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Fruit flavonoids as modulators of norfloxacin resistance in Staphylococcus aureus that overexpresses norA
Accepted Manuscript Fruit flavonoids as modulators of norfloxacin resistance in Staphylococcus aureus that overexpresses norA Helena Tainá Diniz-Silva, Marciane Magnani, Sílvia de Siqueira, Evandro Leite de Souza, José Pinto de Siqueira-Júnior PII:
S0023-6438(16)30187-6
DOI:
10.1016/j.lwt.2016.04.003
Reference:
YFSTL 5391
To appear in:
LWT - Food Science and Technology
Received Date: 18 December 2015 Revised Date:
29 March 2016
Accepted Date: 1 April 2016
Please cite this article as: Diniz-Silva, H.T., Magnani, M., de Siqueira, S., de Souza, E.L., de SiqueiraJúnior, J.P., Fruit flavonoids as modulators of norfloxacin resistance in Staphylococcus aureus that overexpresses norA, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fruit
flavonoids
as
modulators
of
norfloxacin
2
Staphylococcus aureus that overexpresses norA
resistance
in
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Running title: Antibiotic resistance modulatory effects of fruit flavonoids
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Helena Tainá Diniz-Silva1, Marciane Magnani2, Sílvia de Siqueira1, Evandro Leite de
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Souza3, José Pinto de Siqueira-Júnior1
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Laboratório de Genética de Microrganismos, Departamento de Biologia Molecular,
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Universidade Federal da Paraíba, João Pessoa, Paraíba, Brazil
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Alimentos, Universidade Federal da Paraíba, João Pessoa, Paraíba, Brazil
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Federal da Paraíba, João Pessoa, Paraíba, Brazil
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Laboratório de Microbiologia de Alimentos, Departamento de Nutrição, Universidade
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Laboratório de Processos Microbianos em Alimentos, Departamento de Engenharia de
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ACCEPTED MANUSCRIPT Abstract
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This study investigated whether different fruit flavonoids are capable of modulating
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norfloxacin (Nor) resistance in S. aureus SA-1119B, an S. aureus strain that overexpresses
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norA. The minimum inhibitory concentration (MIC) of flavonoids and norfloxacin (Nor)
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were determined using broth microdilution tests. To assess the antibiotic resistance
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modulatory effect, the MIC of Nor was measured in growth media with or without
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flavonoids at a sub-MIC level. The lowest MIC (256 µg/mL) against S. aureus SA-1199B
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was observed for myricetin; all other tested flavonoids produced MICs > 256 µg/mL.
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When tested at sub-MIC levels in combination with Nor, hesperetin reduced the MIC of
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Nor from 128 µg/mL to 8 µg/mL (16-fold), whereas phloretin, diosmetin, and myricitrin
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reduced the MIC of Nor from 128 µg/mL to 32 µg/mL (4-fold), and quercitrin reduced the
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MIC of Nor from 128 µg/mL to 64 µg/mL (2-fold). No modulatory effects were observed
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for flavonoids with 7-O-glycoside residue or for non-glycosylated forms of 3-O-glycoside
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flavonoids. These results suggest that fruit flavonoids in aglycone forms or 3-O-glycoside
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flavonoids are capable of modulating Nor-resistance in S. aureus that overexpresses NorA.
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Key-words: flavonoids, glycosylation, Staphylococcus spp., resistance-modifying agents.
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1. Introduction
Flavonoids are the most abundant polyphenols present in plant foods. They are
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generally defined as dietary antioxidants, and these nonessential nutrients have been
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established as bioactive compounds that benefit human health (Corcoran, McKay, &
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Blumberg, 2012; Choi, Islam, Ali, Ji Kim, Kim, & Jung, 2014). Characterized by a basis
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backbone composed of 15 carbon atoms (C6 - C3 - C6), flavonoids are organized into ten
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different groups (Cushnie & Lamb, 2011; Xiao, Muzashvili, & Georgiev, 2014). These 2
ACCEPTED MANUSCRIPT compounds occur as α- or β-glycosides that include flavonoid glycosides, galactosides,
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rhamnosides, arabinosides and rutinosides. The flavonoid glycosides mainly occur as C-3
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or C-7 O-glycosides, but the C-4, C-5, C-6, and C-8′ positions are sometimes glycosylated
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as well. In some fruit, e.g., apples, flavonoids are usually present in the C-3 position of an
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O-glycosylated form (Cushnie & Lamb, 2011; Barreca, Bellocco, Laganà, Ginestra, &
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Bisignano, 2014). Differences in molecular structure have been commonly related to
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specific biological properties of compounds that comprise the flavonoid group (Choi et al.,
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2014).
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Fruit flavonoids have a variety of biological properties, including antioxidant, anti-
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inflammatory, antidiabetic, hepatoprotective, antiviral and antimicrobial effects (Céliz,
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Daz, & Audisio, 2011; Barreca, Belloco et al., 2014). Some flavonoids widely found in tea,
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such as cathecins, have shown the ability to restore antibiotic sensitivity in methicillin-
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resistant Staphylococcus aureus (MRSA) strains because these compounds possess an
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affinity for binding to bacterial proteins that are involved in the resistance of methicillin or
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other drugs (Hatano et al., 2005, Abreu, McBainband, & Simões, 2012).
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Reports of infections caused by multidrug-resistant S. aureus have increased in
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recent years, especially in developing countries (WHO, 2014). Compounds with the ability
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to enhance the efficacy of existing antibiotics are called modifiers of resistance (resistance-
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modifying agents, RMAs), and discovering new RMAs is considered a strategy to combat
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antibiotic resistance in S. aureus (Gibbons, Oluwatuyi, Veitch, & Gray, 2003; Chan et al.,
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2013). RMAs could provide effective tools to control S. aureus resistance in clinical
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practice, which would make possible the use of a reduced set of classical antibiotics and
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reducing the costs of antibiotic therapy (Abreu, McBainband, & Simões, 2012).
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Studies have reported that some flavonoids are effective at inhibiting S. aureus
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MRSA by acting either directly or by restoring the antibacterial efficacy of antibiotics; this 3
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study aimed to evaluate the modulatory effects of glycosylated and aglycone forms of fruit
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flavonoids on norfloxacin (Nor) resistance in S. aureus that overexpresses the norA gene.
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2. Materials and methods
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2.1 Nor, ethidium bromide and fruit flavonoids
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Nor, flavonoids and ethidium bromide (EtBr) were obtained from Sigma-Aldrich
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(purity ≥ 98%; St. Louis, USA). A stock solution of Nor was prepared in a mixture of 1 M
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NaOH and sterile distilled water (1 : 9 proportion) according to a procedure described
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elsewhere (CLSI, 2012). The stock solution of EtBr was prepared in distilled water. Test
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flavonoids (Table 1) were dissolved in a mixture of DMSO and sterile distilled water (4 :
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1). DMSO at its highest final assayed concentration after dilution in broth (4%) did not
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inhibit bacterial growth.
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2.2 Test strain and growth conditions
S. aureus SA-1199B encoding the NorA efflux protein, which was kindly
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provided by Dr. S. Gibbons (University of London – UK), was used as the test strain. The
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stock cultures were maintained in Brain Heart Infusion (BHI) broth (Himedia, India)
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containing glycerol (15 mL/100 mL) at -20 °C. To obtain the inoculum, the strain was
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grown in BHI broth at 37 °C for 18 - 20 h (until the late exponential growth phase). Then,
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cells were harvested by centrifugation (4500 g, 15 min, 4 °C), washed twice in sterile
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saline solution (NaCl 0.85% w/v) and resuspended to obtain standard cell suspensions. The
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OD reading at 660 nm of cell suspension was 0.1, and it provided viable cell counts of
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approximately 8 log cfu/mL when the sample was pour-plated in BHI agar (Himedia,
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India).
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2.3 Determination of minimum inhibitory concentration (MIC) The MIC of the different flavonoids was determined using broth microdilution tests
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following a standard procedure (CLSI, 2012). The 96-well plates were prepared by
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dispensing 180 µL of Mueller Hinton broth (Himedia, India) containing 1.8 µL of bacterial
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suspension (8 log cfu/mL). Then, 20 µL of the solution of each flavonoid tested (final
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concentration varying from 1024 to 0.25 µg/mL) was added to each well. Each plate
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included positive controls without flavonoids. The plates were statically incubated at 37 °C
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for 24 h. After the incubation period, the MIC value was defined as the lowest
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concentration (µg/mL) of each flavonoid that was capable of visually inhibiting the growth
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of the test bacterial strain.
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2.4 Assessment of antibiotic resistance modulatory effect
The “modulation assay” was used to evaluate the effects of the flavonoids on Nor
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resistance in S. aureus SA-1199B (Gibbons et al., 2003). The MIC of Nor was determined
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in growth media containing and not containing flavonoids at a sub-MIC level (1/4 MIC)
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using the broth microdilution test as previously described (CLSI, 2012). To determine
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whether Nor resistance modulation involved the inhibition of the NorA-efflux pump, the
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MIC of EtBr (indicator of NorA efflux pump inhibition; Gomes et al., 2011) was
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determined in growth media containing or not containing each of the tested flavonoids.
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EtBr is a well-known substrate for efflux proteins, and active efflux is the only known
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mechanism of resistance to this DNA-intercalating dye (Kummar et al., 2008).
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A positive resistance modulatory effect was demonstrated when the incorporation
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of the test flavonoid into bacteria growth media decreased the baseline MIC of Nor or EtBr
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(Gibbons et al., 2003; Stavri, Piddock, & Gibbons, 2007).
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2.4 Statistical analysis The assays were performed in triplicate in three separate experiments. The MIC
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results are expressed as the modal values because the values of the repetitions were the
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same.
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3. Results and discussion
The lowest MIC (256 µg/mL) against S. aureus SA-1199B was observed for
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myricetin (aglycone form); all other flavonoids produced MICs > 256 µg/mL, suggesting a
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weak inhibitory effect (Van Vuuren, 2008). Similarly, the aglycone named narigenin was
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previously found as more effective than its glycosylated form to inhibit S. aureus (Han &
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You, 1988). The attachment of the hydrophilic substituent, 7-O-glycoside, onto the
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flavonoid molecule has been suggested to reduce its interaction with the target bacteria due
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to a lack of affinity with the phospholipid bi-layer or specific receptors located on the cell
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membrane (Gomes et al., 2011). The antimicrobial efficacy of the di-glycosides naringin,
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neohesperidin and neoeriocitrina and their aglycone forms (naringenin, hesperetin and
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eriodictyol, respectively) against Listeria innocua and S. aureus was already verified
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(Mandalari et al., 2007). In these studies, some aglycones were more effective at inhibiting
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both target bacteria than their respective di-glycosides forms. On the contrary, similar
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inhibitory effects of flavonoids in either glycosylated (e.g., naringin and prunin) or
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aglycone (e.g., naringenin) forms against S. aureus have been demonstrated (Céliz, Daz, &
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Audisio, 2011). These results are in agreement with our findings for phloridzin, hesperidin,
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diosmin, quercitrin, naringin and their respective aglycone forms. Therefore, in addition to
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the glycosylation, other aspects of the molecular structure of flavonoids, such as
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hydroxylation, methoxylation and alkylation, may be involved in their efficacy to inhibit
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different bacteria (Gomes et al., 2011; Xiao et al., 2014).
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diosmetin at a sub-MIC level decreased the baseline MIC of Nor from 128 to 32 µg/mL (4-
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fold) against S. aureus SA-1199B, whereas hesperetin decreased the baseline MIC of Nor
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from 128 to 8 µg/mL (16-fold) (Table 2). The incorporation of a sub-MIC of the 7-O-
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glycoside forms of phloridzin, naringin, diosmin or hesperidin into growth media did not
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decrease the MIC of Nor against S. aureus SA-1199. These findings that the aglycones are
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able to modulate Nor-resistance is in accordance with an earlier study that verified that
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acacetin (aglycone flavonoid) modulated Nor-resistance in S. aureus SA-1199B (Gomes et
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al., 2011). Still, the ability of the aglycone diosmetin to modulate resistance to the
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fluoroquinolone ciprofloxacin in the same strain has already been reported (Chan et al.,
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2013).
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Myricitrin and quercitrin (which possess the 3-O-glycoside residue) at sub-MIC
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levels decreased the MIC of Nor from 128 to 32 µg/mL (4-fold) and from 128 to 64 µg/mL
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(2-fold decrease) against S. aureus SA-1199B, respectively (Table 2). Otherwise, the
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incorporation of the aglycone forms of quercetin and myricetin into growth media did not
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decrease the MIC of Nor. Similarly, an earlier study reported a decrease of MIC of
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ciprofloxacin from 16 µg/mL to 1 µg/mL (16-fold) against S. aureus SA-1199B when the
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3-O-glycoside flavonoid kaempferol at a sub-MIC level was incorporated into growth
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media (Falcão-Silva, Silva, Souza, & Siqueira-Júnior, 2009).
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Flavonoids are commonly present in fruits as glycoside conjugates, and structural
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characteristics (e.g., presence of aromatic ring or numbers of hydroxyl and methoxyl
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groups) may influence their effects on membrane permeability and affinity to target sites in
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microbial cells (Mandalari et al., 2007; Cushnie & Lamb, 2011). Because flavonoids
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without a 7-O-glycoside residue and with a 3-O-glycoside residue modulate the Nor
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resistance in S. aureus SA-1199B, further investigations into whether these effects were 7
ACCEPTED MANUSCRIPT related to the NorA-efflux pump inhibition were carried out using EtBr. The MIC of EtBr
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against S. aureus SA-1199B decreased from 32 µg/mL to 8 µg/mL (4-fold) when
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naringenin was incorporated into growth media at sub-MIC level (Table 2). Decreases in
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MIC of EtBr from 32 µg/mL to 16 µg/mL (2-fold) were verified when a sub-MIC level of
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phloretin, hesperetin or myricitrin was incorporated into growth media (Table 2). These
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decreases in the MIC of EtBr suggest that the putative modulation of Nor-resistance in S.
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aureus SA-1199B involved a disturbance in the function of the NorA gene (Falcão-Silva,
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Silva, Souza, & Siqueira-Júnior, 2009). However, quercitrin (3-O-glycoside form) and
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diosmetin (aglycone) decreased the MIC of Nor but not the MIC of EtBr, suggesting that
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these flavonoids may modulate Nor-resistance in S. aureus SA-1199B by other
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mechanisms. Interestingly, the inhibition of topoisomerase, a well-known target of
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quinolones (Monte et al., 2014), e.g., norfloxacin, has also been suggested to participate in
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the antibiotic resistance modulatory activity of flavonoids. Considering that a specific
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mechanism does not fully account for the resistance modulatory activity of flavonoids, it
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has been proposed that these compounds may exert these effects through multiple
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mechanisms (Cushnie & Lamb, 2011).
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4. Conclusions
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The results of this study demonstrate that fruit flavonoids in aglycone forms or 3-O-
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glycoside flavonoids can modulate Nor-resistance in S. aureus that overexpresses norA by
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acting as RMAs through the inhibition of the efflux system and/or other mechanisms
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involved in Nor-resistance.
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Acknowledgements
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The authors would like to thank Prof. Simon Gibbons (University of London, UK)
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for his valuable and kind cooperation, CNPq (Brazil) for the financial support and CAPES
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(Brazil) for a scholarship awarded to the first author (H.T. Diniz-Silva).
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of C-glycosylation on anti-diabetic, anti-Alzheimer’s disease and anti-inflammatory potential of apigenin. Food and Chemical Toxicology, 64, 27–33.
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Informational Supplement. CLSI document M100-22. Wayne, PA: Clinical and
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Laboratory Standards Institute; 2012a. p.151.
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Corcoran, M. P., McKay, D. L., & Blumberg, J. B. (2012). Flavonoid basics: chemistry,
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Geriatrics, 31,176–89.
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Cushnie, T.T.P. & Lamb, A.J. (2011). Recent advances in understanding the antibacterial
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Gibbons, S., Oluwatuyi, M., Veitch, N.C., & Gray, A.I. (2003). Bacterial resistance
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V.S., & Siqueira-Junior, J.P. (2011). Phenolic compounds from Sidastrum micranthum
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Mirza, Z.M., Kumar, M., Sangwan, P.L., Gupta, P., Thota, N., & Qazi, G.N. (2008).
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Table 1. Chemical classification of fruit flavonoids tested to evaluate the Nor-resistance
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modulatory effects in Staphylococcus aureus strain that overexpresses the norA gene (S.
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aureus SA-1199B). Fruit flavonoids 7-O-glycoside
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3-O-glycoside Aglycone
Glycone
Phloridzin
Phloretin
Myricitrin
Naringin
Naringenin
Quercitrin
Diosmin
Diosmetin
Hesperidin
Hesperetin
Myricetin Quercetin
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Aglycone
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Glycone
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Table 2. Minimum inhibitory concentration of norfloxacin and ethidium bromide in the
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absence and presence of flavonoids (at ¼ MIC) against the Staphylococcus aureus strain
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that overexpresses the norA gene (S. aureus SA-1199B).
ethidium bromide
128 32 (4)* 128 32 (4)* 128 32 (4)* 128 8 (16)* 128 128 32 (4)* 128 64 (2)*
32 8 (4)* 16 (2)*
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Alone +Naringenin +Naringin +Phloretin +Phloridzin +Diosmetin +Diosmin +Hesperetin +Hesperidin +Myricetin +Myricitrin +Quercetin +Quercitrin
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268 269
32 16 (2)* 16 (2)* 32
* fold reduction in MIC
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Fruit flavonoids presented weak or no inhibitory effect against S. aureus;
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Aglycone forms and 3-O-glycoside flavonoids modulated the norfloxacin resistance;
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Structural characteristics influenced the modulatory effects of flavonoids;
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Some fruit flavonoids might be promising resistance-modifying agents.
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S0023-6438(16)30187-6
DOI:
10.1016/j.lwt.2016.04.003
Reference:
YFSTL 5391
To appear in:
LWT - Food Science and Technology
Received Date: 18 December 2015 Revised Date:
29 March 2016
Accepted Date: 1 April 2016
Please cite this article as: Diniz-Silva, H.T., Magnani, M., de Siqueira, S., de Souza, E.L., de SiqueiraJúnior, J.P., Fruit flavonoids as modulators of norfloxacin resistance in Staphylococcus aureus that overexpresses norA, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Fruit
flavonoids
as
modulators
of
norfloxacin
2
Staphylococcus aureus that overexpresses norA
resistance
in
3
Running title: Antibiotic resistance modulatory effects of fruit flavonoids
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Helena Tainá Diniz-Silva1, Marciane Magnani2, Sílvia de Siqueira1, Evandro Leite de
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Souza3, José Pinto de Siqueira-Júnior1
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Laboratório de Genética de Microrganismos, Departamento de Biologia Molecular,
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Universidade Federal da Paraíba, João Pessoa, Paraíba, Brazil
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2
12
Alimentos, Universidade Federal da Paraíba, João Pessoa, Paraíba, Brazil
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3
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Federal da Paraíba, João Pessoa, Paraíba, Brazil
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Laboratório de Microbiologia de Alimentos, Departamento de Nutrição, Universidade
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Laboratório de Processos Microbianos em Alimentos, Departamento de Engenharia de
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1
ACCEPTED MANUSCRIPT Abstract
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This study investigated whether different fruit flavonoids are capable of modulating
27
norfloxacin (Nor) resistance in S. aureus SA-1119B, an S. aureus strain that overexpresses
28
norA. The minimum inhibitory concentration (MIC) of flavonoids and norfloxacin (Nor)
29
were determined using broth microdilution tests. To assess the antibiotic resistance
30
modulatory effect, the MIC of Nor was measured in growth media with or without
31
flavonoids at a sub-MIC level. The lowest MIC (256 µg/mL) against S. aureus SA-1199B
32
was observed for myricetin; all other tested flavonoids produced MICs > 256 µg/mL.
33
When tested at sub-MIC levels in combination with Nor, hesperetin reduced the MIC of
34
Nor from 128 µg/mL to 8 µg/mL (16-fold), whereas phloretin, diosmetin, and myricitrin
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reduced the MIC of Nor from 128 µg/mL to 32 µg/mL (4-fold), and quercitrin reduced the
36
MIC of Nor from 128 µg/mL to 64 µg/mL (2-fold). No modulatory effects were observed
37
for flavonoids with 7-O-glycoside residue or for non-glycosylated forms of 3-O-glycoside
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flavonoids. These results suggest that fruit flavonoids in aglycone forms or 3-O-glycoside
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flavonoids are capable of modulating Nor-resistance in S. aureus that overexpresses NorA.
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Key-words: flavonoids, glycosylation, Staphylococcus spp., resistance-modifying agents.
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1. Introduction
Flavonoids are the most abundant polyphenols present in plant foods. They are
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generally defined as dietary antioxidants, and these nonessential nutrients have been
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established as bioactive compounds that benefit human health (Corcoran, McKay, &
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Blumberg, 2012; Choi, Islam, Ali, Ji Kim, Kim, & Jung, 2014). Characterized by a basis
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backbone composed of 15 carbon atoms (C6 - C3 - C6), flavonoids are organized into ten
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different groups (Cushnie & Lamb, 2011; Xiao, Muzashvili, & Georgiev, 2014). These 2
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rhamnosides, arabinosides and rutinosides. The flavonoid glycosides mainly occur as C-3
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or C-7 O-glycosides, but the C-4, C-5, C-6, and C-8′ positions are sometimes glycosylated
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as well. In some fruit, e.g., apples, flavonoids are usually present in the C-3 position of an
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O-glycosylated form (Cushnie & Lamb, 2011; Barreca, Bellocco, Laganà, Ginestra, &
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Bisignano, 2014). Differences in molecular structure have been commonly related to
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specific biological properties of compounds that comprise the flavonoid group (Choi et al.,
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2014).
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Fruit flavonoids have a variety of biological properties, including antioxidant, anti-
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inflammatory, antidiabetic, hepatoprotective, antiviral and antimicrobial effects (Céliz,
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Daz, & Audisio, 2011; Barreca, Belloco et al., 2014). Some flavonoids widely found in tea,
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such as cathecins, have shown the ability to restore antibiotic sensitivity in methicillin-
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resistant Staphylococcus aureus (MRSA) strains because these compounds possess an
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affinity for binding to bacterial proteins that are involved in the resistance of methicillin or
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other drugs (Hatano et al., 2005, Abreu, McBainband, & Simões, 2012).
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Reports of infections caused by multidrug-resistant S. aureus have increased in
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recent years, especially in developing countries (WHO, 2014). Compounds with the ability
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to enhance the efficacy of existing antibiotics are called modifiers of resistance (resistance-
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modifying agents, RMAs), and discovering new RMAs is considered a strategy to combat
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antibiotic resistance in S. aureus (Gibbons, Oluwatuyi, Veitch, & Gray, 2003; Chan et al.,
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2013). RMAs could provide effective tools to control S. aureus resistance in clinical
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practice, which would make possible the use of a reduced set of classical antibiotics and
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reducing the costs of antibiotic therapy (Abreu, McBainband, & Simões, 2012).
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Studies have reported that some flavonoids are effective at inhibiting S. aureus
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MRSA by acting either directly or by restoring the antibacterial efficacy of antibiotics; this 3
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study aimed to evaluate the modulatory effects of glycosylated and aglycone forms of fruit
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flavonoids on norfloxacin (Nor) resistance in S. aureus that overexpresses the norA gene.
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2. Materials and methods
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2.1 Nor, ethidium bromide and fruit flavonoids
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Nor, flavonoids and ethidium bromide (EtBr) were obtained from Sigma-Aldrich
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(purity ≥ 98%; St. Louis, USA). A stock solution of Nor was prepared in a mixture of 1 M
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NaOH and sterile distilled water (1 : 9 proportion) according to a procedure described
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elsewhere (CLSI, 2012). The stock solution of EtBr was prepared in distilled water. Test
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flavonoids (Table 1) were dissolved in a mixture of DMSO and sterile distilled water (4 :
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1). DMSO at its highest final assayed concentration after dilution in broth (4%) did not
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inhibit bacterial growth.
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2.2 Test strain and growth conditions
S. aureus SA-1199B encoding the NorA efflux protein, which was kindly
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provided by Dr. S. Gibbons (University of London – UK), was used as the test strain. The
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stock cultures were maintained in Brain Heart Infusion (BHI) broth (Himedia, India)
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containing glycerol (15 mL/100 mL) at -20 °C. To obtain the inoculum, the strain was
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grown in BHI broth at 37 °C for 18 - 20 h (until the late exponential growth phase). Then,
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cells were harvested by centrifugation (4500 g, 15 min, 4 °C), washed twice in sterile
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saline solution (NaCl 0.85% w/v) and resuspended to obtain standard cell suspensions. The
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OD reading at 660 nm of cell suspension was 0.1, and it provided viable cell counts of
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approximately 8 log cfu/mL when the sample was pour-plated in BHI agar (Himedia,
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India).
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2.3 Determination of minimum inhibitory concentration (MIC) The MIC of the different flavonoids was determined using broth microdilution tests
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following a standard procedure (CLSI, 2012). The 96-well plates were prepared by
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dispensing 180 µL of Mueller Hinton broth (Himedia, India) containing 1.8 µL of bacterial
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suspension (8 log cfu/mL). Then, 20 µL of the solution of each flavonoid tested (final
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concentration varying from 1024 to 0.25 µg/mL) was added to each well. Each plate
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included positive controls without flavonoids. The plates were statically incubated at 37 °C
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for 24 h. After the incubation period, the MIC value was defined as the lowest
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concentration (µg/mL) of each flavonoid that was capable of visually inhibiting the growth
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of the test bacterial strain.
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2.4 Assessment of antibiotic resistance modulatory effect
The “modulation assay” was used to evaluate the effects of the flavonoids on Nor
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resistance in S. aureus SA-1199B (Gibbons et al., 2003). The MIC of Nor was determined
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in growth media containing and not containing flavonoids at a sub-MIC level (1/4 MIC)
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using the broth microdilution test as previously described (CLSI, 2012). To determine
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whether Nor resistance modulation involved the inhibition of the NorA-efflux pump, the
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MIC of EtBr (indicator of NorA efflux pump inhibition; Gomes et al., 2011) was
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determined in growth media containing or not containing each of the tested flavonoids.
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EtBr is a well-known substrate for efflux proteins, and active efflux is the only known
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mechanism of resistance to this DNA-intercalating dye (Kummar et al., 2008).
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A positive resistance modulatory effect was demonstrated when the incorporation
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of the test flavonoid into bacteria growth media decreased the baseline MIC of Nor or EtBr
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(Gibbons et al., 2003; Stavri, Piddock, & Gibbons, 2007).
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2.4 Statistical analysis The assays were performed in triplicate in three separate experiments. The MIC
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results are expressed as the modal values because the values of the repetitions were the
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same.
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3. Results and discussion
The lowest MIC (256 µg/mL) against S. aureus SA-1199B was observed for
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myricetin (aglycone form); all other flavonoids produced MICs > 256 µg/mL, suggesting a
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weak inhibitory effect (Van Vuuren, 2008). Similarly, the aglycone named narigenin was
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previously found as more effective than its glycosylated form to inhibit S. aureus (Han &
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You, 1988). The attachment of the hydrophilic substituent, 7-O-glycoside, onto the
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flavonoid molecule has been suggested to reduce its interaction with the target bacteria due
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to a lack of affinity with the phospholipid bi-layer or specific receptors located on the cell
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membrane (Gomes et al., 2011). The antimicrobial efficacy of the di-glycosides naringin,
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neohesperidin and neoeriocitrina and their aglycone forms (naringenin, hesperetin and
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eriodictyol, respectively) against Listeria innocua and S. aureus was already verified
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(Mandalari et al., 2007). In these studies, some aglycones were more effective at inhibiting
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both target bacteria than their respective di-glycosides forms. On the contrary, similar
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inhibitory effects of flavonoids in either glycosylated (e.g., naringin and prunin) or
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aglycone (e.g., naringenin) forms against S. aureus have been demonstrated (Céliz, Daz, &
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Audisio, 2011). These results are in agreement with our findings for phloridzin, hesperidin,
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diosmin, quercitrin, naringin and their respective aglycone forms. Therefore, in addition to
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the glycosylation, other aspects of the molecular structure of flavonoids, such as
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hydroxylation, methoxylation and alkylation, may be involved in their efficacy to inhibit
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different bacteria (Gomes et al., 2011; Xiao et al., 2014).
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diosmetin at a sub-MIC level decreased the baseline MIC of Nor from 128 to 32 µg/mL (4-
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fold) against S. aureus SA-1199B, whereas hesperetin decreased the baseline MIC of Nor
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from 128 to 8 µg/mL (16-fold) (Table 2). The incorporation of a sub-MIC of the 7-O-
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glycoside forms of phloridzin, naringin, diosmin or hesperidin into growth media did not
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decrease the MIC of Nor against S. aureus SA-1199. These findings that the aglycones are
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able to modulate Nor-resistance is in accordance with an earlier study that verified that
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acacetin (aglycone flavonoid) modulated Nor-resistance in S. aureus SA-1199B (Gomes et
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al., 2011). Still, the ability of the aglycone diosmetin to modulate resistance to the
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fluoroquinolone ciprofloxacin in the same strain has already been reported (Chan et al.,
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2013).
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Myricitrin and quercitrin (which possess the 3-O-glycoside residue) at sub-MIC
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levels decreased the MIC of Nor from 128 to 32 µg/mL (4-fold) and from 128 to 64 µg/mL
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(2-fold decrease) against S. aureus SA-1199B, respectively (Table 2). Otherwise, the
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incorporation of the aglycone forms of quercetin and myricetin into growth media did not
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decrease the MIC of Nor. Similarly, an earlier study reported a decrease of MIC of
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ciprofloxacin from 16 µg/mL to 1 µg/mL (16-fold) against S. aureus SA-1199B when the
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3-O-glycoside flavonoid kaempferol at a sub-MIC level was incorporated into growth
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media (Falcão-Silva, Silva, Souza, & Siqueira-Júnior, 2009).
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Flavonoids are commonly present in fruits as glycoside conjugates, and structural
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characteristics (e.g., presence of aromatic ring or numbers of hydroxyl and methoxyl
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groups) may influence their effects on membrane permeability and affinity to target sites in
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microbial cells (Mandalari et al., 2007; Cushnie & Lamb, 2011). Because flavonoids
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without a 7-O-glycoside residue and with a 3-O-glycoside residue modulate the Nor
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resistance in S. aureus SA-1199B, further investigations into whether these effects were 7
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against S. aureus SA-1199B decreased from 32 µg/mL to 8 µg/mL (4-fold) when
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naringenin was incorporated into growth media at sub-MIC level (Table 2). Decreases in
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MIC of EtBr from 32 µg/mL to 16 µg/mL (2-fold) were verified when a sub-MIC level of
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phloretin, hesperetin or myricitrin was incorporated into growth media (Table 2). These
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decreases in the MIC of EtBr suggest that the putative modulation of Nor-resistance in S.
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aureus SA-1199B involved a disturbance in the function of the NorA gene (Falcão-Silva,
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Silva, Souza, & Siqueira-Júnior, 2009). However, quercitrin (3-O-glycoside form) and
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diosmetin (aglycone) decreased the MIC of Nor but not the MIC of EtBr, suggesting that
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these flavonoids may modulate Nor-resistance in S. aureus SA-1199B by other
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mechanisms. Interestingly, the inhibition of topoisomerase, a well-known target of
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quinolones (Monte et al., 2014), e.g., norfloxacin, has also been suggested to participate in
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the antibiotic resistance modulatory activity of flavonoids. Considering that a specific
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mechanism does not fully account for the resistance modulatory activity of flavonoids, it
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has been proposed that these compounds may exert these effects through multiple
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mechanisms (Cushnie & Lamb, 2011).
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4. Conclusions
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The results of this study demonstrate that fruit flavonoids in aglycone forms or 3-O-
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glycoside flavonoids can modulate Nor-resistance in S. aureus that overexpresses norA by
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acting as RMAs through the inhibition of the efflux system and/or other mechanisms
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involved in Nor-resistance.
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Acknowledgements
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The authors would like to thank Prof. Simon Gibbons (University of London, UK)
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for his valuable and kind cooperation, CNPq (Brazil) for the financial support and CAPES
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(Brazil) for a scholarship awarded to the first author (H.T. Diniz-Silva).
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References
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Table 1. Chemical classification of fruit flavonoids tested to evaluate the Nor-resistance
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modulatory effects in Staphylococcus aureus strain that overexpresses the norA gene (S.
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aureus SA-1199B). Fruit flavonoids 7-O-glycoside
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3-O-glycoside Aglycone
Glycone
Phloridzin
Phloretin
Myricitrin
Naringin
Naringenin
Quercitrin
Diosmin
Diosmetin
Hesperidin
Hesperetin
Myricetin Quercetin
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Aglycone
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Table 2. Minimum inhibitory concentration of norfloxacin and ethidium bromide in the
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absence and presence of flavonoids (at ¼ MIC) against the Staphylococcus aureus strain
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that overexpresses the norA gene (S. aureus SA-1199B).
ethidium bromide
128 32 (4)* 128 32 (4)* 128 32 (4)* 128 8 (16)* 128 128 32 (4)* 128 64 (2)*
32 8 (4)* 16 (2)*
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32 16 (2)* 16 (2)* 32
* fold reduction in MIC
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Fruit flavonoids presented weak or no inhibitory effect against S. aureus;
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Aglycone forms and 3-O-glycoside flavonoids modulated the norfloxacin resistance;
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Structural characteristics influenced the modulatory effects of flavonoids;
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Some fruit flavonoids might be promising resistance-modifying agents.
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