
2.00+1.25
1.00
1.03
0.24
2.00
1.00
0.26
1.12
Figure 2 shows representative images of checkerboard assays and the respective isobolograms
combining different concentrations of RSV-3 or RSV-4 and LVX against two strains: the resistant
H. pylori 7A/12 and the MDR H. pylori 13A/13. RSV, RSV-3, and RSV-4 are able to restore the efficacy of
LVX in the H. pylori strains.
Figure 2. Checkerboard assay and isobolograms showing the synergism between RSV, RSV-3, or RSV-4
with LVX against resistant H. pylori 7A/12 and MDR H. pylori 13A/13 strains. On the left, representative
checkerboard assays, the grey zone represents the bacterial growth and the white zone represents
the growth inhibition in the presence of both RSV-3, RSV-4, and LVX. On the right, the isobolograms
illustrate the related synergistic curves. The x-axis represents the dose of LVX and the y-axis represents
the dose of RSV-3 and RSV-4. The imaginary straight line connecting the intercept points represents
no interaction. Between this line and the synergistic curve, there is the synergistic area (FIC I
≤
0.5)
and additive area (FIC I > 0.5–4.0) interactions. Values above the straight line represent antagonistic
interactions (FIC I ≥ 4.0).
To evaluate the anti-virulence action of RSV, RSV-3, and RSV-4, the anti-biofilm effects of sub-MIC
concentrations of RSV, RSV-3, and RSV-4 alone and combined with LVX at sub-synergistic concentrations
were also evaluated for the resistant H. pylori 7A/12 and the MDR H. pylori 13A/13 for which the LVX
restoring effect was observed.
As shown in Figure 3A, for H. pylori 7A/12, RSV induced a moderate biofilm reduction not
exceeding 20.8%
RSV-4 produced the highest biofilm inhibition rates with 84.8%
at 1/2, 1/4, and 1/8 MIC, respectively, with respect to the control (p
the biofilm formation with a reduction rate of 49.6% 3.3, 39.2%
±
3.9 (1/2MIC). Higher reduction rates were induced by RSV-3 and RSV-4. In particular,
0.9, 53.4% 7.1, and 46.7% 8.4
0.05). RSV-3 was able to reduce
11.4, and 23.2%
13.3 at 1/2, 1
±
±
±
≤
±
±
±
⁄4,

Antibiotics 2020, 9, 891
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and 1/8 MIC, respectively. Regarding the sub-synergistic concentrations, the reduction trend in biofilm
formation for RSV, RSV-3, or RSV-4 with LVX was similar (from 30.2% 2.2 to 20.3% 2.7 for RSV-3
8 for RSV-4 and LVX and from 30.1% 5.5 to 13.8% 3.3 for
±
±
and LVX, from 35.6%
RSV and LVX).
±
11.3 to 23.3%
±
±
±
Figure 3. Effect of RSV, its derivatives (RSV-3 and RSV-4), and LVX at sub-inhibitory concentration
(1/2, 1/4, 1/8MIC) and their combinations at sub-synergistic concentrations (SbC), against H. pylori
7A/12 and H. pylori 13A/13 biofilm formation. (
7A/12 and MDR H. pylori 13A/13 after treatments. * Statistically significant values with respect to the
control. ( ) Representative fluorescence (after Live/Dead staining) and phase contrast light microscopy
A) Percentage of biofilm reduction of resistant strain
B
images of H. pylori 13A/13 biofilm treated with 1/4 MIC LVX, 1/4 MIC RSV, 1/4 MIC RSV-3, and 1/4
MIC RSV-4 and 1/4 sub-synergistic combinations of RSV, RSV-3, or RSV-4 and LVX and the untreated
sample (control). Viable cells exhibit green fluorescence while dead cells exhibit red fluorescence.
Arrows indicate the elongated forms of H. pylori cells after treatment with RSV, RSV-3, and RSV-4 alone
and combined with LVX. Original magnification 1000× (scale bar: 5 µm).
For H. pylori 13A/13, as shown in Figure 3A, RSV induced a moderate biofilm reduction with
a percentage of a biofilm reduction up to 24.8%
H. pylori 7A/12, RSV-4 expressed the highest H. pylori 13A/13 biofilm inhibition rate with respect to the
control (65.5% 4.0, 61.4% 5.0, and 43.7% 8.0 at 1/2, 1/4, and 1/8 MIC). Regarding the sub-synergistic
±
7.0 (1/2MIC). Similarly to the behaviour toward
±
±
±
concentrations, the reduction trend in biofilm formation for RSV, RSV-3, or RSV-4 with LVX was similar,
with a higher percentage of biofilm formation reduction at 1/2 sub-inhibitory concentration (Figure 3A).
Representative Live/Dead staining (Figure 3B, up) and phase contrast (down) images of
multidrug-resistant (MDR) H. pylori 13A/13 biofilm after treatment with 1/4 MIC LVX, 1/4 MIC
RSV, 1/4 MIC RSV-3, and 1/4 MIC RSV-4 and 1/4 sub-synergistic combinations of RSV, RSV-3, or RSV-4
and LVX. A general analysis of the detected microbial biofilms revealed a modification on the
aggregation and compactness of treated biofilms with respect to the control through viable staining.
This anti-adhesive effect was more pronounced in the presence of RSV-4 and RSV-4 plus LVX. Moreover,

Antibiotics 2020, 9, 891
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samples treated with LVX showed a large amount of red (dead) cells whereas, in all other conditions,
green (live) cells were detected underlying the bacteriostatic effect of RSV, RSV-3, and RSV-4.
Micrographs in a phase contrast showed the interesting effect of RSV, RSV-3, and RSV-4 and their
combination with LVX in terms of the elongated forms’ presence (Figure 3B, down).
The soft agar motility assay was performed to evaluate the effect of RSV, RSV-3, and RSV-4 on
H. pylori swarming motility. For H. pylori 7A/12 and H. pylori 13A/13, the RSV-3 and RSV-4, included in
the soft agar at 1/4 MIC and 1/8 MIC, induced the loss of motility by a smaller diameter of growth in
comparison with the control and RSV (p < 0.05). Figure 4A shows representative images regarding the
loss of H. pylori 13A/13 motility in the presence of RSV-3 and RSV-4 at 1/4 MIC concentration. The mean
halo diameters for the two analysed strains were 15 mm
±
2 mm for the control, 13 mm
±
2 mm in the
1 mm in the
presence of 1/4 MIC RSV, 8 mm
presence of 1/4 MIC RSV-4.
±
0.5 mm in the presence of 1/4 MIC RSV-3, and 5 mm
±
Figure 4. Effect of RSV, RSV-3, and RSV-4 at 1/4 MIC concentrations on the H. pylori motility and on
the expression of flaA gene. ( ) Representative images of H. pylori 13A/13 motility on soft agar 0.5%.
RSV-3 and RSV-4 induced a significant loss of motility with respect to RSV and the untreated sample
(control), as shown by a smaller diameter of growth on soft agar plates. ( ) Relative gene expression
A
B
of H. pylori flaA gene in the presence of RSV, RSV-3, and RSV-4. * Statistically significant values with
respect to the control.
In order to clarify the effect of RSV, RSV-3, and RSV-4 on H. pylori motility, the expression of the
flaA gene was performed, which represents the predominant subunits subtype of H. pylori flagellum.
The expression of flaA was induced with response to RSV, RSV-3, RSV-4, and control. FlaA gene
expression, in the two analysed strains, was significantly increased by RSV (1.86
and RSV-3 (1.62 0.31, fold change), (p < 0.05) and slightly increased by RSV-4 (1.25
(Figure 4B) in apparent contrast with the detected swarming motility.
±
0.43, fold change)
0.28, fold change),
±
±
The RSV, RSV-3, and RSV-4 toxicity was evaluated in vivo in G. mellonella model. The G. mellonella
survival percentage at 1000 mg/kg of RSV was 100% after 9 days. For RSV-3, the survival percentage
was 90% after 1 day, 80% after 2 days, and 70% after 9 days. A similar RSV-3 trend of survival rate
was recorded for RSV-4 with a lower survival rate after 9 days (60%). The survival percentages

Antibiotics 2020, 9, 891
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of G. mellonella larvae of the control group and treated with PBS until 8 days were 80% and 100%,
respectively (Figure 5). Although the percentage of G. mellonella survival, after RSV-3 and RSV-4
treatments, was lower than the RSV treatment, RSV-3 and RSV-4 can be considered non-toxic [40].
Figure 5. Survival of Galleria mellonella larvae in the presence of PBS, RSV, RSV-3, RSV-4 for 9 days,
for the toxicity evaluation.
Regarding the G. mellonella in vivo infection assay, the survival rate of infected larvae and treated
with MIC LVX, RSV-3, and RSV-4, and the best combination of RSV-3 or RSV-4 and LVX (0.09
µg/mL
RSV-3 or RSV-4 + 0.25 g/mL LVX) was monitored every day until the sixth day. After H. pylori infection,
µ
the treatment with LVX rescued larvae injected with a survival rate between 92% and 75% until 6 days.
The treatment with RSV-3 rescued larvae from H. pylori infection with 83% of survival until 2 days
and 75% at 6 days. The best synergistic combination of RSV-3 plus LVX showed a protective effect
against H. pylori infection with a larvae survival rate of 100% after 1 day, 83% after 2 days, and 75%
after 6 days (Figure 6A). The differences were compared with a Long-rank test and the survival curves
were statistically significant.
For RSV-4, the treatment rescued larvae from H. pylori infection with an 83% of survival rate after
2 days and 67% after 6 days. The best synergistic combination of RSV-4 plus LVX showed a protective
effect against H. pylori infection with a larvae survival rate of 100% until 5 days, 75% at 6 days.
RSV-3 and RSV-4 showed in vivo protective effects against an H. pylori infection over time,
alone and combined with LVX.
The H. pylori ability to infect G. mellonella larvae was analyzed over time by CFU determination.
As shown in Figure 6B, H. pylori 13A/13 was able to infect G. mellonella larvae and grow over time.
In the presence of LVX, the high survival rate of G. mellonella larvae was related to the reduced H. pylori
survival rate, shown by the lower CFU values detected with respect to the sham injection and PBS
treatment (reduction in CFUs of 98% with respect to sham injection and 94% with respect to treatment
with PBS) after 5 days (p
≤
0.05). RSV-3 and RSV-4 showed their protective effect in G. mellonella larvae
reducing significantly the H. pylori CFUs of 97.2% with respect to sham injection and 92.4% with respect
to PBS treatment for 5 days for both derivatives. In the presence of the best synergistic combinations
RSV-3 or RSV-4 and LVX, an interesting H. pylori CFU/larva reduction with 92.4% (for RSV-3 and LVX)
and 90% (for RSV-4 and LVX) until 5 days (p ≤ 0.05) was detected (Figure 6B).

Antibiotics 2020, 9, 891
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Figure 6. In Vivo infection assay in Galleria mellonella larvae. (
G. mellonella infected with H. pylori 13A/13 strain for 2 h, then treated with PBS, LVX, RSV-3, RSV-4,
and LVX+RSV-3, LVX+RSV-4 at the best synergistic combination. ( ) Recovery of H. pylori 13A/13
A) Kaplan Meyer survival curves of
B
CFU/larva in G. mellonella at different time points and different conditions (sham injection, PBS, LVX,
RSV-3, and RSV-4 and the best synergistic combination of RSV-3/LVX, RSV-4/LVX). ***** Statistically
significant values with respect to the control.
3. Discussion
Resveratrol is a naturally occurring polyphenolic antioxidant, belonging to the stilbene family,
produced by the plants in response to a microbial attack. The antibacterial activity of RSV has been
described, based on more than a single mechanism, such as alteration of the lipid bilayer of the
membrane and its permeability, changes in intracellular functions induced by hydrogen bonding of
the phenolic group in 4-position to enzymes, and antioxidant-scavenging activity that could inhibit the
generation of reactive oxygen species, reducing the redox potential of the growth medium [41–44].
The antibacterial and anti-virulence activities of RSV and newly synthetized RSV derivatives,
chosen to overcome the limitation of the poor bioavailability of RSV combined with LVX against
resistant H. pylori clinical strains, were investigated.
Our results show an antimicrobial activity of RSV against resistant H. pylori strains similar
to previous studies [45–49]. In particular, Paulo et al. [28] suggested a possible mechanism of
RSV action related to the inhibition of H. pylori urease activity, preventing the production of the
alkaline environment around bacterial cells, allowing the microorganism to survive the stomach acidic
conditions. However, RSV seems to be less active against Gram-negative bacteria than Gram-positive.
Likely, the presence of efflux pump systems in Gram-negative may be responsible for their decreasing
susceptibility to this natural stilbenoid, as documented by experiments performed with mutants or
in the presence of efflux pumps inhibitors [50–52]. This observation leads us to hypothesize that the
antibacterial action of RSV could be due to its interaction with targets present inside cytoplasmic or in
periplasmic sites in Gram-negative bacteria [19].
As known, RSV shows low bioavailability so, in this work, newly synthetized RSV derivatives
were investigated for their antibacterial properties. To this aim, selected derivatives 1–5 were chosen.

Antibiotics 2020, 9, 891
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They kept the 40-hydroxyl group because its importance for biological activity and the 3,5-hydroxy
group was replaced with a substituent in the 4-position.
Regarding the newly synthetized RSV derivatives, the RSV-3 (R = Me) and RSV-4 (R = Cl)
showed the best antibacterial action at very low concentrations. These RSV derivatives show greater
effectiveness than the lead compound, RSV. The MIC of RSV-4 was 64-fold lower than RSV against the
H. pylori strains, suggesting the improved antibacterial activity in RSV derivatives. In compounds
RSV-3 and RSV-4, the 3,5-dihydroxy motif of RSV was substituted with a more lipophilic methyl (RSV-3)
and chlorine (RSV-4), keeping the unchanged OH-group in the 40-position. It can be hypothesized that,
in this way, the mechanism of antimicrobial action of RSV remains unchanged due to the presence of
the OH-group in the 40-position. However, the loss of two hydrophilic OH-groups and the introduction
of a lipophilic group could be beneficial for overcoming the cell membrane in a passive way and
inducing antibacterial action inside cells. Likely, the simpler and more lipophilic structures of RSV-3
and RSV-4 could promote their entry into the bacterial cell and induce its antibacterial action inside the
cells. In order to elucidate the possible mechanism of action, further studies are required.
The capability of RSV, RSV-3, and RSV-4 to synergize with LVX to restore the antibiotic efficacy
and reduce its MIC values under the breakpoint was also evaluated. To the best of our knowledge,
this is the first study in which the capability of RSV and RSV derivatives to synergize with antibiotics
was evaluated. Xia et al. [53] showed the RSV synergism when combined with alcohol, underlying that
the anti-H. pylori mechanism of RSV is linked to its inhibitory effect on translation, outer membrane
protein production, ATP synthase, and transports.
To explore the anti-virulence activities of RSV, RSV-3, and RSV-4, we evaluated their effect on
biofilm formation and H. pylori motility.
Regarding the effect on microbial biofilm formation, RSV, RSV-3, and RSV-4 were detected alone
and in combination with LVX at sub-synergistic concentrations. Our results highlighted a moderate RSV
antibiofilm effect on resistant and MDR H. pylori strains. RSV-3 and RSV-4 exhibited a higher antibiofilm
activity, likely due to their higher bioavailability and ability to interact with the bacterial cells. It was
proposed that inducing the down regulation of outer membrane proteins, RSV, and likely RSV-3 and
RSV-4 affect the bacterial adhesion and colonization [52,54]. A study by Klancnik et al. [55] evaluated
the effect of RSV on the biofilm formation in Campylobacter jejuni together with the culturability and
viability of the adhered cells post-treatment with RSV. Sub-inhibitory concentration of RSV induced
40% inhibition of C. jejuni biofilm formation interfering in the quorum sensing system [55].
Regarding the anti-virulence activity of RSV, RSV-3, and RSV-4, we evaluated their effect on
H. pylori motility. As well known, the flagellar motility represents for H. pylori an important virulence
factor for successful colonization in vivo of gastric mucosa, for chemotaxis, and for its movement
toward the gastric mucus within the stomach [51]. From our results, on soft agar, it was possible to
observe the loss of H. pylori motility in the presence of RSV-3 and RSV-4 at sub-MIC concentrations and
a moderate anti-motility action in the presence of RSV. As reported by others [55–57], at sub-inhibitory
concentrations, RSV reduced the swarming motility in a dose-dependent manner in Proteus mirabilis,
the swimming and swarming motility in E. coli as well as in Vibrio vulnificus.
In this work, RSV, RSV-3, and, to a lesser extent, RSV-4, induced the over-expression of the flaA
gene. This result could seem an apparent contradiction when looking the phenotypic motility on a
soft agar plate. Since the control system for the flagellar motility in H. pylori is an intricate process not
dependent only on FlaA production [58,59] and considering that, in E. coli and M. smegmatis, RSV is
able to inhibit ATP synthesis [24 28]. We can hypothesize that RSV and RSV derivatives, affecting the
,
flagellar hook and its energy production system could compromise the flagellar functionality. We can
suppose that H. pylori cells, in the presence of RSV, RSV-3, or RSV-4 are characterised by a major
production of flagella but their efficacy is reduced by an insufficient ATP synthesis system resulting in
a reduced swarming motility. More studies are required to explore this complex mechanism.
RSV, RSV-3, and RSV-4 were able to induce an elongated morphology in H. pylori cells, as reported,
for RSV, in other Gram negative [29,30]. This atypical filamentous morphology might be due to an

Antibiotics 2020, 9, 891
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alteration during the regular cell division and to the FtsZ ring uncorrected production compromising
the microbial division.
Moreover, infecting G. mellonella larvae with H. pylori MDR strain and treating, then, with RSV-3
and RSV-4 alone and combined with LVX at the best synergistic combinations, an interesting protective
effect against H. pylori was observed, confirming the in vitro results. The proven effectiveness of these
compounds, in the in vivo model, allow us to emphasize the RSV derivatives’ possible role in the
management of H. pylori infection.
Despite the limitation of this study related to a few numbers of resistant H. pylori detected
micro-organisms, we can conclude that, on the basis of the interesting antibacterial action, and their
effects on biofilm formation and on bacterial motility, RSV–phenol derivatives could be considered
interesting anti-H. pylori candidates for innovative, therapeutic schemes to tackle the H. pylori
antibiotic resistance.
4. Materials and Methods
4.1. Chemistry
The synthesis of compounds RSV-1–5 was carried out as previously reported [36,37].
The 4-hydroxybenzaldehyde and the appropriate aryl acetic acid were mixed in the presence
of piperidine at 130◦C. The usual aqueous work-up and purification using silica gel column
chromatography produced the desired phenols. The chemical stability of all compounds was evaluated
before and after the sterilization in autoclave (Fedegari Autoclavi S.p.a., Albuzzano (PV), Italy)
comparing the 1H-NMR signals on a Varian Mercury 300 spectrometer (Varian, Palo Alto, CA, USA).
4.2. Bacterial Cultures
Seven clinical H. pylori isolated strains, coming from the private collection of Bacteriological
Laboratory of the Pharmacy Department, University “G. d’Annunzio” Chieti-Pescara, were used in
the experiments. Strains were previously characterized for their antimicrobial susceptibility profiles
against the antibiotic commonly used in therapy: clarithromycin, metronidazole, levofloxacin (LVX),
moxifloxacin, ciprofloxacin, rifabutin, tetracycline, ampicillin, and amoxicillin (Table S1) [13]. All strains
were resistant to LVX (H. pylori 2A/12 was also included despite its borderline MIC LVX values between
0.5–1.00 µg/mL). Helicobacter pylori 11F/11, H. pylori 2A/12, and H. pylori 7A/12 were resistant to at
least three antibiotics, whereas H. pylori 5A/13, H. pylori 13A/13, and H. pylori 26A/13 were multi-drug
resistant (MDR) strains with resistance profiles of at least three antimicrobial classes. The reference
strain H. pylori ATCC 43629 was also included. The study was approved by the Inter Institutional Ethic
Committee of University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy (ID Number RICH9RTLH).
Bacteria were cultured on Columbia agar base (CA, Oxoid, Milan, Italy) with 10% (v/v) lacked
horse blood plus IsoVitalex 1% (v/v) (BBL, Microbiology System, Milan, Italy). For the experiments,
the bacterial suspensions were prepared in Brucella Broth (BB) plus 2% foetal calf serum (FS) (Biolife
Italiana, Milan, Italy) and adjusted to an optical density at 600 nm (OD600) of 0.2 corresponding to
mboxtextasciitilde1.8 × 106 Colony Forming Unit (CFU)/mL
4.3. Antibacterial Susceptibility Assay
The determination of Minimum Inhibitory Concentration (MIC) values of RSV and new synthesized
RSV-1–5 against standardized broth cultures of H. pylori strains was performed by a microdilution
method assay in 96-wells-microtitre plates (Nunc, Euro Clone SpA, Life Sciences-Division, Milan, Italy).
Two-fold dilutions of RSV and RSV-1–5 stock solution, ranging from 800 to 6.25
in BB plus 2% FS. Levofloxacin (Sigma Aldrich S.R.L, Milan, Italy) was prepared in BB plus 2% FS in
two-fold dilutions from 2.00 to 0.01 g/mL. One hundred L of RSV or each RSV derivative or 100
LVX and 100 L of standardized bacteria were dispensed in each well of 96-wells-microtitre plate and
incubated in micro-aerobic condition (5% O2, 10% CO2, 85% N2) for 3 days at 37 ◦C.
µg/mL, were prepared
µ
µ
µL
µ

Antibiotics 2020, 9, 891
11 of 16
MIC values were measured by determining the lowest concentration of substances able to
inhibit the visible growth of the microorganisms. Minimum Bactericidal Concentrations (MBCs) were
determined by sub-cultivation of 10 µL of suspensions from the non-turbid wells on CA and incubated
as describe above. The MBC represents the lowest concentration of each substance that inhibited the
bacterial growth on plates. The MBC values were also confirmed by an iodo-nitro tetrazolium violet
assay (INT, Sigma-Aldrich) following the addition (40 µL) of 0.2 mg/mL of INT and incubation at
37 ◦C for 2 h. Viable bacteria reduce the yellow dye to a pink-purple and dead cells do not produce a
color change.
4.4. Checkerboard Assay
The checkerboard test was performed to evaluate the synergism between RSV or the most
promising antibacterial derivatives (RSV-3 or RSV-4) and LVX against H. pylori strains.
Dilutions of the RSV or RSV-3, RSV-4, and LVX from MIC values to serial dilution below were
inoculated in 96-well microtiter plates and incubated as described above. The checkerboard test was
used to calculate the Fractional Inhibitory Concentration (FIC) equal to MICAB/MICA + MICBA/MICB,
where MICAB is the MIC of compound A in the presence of compound B. MICBA is MIC of B in the
presence of A. FIC Index (FIC I) values were interpreted according to Odds [60] as follows: synergism
FIC I
≤
0.5, antagonism FIC I
≥
4.0, and additive FIC I > 0.5–4.0. All studied substances (RSV, RSV-3,
RSV-4) and LVX were assayed alone as a control. The results were also reported as isobolograms
constructed by plotting synergistic concentrations [13].
4.5. Biofilm Biomass Quantification and Microscopic Analysis
The anti-biofilm activity of RSV, RSV-3, and RSV-4 (at sub-MIC values) were evaluated on the
biofilm formation of selected H. pylori strains: resistant H. pylori 7A/12 strain and MDR H. pylori
13A/13 strain, toward which the synergistic action with LVX was observed. Broth cultures of H. pylori,
harvested in BB with 2% (w/v) FS and 0.3% (w/v) glucose, were gently shaken and incubated overnight
at 37 ◦C in a micro-aerobic atmosphere. After incubation, each broth culture was adjusted to OD600~0.2
and 100
µL were inoculated on flat-bottomed 96-wells-polystyrene-microtiter plates with a sub-MIC
concentration of RSV or RSV-3-4 (100
µ
L), LVX (100 L), and the best synergistic combinations at a
µ
sub-synergistic concentration. After incubation at 37 ◦C in microaerobic condition for 48 h, the produced
biomasses of the treated and untreated biofilms were quantified by a safranin staining method [13].
For the cell viability evaluation, biofilms were grown as described above. Briefly, 1 mL of sub-MIC
RSV, or sub-MIC of RSV-3, RSV-4, or 1 mL of sub-MIC LVX, or 1 mL of the best synergistic combinations
at a sub-synergistic concentration and 1 mL of standardized broth cultures of each H. pylori strains
were inoculated in a Petri dish (3.5 cm) and incubated at 37 ◦C in a microaerobic condition for 48 h.
After incubation, the planktonic phase was removed and the sessile bacterial populations were washed
with PBS and stained with Backlight Live/Dead Viability staining (Molecular Probes, Invitrogen
detection technologies, USA) as indicated by the manufacturer [61]. The images were observed at
Leica 4000 DM fluorescent microscopy (Leica Microsystems, Milan, Italy), and more fields of view
were examined randomly [14]. Moreover, the samples were also observed under a phase contrast light
microscope (Leica 4000 DM) to observe the H. pylori morphology.
4.6. Motility and flaA Gene Expression Assays
The effect of RSV, RSV-3, and RSV-4 on the resistant H. pylori 7A/12 strain and MDR H. pylori 13A/13
strain motility was evaluated following the Ciccaglione et al. [4] methodology with some modifications.
Briefly, H. pylori clinical strains motility was analysed by using soft agar plates composed by BB plus
10% FS, 0.5% agar bacteriological, and sub-MIC concentration (1/4MIC, 1/8MIC) of RSV, RSV-3 and
RSV-4. Ten microliters of overnight broth cultures standardized at 600 nm (OD600) of 0.4 in BB, plus 10%
FS, were inoculated into the thickness of the soft agar with a sterile tip. Plates were incubated at 37 ◦C
under microaerobic conditions for 5–6 days. Then the bacterial halo was recorded.

Antibiotics 2020, 9, 891
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For the evaluation of flaA gene expression, H. pylori broth cultures in 40 mL of BB plus 2% FS at
OD600 ~0.2 were incubated in the presence of 1/4 MIC of RSV or 1/4 MIC of RSV-3 or RSV-4 at 37 ◦C for
3 days in a micro-aerobic condition. Subsequently, H. pylori cells were harvested by centrifugation at
5000× g for 10 min at 4 ◦C and washed three times with sterile PBS.
Total RNA extraction was performed using the RNeasy mini kit (Qiagen, Milan, Italy), according to
the manufacturer’s instructions. cDNA was generated using the iScript cDNA Synthesis Kit (Bio-Rad,
◦
Milan, Italy) and then stored at
−
20 C until use. For the quantitative PCR, the oligonucleotide
primers used were the flaA gene sequence Fwd: 50-CAGTATAGATGGTCGTGGGATTG-30,
Rvs: 50-GAGAGAAAGCCTTCCGTAGTTAG-30; the housekeeping gene 16SrRNA Fwd: 50-
GGAGTACGGTCGCAAGATTAAA-30 Rsv: 50-CTAGCGGATTCTCTCAATGTCAA-30
[
62].
95% efficiency values). The quantitative PCR reactions were performed
in 96-well microtiter plates (Bio-Rad, Milan, Italy) using 10 L of SsoAdvanced universal SYBR
Green supermix 2X (Bio-Rad, Milan, Italy), 0.6 M forward/reverse primer mix, and 2 L of cDNA,
in free-nucleases water to a final volume of 20 L. The qPCR reactions were placed into a CFX96 Real
(The primers showed
≥
µ
µ
µ
µ
Time system, C1000 Touch, Thermal Cycler (Bio-Rad, Milan, Italy).
Thermal cycling conditions were as follows: 3 min at 95 ◦C for initial denaturation followed by
39 cycles of 95 ◦C for 15 s and 55 ◦C for 40 s. After a finalization step of 15 s at 95 ◦C and 55 ◦C for
40 s, a melting curve analysis was performed with a temperature range between 65 ◦C and 95 ◦C and
an increment of 0.5 ◦C for 15 s followed by the plate read out. A melting curve was used at the end
to confirm only one peak and only one product. The values of the threshold cycle (Ct) and relative
expression level were normalized by the ∆∆CT method. Results were analysed using the Bio-Rad CFX
Manager Software, version 3.1 (Bio-Rad Laboratories).
4.7. Toxicity Test in the Galleria mellonella Model
The toxicity of RSV, RSV-3, and RSV-4 was evaluated by using wax moth G. mellonella larvae.
Stock solutions of RSV, RSV-3, and RSV-4 were diluted in PBS to obtain the final concentration of
1000 mg/Kg. Five groups of 10 randomly-selected G. mellonella larvae, choosing with a weight ranging
between 0.2–0.3 g, were treated as follows: three groups were injected in the last left proleg with
10 µL of RSV, 10 µL of RSV-3, 10 µL of RSV-4, one group was injected with 10 µL of PBS by using
0.3 mL micro-fine needle insulin syringes (BD, Milan, Italy), and one group was un-injected. A total of
50 larvae were incubated at 37 ◦C in Petri dishes in the dark for nine days. The wax moth survival was
monitored over nine days, every 24 h. The larvae were considered dead when they were unresponsive
to touch [13]. During assays, larvae did not receive nutrition.
4.8. In Vivo G. mellonella Infection Assay
The activity of RSV-3 and RSV-4 against H. pylori 13A/13 infection was evaluated by the in vivo model
of G. mellonella larvae, which represents a recognized model for H. pylori infection for which no ethical
approval is required. H. pylori studied strain 13A/13, standardized at OD600 = 0.2 (~1.8 × 106 CFU/mL),
was chosen for the experiments. Seven groups of 10 randomly selected G. mellonella larvae were
injected with 10
µL of H. pylori broth culture in the last left proleg of each larva for a total of 70 larvae.
One group of 10 larvae was not infected. After 2 h of infection, 10 larvae were treated with 10
µL
of LVX at the MIC value, 10 larvae were treated with RSV-3 or RSV-4 at 1000 mg/Kg, and 10 larvae
were treated with the best synergistic combination of RSV-3 or RSV-4 plus LVX on the last right proleg.
A control group of 10 larvae was treated with 10 µL of PBS and 10 larvae with a sham injection
(larvae were nicked with a sterile syringe to evaluate the effect of the larval puncture). G. mellonella
larvae were incubated at 37 ◦C in the dark for 6 days. During assays, larvae did not receive nutrition.
The G. mellonella survival was controlled every day. Dead larvae were unresponsive to touch.
To evaluate the H. pylori 13A/13 survival rate in G. mellonella larvae after 1-, 2-, 3-, 4-, 5-, and 6-days
post-infection, seven groups of 10 randomly selected G. mellonella larvae were infected as described
above. After incubation, for each group, three larvae were chilled on ice for 10 min, and the haemocoel

Antibiotics 2020, 9, 891
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was serially diluted in sterile PBS and the H. pylori cells were quantified by CFUs determination on
Campylobacter selective agar (CP Dent) with 7% defibrinated horse blood and 0.4% of Dent supplement
(Oxoid) and incubated in a microaerobic condition at 37 ◦C. The CFU/larva were counted after 3 days.
4.9. Statistical Analysis
Data is obtained from at least three independent experiments performed in triplicate. Data is
shown as the means ± standard deviation.
Differences between groups were assessed with a paired Student’s t-test. p values
≤
0.05 were
considered statistically significant. Survival curves were plotted using the Kaplan-Meier method,
and survival differences were calculated using the Long-rank test for multiple comparisons. GraphPad
Prism 6 was used to fit a curve to the infection data.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/12/891/s1,
Table S1: Antimicrobial susceptibility panel of H. pylori clinical strains used in this study.
Author Contributions: Conceptualization, R.A., B.D.F., L.C., and M.D.G. Methodology, P.D.F., S.D.L., S.D.,
and E.D.C. Validation and data curation, S.D.L., S.D., and E.D.C. Investigation, P.D.F. and S.D.L. Writing—original
draft preparation, P.D.F., B.D.F., and M.D.G. Writing—review and editing, R.A., L.C., and M.D.G. Project
administration, L.C. Funding acquisition, M.D.G. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was funded by a Research Grant PRIN 2017 SFBFER from MIUR, Italy.
Conflicts of Interest: The authors declare no conflict of interest.
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