Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureus

Accepted Manuscript Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureus Sara Silva , Eduardo M. Costa , Mª Rosário Costa , Miguel F. Pereira , Joana O. Pereira , José C. Soares , Prof. Dr. Mª Manuela Pintado PII:

S0956-7135(14)00683-5

DOI:

10.1016/j.foodcont.2014.11.040

Reference:

JFCO 4192

To appear in:

Food Control

Received Date: 16 September 2014 Revised Date:

18 November 2014

Accepted Date: 25 November 2014

Please cite this article as: Silva S., Costa E.M, Costa M.R., Pereira M.F, Pereira J.O, Soares J.C & Pintado M.M., Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureus, Food Control (2015), doi: 10.1016/j.foodcont.2014.11.040. 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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Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureus

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Sara Silvaa, Eduardo M Costaa, Mª Rosário Costaa, Miguel F Pereiraa, Joana O Pereiraa, José C Soaresa and Mª Manuela Pintadoa*

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Correspondence

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Prof. Dr. Mª Manuela Pintado, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto, Portugal. E-mail: [email protected]; Phone: +351225580097; Fax: +351225090351

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CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Arquiteto Lobão Vital, Apartado 2511, 4202-401 Porto, Portugal.

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Abstract

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Staphylococcus aureus has been established has one of the most common pathogens

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causing nosocomial infections worldwide and

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outbreaks related to the consumption of contaminated foodstuffs. More recently, the

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discovery of methicillin resistant S. aureus (MRSA) in raw and processed foods

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increased the risk factor associated with S. aureus associated foodborne diseases and

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has led to the search for new sources of antimicrobial agents. Therefore the aim of this

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study is to evaluate, for the first time, the impact of Vaccinium corymbosum L., fruit and

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leaf, infusions and decoctions upon methicillin resistant (MRSA) and sensitive S.

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aureus (MSSA). In order to accomplish these objectives V. corymbosum extracts were

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characterized and inhibition halos, inhibitory concentrations, impact upon enzymatic

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activity and biofilm formation were assessed. The results obtained showed that major

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compounds present in V. corymbosum constitution were quercetin-3-glucoside,

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chlorogenic and cafeic acids. MRSA and MSSA growth was inhibited at 12.5 mg/mL,

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for leaf, and 50 mg/mL, for fruit, and sub-MIC concentrations presented inhibition

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percentages as high as 3 log of viable cells and 47% of biomass. Furthermore DNase

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and coagulase were also inhibited at sub-MIC concentrations of the extracts. The results

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obtained imply an effective antibacterial and antibiofilm activity of these extracts

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towards MRSA and MSSA, thus revealing an interest potential for application in the

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food industry either as a functional ingredient or a preservative.

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also playing a role in for several

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Keywords

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Vaccinium corymbosum, MRSA, MSSA, antibiofilm, enzymatic activity

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ACCEPTED MANUSCRIPT Introduction

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Bacterial contamination of food-contact surfaces is a major factor of pathogen

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persistence in food processing environments and, with the increase of food-borne

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diseases in industrialized countries, it is believed to have a significant public health

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impact (Bridier et al.).Throughout the food chain, wet surfaces provide a substrate for

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the development and persistence of bacterial ecosystems called biofilms, which may

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contain pathogenic microorganisms. In fact, most of the pathogenic microorganisms

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involved in food-borne diseases are able to adhere to and grow on food surfaces,

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equipment and processing environments and form biofilms. Therefore, the existence of

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pathogenic bacteria on foods and food-contact surfaces increases the safety risk, in fact

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several potentially pathogenic microorganisms, among which is Staphylococcus aureus,

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have already been isolated from biofilms established in dairy, egg and seafood

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processing industries (Meira, Barbos, Athayde, de Siqueira, & de Souza, 2012; Sharma

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& Anand, 2002; Shi & Zhu, 2009). Furthermore, as biofilms have greater resistance to

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the environmental stress than their planktonic counterparts, they are more resistant to

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sanitizers, trait that is also true for bacterial aggregates that are detached from it. (Fux,

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Wilson, & Stoodley, 2004; Meira et al., 2012; Spoering & Lewis, 2001). Considering

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the increased resistance of biofilms and the potential risk they represent for the food

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industry, the mechanisms of microbial biofilm formation and control in food-processing

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have become a hot topic in the past several years (Shi & Zhu, 2009).

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Staphylococcus aureus, though typically a commensal microorganism, has been

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regularly found, worldwide, in surfaces of food processing plants responsible for

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outbreaks of foodborne diseases (Meira et al., 2012; Normanno et al., 2007; Nostro,

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Bisignano, et al., 2001; Nostro et al., 2012). Food poisoning caused by S. aureus

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depends on several factors among which is the ability of the strain to survive in/on a

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colonized substrate and effectively infecting the host or its capacity to produce

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extracellular substances (virulence factors) that aid in the colonisation and survival of

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the microorganisms, such as gelatinase, hemolysins, coagulase, DNase, lipases, catalase

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and enterotoxins (Kamal, Bayoumi, & Abd El Aal, 2013; Pastoriza, Cabo, Bernárdez,

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Sampedro, & Herrera, 2002; Peacock et al., 2002; Pereira et al., 2009; Sandel &

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McKillip, 2004). Among S. aureus, methicillin-resistant strains (MRSA), have emerged

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as a potential public health risk due to their lack of response to antimicrobials, fact that

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ACCEPTED MANUSCRIPT is accentuated, by the rising prevalence of MRSA in ovine and bovine food products

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(Crago et al., 2012; Graveland et al., 2012; Normanno et al., 2007).

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Natural antimicrobials stand as an interesting solution for the treatment/prevention of

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biofilm formation upon foodstuffs and processing surfaces. Particularly extracts

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produced using edible plant tissues, as their toxicity is perceived as almost inexistent.

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Vaccinium corymbosum’s fruits (blueberries) and leaves have long since been reported

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as being rich in phenolics (particularly anthocyanins and phenolic acids), compounds

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that are known for their antimicrobial potential (Riihinen, Jaakola, Kärenlampi, &

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Hohtola, 2008; Silva, Costa, Pereira, Costa, & Pintado, 2013). In fact, several authors

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have demonstrated the antimicrobial potential of V. corymbosum’s extracts against

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potentially pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas

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aeruginosa, Bacillus cereus, Listeria innocua, Salmonella enteritidis, Enterococcus

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faecalis, Citrobacter freundii, Escherichia coli, Salmonella typhymurium and Listeria

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monocytogenes (Burdulis et al., 2009; Deng et al., 2014; Silva et al., 2013). However,

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most authors report inhibition of cells in planktonic state disregarding their potential

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impact upon sessile cells (biofilms) and upon virulence factors. As phenolics are known

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for their interactions with proteins (that can be mediators of cellular adhesion or act as

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virulence factors) it is possible that their antimicrobial potential goes beyond their

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capacity to interfere with cellular growth (Naczk, Grant, Zadernowski, & Barre, 2006;

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Rawel, Czajka, Rohn, & Kroll, 2002). As such, the aim of this work was to characterize,

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for the first time, the antimicrobial and antibiofilm potential of blueberry leaf and fruit

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extracts against methicillin resistant and methicillin sensitive Staphylococcus aureus

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and assess their capacity to inhibit several staphylococcal associated virulence factors.

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2. Materials and methods

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2.1. Sample origin and extracts

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A commercially available infusion of Vaccinium corymbosym L leaves (Mirtilusa, Sever

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do Vouga, Portugal) was powdered and used to prepare the leaf extracts. While blueberries, collected from V. corymbosym L bushes grown in Sever do Vouga, were

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kindly provided by the producer’s association Mirtilusa (Sever do Vouga, Portugal).

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Blueberries were frozen, dried using a food dehydrator N3040 (Tellier SA, France) and

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powdered using an appliance mill.

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The extracts were produced as described by (Silva et al., 2013). Briefly, fruit (5% (w/v)

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and leaf (2% (w/v) decoctions were made by heating each mixture to 100 °C for 15 or

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30 min, respectively. To prepare leaf (2% (w/v)) and fruit (5% (w/v)) infusions, water

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(100 °C) was added to the samples and stirred for 45 or 15 min, respectively. All

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extracts were freeze dried (Heto Drywinner, Cambridge Biosystems, Cambridge, United

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Kingdom) and the powder yield was calculated.

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2.2. Compound identification

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Putative phenolic compound identification was carried out using a HPLC-DAD system

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(Waters Series 600, Mildford MA, USA) measuring, in 2 nm intervals, from 200 to 600

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nm. Separation was adapted from Oliveira, Almeida, and Pintado (2013), using the

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gradient described in Table 1, an injection volume of 40 µL and analysis of the spectra

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obtained at 320 nm (phenolic acids) and 520 nm (anthocyanins). Several pure standards

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were used, delphinidin-3-O-glucoside chloride, peonidin-3-O-glucoside chloride,

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cyanidin-3-O-arabinoside chloride, cyanidin-3-O-galactoside chloride; cyanidin-3-O-

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glucoside

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chloride, hydroxycinnamic acid – (Extrasynthese, Lyon, France), petunidin-3-O-

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glucoside chloride – (Polyphenols, Sandnes, Norway), delphinidin-3-O-galactoside

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chloride – (Applichem, Darmstadt, Germany), chlorogenic, caffeic, gallic, ferulic,

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protocatechuic and p-coumaric acids, myricitrin, quercetin-3-D-galactoside, quercetin-

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3-D-glucoside – (Sigma, St. Louis, USA). Quantification was performed using

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calibration curves of the presumed compounds. Three independent analyses were

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performed for each of the triplicate extracts obtained per treatment.

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2.3. Microorganisms

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chloride;

malvidin-3-O-galactoside

chloride,

malvidin-3-O-glucoside

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ACCEPTED MANUSCRIPT Two isolates of S. aureus were used namely, MRSA LMG 15975 and Methicillin

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Sensitive Staphylococcus aureus (MSSA) ATCC 6538.

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2.4 Well diffusion assay

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Initial screening of antimicrobial activity was performed by well diffusion assay.

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Twenty milliliter Muller-Hinton agar (MHA) (Biokar Diagnostics, Beauvais, France)

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plates were seeded with a 0.5 McFarland scale bacterial suspension. Wells with 4 mm in

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diameter were punctured into the plates and filled with 40 µL of an aqueous saturated

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solution of the freeze dried powders (0.5 or 0.25 g/mL solution of freeze dried powder

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for fruit and leaf respectively). Plates were then incubated for 24 h at 37 °C. All assays

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were done in triplicate.

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2.5 Minimum inhibitory and bactericidal concentration

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The minimum inhibitory concentration (MIC) determination was performed according

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to M07-A8 (2009). Test solutions at 25, 12.5 and 6.25 mg/mL (for fruit extracts 50

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mg/mL was also tested) were prepared, inoculated at 1% (v/v) with an inoculum of ca.

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108 CFU/mL and incubated for 24 h at 37 °C. The MIC was determined by observing

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the lowest concentration of extract that completely inhibited bacterial growth. The

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minimum bactericidal concentration (MBC) was determined as described by Costa,

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Silva, Pina, Tavaria, and Pintado (2012). Briefly, MIC and higher concentrations were

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plated in plate count agar (PCA) (Biokar Diagnostics, Beauvais, France) and incubated

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at 37 °C. The MBC concentration was considered when no bacteria were detected after

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24 h incubation. Sterile MHB was used as negative control. All assays were done in

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triplicate.

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2.6 Growth inhibition curves

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Solutions, at MIC concentration, were prepared and inoculated at 1% (v/v) using an

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inoculum of ca. 108 CFU/mL and incubated at 37 ºC for 24 h. Viable counts were

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determined at 0, 0.5, 1, 2, 4, 7, 12 and 24 h, using decimal dilutions and plating using

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the drop method as described by Miles, Misra, and Irwin (1938). A positive control was

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drawn, for comparison purposes, using inoculated MHB without extract. The PCA

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plates used for the viable counts were then incubated for 24 h at 37 °C. Results were

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given by plotting the log CFU versus time. All assays were done in quadruplicate.

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ACCEPTED MANUSCRIPT 2.7. Biofilm production

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2.7.1. Biomass determination

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Microtiters for biofilm assessment were prepared using solutions at sub-MIC

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concentrations (half, one fourth and one eighth of the MIC – 25, 12.5 and 6.25 mg/mL

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for fruit extracts and 6.25, 3.125 and 1.56 mg/mL for leaf extracts). The solutions were

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prepared by diluting the powder in Tryptic soy broth (TSB) (Biokar Diagnostics,

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Beauvais, France) supplemented with 1% (w/v) glucose (Sigma, St. Louis, USA).

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Biomass was determined as described by Costa et al. (2014). Briefly, the test solutions

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were inoculated at 2% (v/v) using an overnight inoculum (ca. 108) and transferred into

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96 wells microtiters (Nunc, Darmstad, Germany). The plates were incubated for 48 h at

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38 °C. Afterwards, the contents of the plate were discarded, each well was carefully

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washed to remove non-adhered cells and the biofilms were stained using crystal violet.

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All assays were done in triplicate a positive control was drawn using inoculated culture

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media and a negative control was prepared using TSB with 1% (w/v) glucose. The

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results were given in biomass formation inhibition percentage, calculated according to

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the following formula:

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% biomass formation inhibition = 100 – (ODassay/ODpositive control) x 100

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2.7.2. Viable cell determination

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Biofilm embedded bacteria were quantified through the adaptation of the method

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described by Costa, Silva, Tavaria, and Pintado (2013). Briefly, the biofilm assay was

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performed as described in the previous section (2.7.1.) up until the removal of non-

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adhered cells. At this point the content of each well was scraped and suspended in 200

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µL of peptone water and, after serial dilutions, the total viable counts were determined

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by the drop method as described by Miles et al. (1938). A positive control was

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evaluated using inoculated TSB with 1% (w/v) glucose a negative control was drawn

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using only the culture media. All assays were done in triplicate. Results were given in

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biofilm formation inhibition percentage, calculated through the following formula:

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% biofilm formation inhibition = 100 – (CFUassay/CFU positive control) x 100

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2.8. Enzymatic activity (DNase and coagulase)

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The powdered extracts were dissolved in TSB at sub-MIC concentrations (half, one

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fourth and one eighth of the MIC – 25, 12.5 and 6.25 mg/mL for fruit extracts and 6.25,

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ACCEPTED MANUSCRIPT 3.125 and 1.56 mg/mL for leaf extracts), plain or supplemented with 0.1% (w/v) of

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DNA sodium salt from calf thymus (Sigma, St. Louis, USA) for the DNase assay. The

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solutions were inoculated using a 108 CFU/mL overnight inoculum and incubated at 37

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°C. After 24 h, the samples were centrifuged at 1600g for 30 min (M-240 centrifuge,

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Boeco, Germany) and the supernatant was used to test the enzymatic activity (Fletcher,

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Parker, & Hassett, 1974; Nostro, Angela Cannatelli, Crisafi, & Alonzo, 2001).

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DNase production was assayed using DNase test agar (DTA) (Pronadisa, Madrid,

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Spain). Briefly, 4 mm wells were punctured into 20 mL DTA plates and filled with 40

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µL of supernatant. Plates were then incubated at 37 °C for 24 h. The degradation halo

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was evaluated after the addition of 1 M HCl (Merck, Darmstadt Germany). A negative

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control was drawn using TSB supplemented with DNA. The tests were performed in

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quadruplicate (Nostro, Angela Cannatelli, et al., 2001).

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For the evaluation of coagulase activity, the supernatant was added to rabbit plasma

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(1:1) (Biokar Diagnostics, Beauvais, France) and incubated at 37 °C for 24 h to evaluate

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clot formation. A positive result was considered when a clot was observed. The negative

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control was drawn using water and the positive controls were prepared using inoculated

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TSB. The tests were performed in triplicate (Fletcher et al., 1974; Nostro, Bisignano, et

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al., 2001).

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2.9. Statistical analysis

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Statistical differences were evaluated using IBM SPSS Statistics 21 (New York, USA).

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Normality was determined using the Kolmogorov-Smirnov test. The differences were

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evaluated using One-way ANOVA and Scheffe’s (for normal distributions) or Mann-

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Whitney tests for the rest. Differences were considered significant when p ≤ 0.05.

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Correlations were determined using Spearman’s test and were considered significant for

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confidence values above 95%.

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ACCEPTED MANUSCRIPT Results and Discussion

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3.1. Extract characterization

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The main compound found in all extracts was chlorogenic acid ranging from 0.8 to 1.0

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µg/mg of freeze dried powder in fruit and 15.0 to 16.8 µg/mg of freeze dried powder in

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leaf extracts.

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In leaf there are two other predominant compounds, caffeic acid and quercetin-3-

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glucoside (Table 2). These results are in line with those previously reported by Kim,

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Shang, and Um (2010) who stated that chlorogenic acid was one of the main

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compounds found in blueberry. The presence of anthocyanins in leaf extracts could be

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somewhat unexpected but, previous work by Riihinen et al. (2008) showed that, despite

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not being present in fresh leaves, senescent leaves possessed anthocyanins. Considering

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that the leaves used in this work were collected post-harvest this a likely explanation for

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the presence of these compounds.

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When considering fruit extracts (Table 2) fewer compounds, and in lower

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concentrations, were identified. A possible explanation for this phenomenon is the

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processing procedure (drying of the fruits and heat present in the extraction) that may

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degrade the compounds, particularly the anthocyanins, leading to the lack of compounds

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identified (Eva Sadilova, Carle, & Stintzing, 2007; E. Sadilova, Stintzing, & Carle,

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2006). However, the lack of previous work focused on the chemical composition of dry

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blueberry fruit extracts does not allow for the comparison and validation of these

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results.

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3.2 Well diffusion assay

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The initial screening of the antimicrobial activity showed that both of the tested strains

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appeared to be sensitive to the extracts, with no differences being apparent between

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MRSA and MSSA (Figure 1). However, it is interesting to note that, while the

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extraction protocol appears to be inconsequential, statistically significant differences in

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activity were found between fruit and leaves. In this case, the overall phenolic content

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of the extracts appeared to be a determinant factor for the antimicrobial activity. Fact

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that is further supported by the strong correlations found between phenolic content and

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antimicrobial activity for both fruit and leaf extracts (0.832 and 0.765, respectively,

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values significant at the 0.01 level).

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The results observed stand in line with those reported by Burdulis et al. (2009) and

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Lacombe, Wu, White, Tadepalli, and Andre (2012) for Vaccinium corymbosum and

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angustifolium extracts which presented inhibition halos for several bacteria among

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which S. aureus.

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3.3 MIC and MBC determination

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The results for MIC and MBC determinations are displayed in Table 3 and they

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corroborate those found in the well diffusion screening, i.e. no significant differences

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were found between either the microorganisms or the extraction type but leaf extracts,

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as can be seen from Table 3, presented significantly lower MIC concentrations and

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were the only to present MBC values at the concentrations tested. Since leaf extracts are

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richer in phenolic compounds than fruits, it stands to reason that this difference is, at

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least, partially responsible for the differences observed. Additionally, the high sugar

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content of fruits (easily extracted by water) may help to reduce the activity observed for

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fruit extracts by either promoting bacterial growth or even direct interaction with the

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bioactive compounds (Sousa, Curado, Vasconcellos, & Trigo, 2007).

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When comparing our MIC results with those observed, for a Staphylococcus aureus (S.

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aureus) resistant to several antibiotics, by Nascimento, Locatelli, Freitas, and Silva

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(2000) it can be seen that our values are significantly lower. In fact of the 10 different

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extracts considered in that study (most rich in flavonoids, phenolic acids and tannins)

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the only one that exhibited activity against S. aureus, was an extract of jambolan leaves,

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and it had an MIC of 300 mg/mL (vs. the 50 and 25 mg/mL for fruit and leaves,

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respectively, registered for the studied blueberry extracts) (Nascimento et al., 2000).

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Furthermore, these results come in line with those reported in an earlier study (Silva et

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al., 2013) performed by this group, where these aqueous extracts showed significant

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activity against gram positive bacteria.

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3.4 Growth inhibition curves

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The analysis of the impact of the MIC upon microbial growth, over a 24 h period,

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showed that all extracts hampered microbial growth. As can be seen in Figure 2, for

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MSSA infusions (both fruit and leaf) were capable of producing a 3 log reduction after

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24 h while decocted leaf extracts caused a 2 log reduction over the same period.

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Decocted fruit extracts presented no significant decrease in viable counts levels but

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ACCEPTED MANUSCRIPT inhibited growth nevertheless. On the other hand, for MRSA, both leaf extracts lead to a

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3 log reduction after 12 h while fruit extracts showed no decrease of viable counts.

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Chlorogenic acid affects membrane fluidity and fatty acid profile (which may lead to

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membrane disruption) therefore its presence in the extracts may explain the reductions

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in total viable counts (Baba-Moussa et al., 2008; Riihinen et al., 2008). Additionally, the

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higher phenolic levels observed in leaf extracts may provide an explanation for the

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results observed for MRSA (higher inhibition). However, this does not explain the

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results observed for MSSA (higher activity of fruit infusion than decocted leaf),

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therefore demonstrating that other compounds (phenolic or not) may also play an

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important role explaining the results observed.

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3.5 Biofilm production

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In the biofilm assays, both microorganisms exhibited varying behaviours when in the

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presence of the different extracts (Figure 3 and Figure 4).

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Regarding the effects upon biofilm vitality/viability the results showed (Figure 3) that

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while all extracts were capable of significantly (p < 0.05) reducing MRSA biofilm

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viable counts, at ½ and ¼ of the MIC, for MSSA this effect was only observed at ½ of

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the MIC. Additionally, while the highest reduction of total viable counts registered for

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MRSA was above 3 log of CFU (ca. 99.9% reduction percentage), for MSSA it was

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only 2.6 log of CFU (ca. 99% reduction percentage). Significant differences were also

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found between leaf and fruit extracts with leaf infusions being, for both

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microorganisms, the only extract active at all tested concentrations. This is particularly

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evident for MRSA where leaf infusion presented reduction values, in average, 2×

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superior to those of the remaining extracts.

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When considering the effects upon biomass production (Figure 4) it is possible to see a

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clear difference between MRSA and MSSA, with biomass inhibition reaching as high as

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60% for the first and only 20% for the later. While this may indicate a strain depended

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effect, one has to consider that MSSA has been previously described a typically weaker

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biofilm producer (Croes et al., 2009). For MRSA, and contrary to what was observed in

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the previous assay, the highest inhibition percentages were obtained for fruit extracts

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with a reverse relation between extract concentration and inhibition percentage being

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observed. Inhibition percentages varied between 42 (½ of the MIC) and 60% (⅛ of the

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MIC) for fruit extracts and between 51% (½ of the MIC) and 22% (⅛ of the MIC) for

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ACCEPTED MANUSCRIPT leaf extracts. On the other hand, for MSSA inhibition percentages varied between 5%

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and 16% for fruit extracts and 25% and 6% for leaf extracts.

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When analysing these results several hypothesis arise as to potentially explain these

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behaviours. One possible explanation as to why MRSA was the preferred target of these

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extracts may be the simple fact that of the two, MRSA is a stronger biofilm former and

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therefore the effect may be felt more intensively than in MSSA (Croes et al., 2009;

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Kwon et al., 2008; Schlievert, Strandberg, Lin, Peterson, & Leung, 2010). As to why

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there were differences in activity between the extracts a possible explanation lays within

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their composition and in the higher concentration of phenolic compounds reported for

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the leaf extracts. Supporting this hypothesis we have the previous work of several

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authors which showed that individual phenolic compounds were capable of interfering

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with biofilm formation; Fiamegos et al. (2011) showed that chlorogenic acid was

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capable of inhibiting S. aureus biofilm formation while Luís, Silva, Sousa, Duarte, and

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Domingues (2013) showed that 4 mg/mL of gallic, caffeic and chlorogenic acids

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reduced biofilm biomass by 80%. In comparison, the results reported in this work are

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inferior to those registered by Luís et al. (2013) however, as they used pure compounds,

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free of interferents (such as the sugars present in the fruit extracts) and the

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concentrations of compounds tested were significantly higher than those present in the

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extracts, the comparison drawn between the results must be considered carefully.

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3.6 Enzymatic activity

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Staphylococcus aureus pathogenicity is greatly enhanced by its virulence factors that,

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ranging from toxins to adhesion factors, help S. aureus induce infections (Baba-Moussa

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et al., 2008). The results of the impact of extracts upon the enzymatic activity (Table 4)

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show that both strains were capable of producing DNase and coagulase. Overall,

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MRSA’s DNase activity suffered little to no inhibition but, for MSSA a significant

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reduction in DNase activity was found for all extracts, particularly at ½ and ¼ of the

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MIC.

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As for coagulase, only fruit extracts appeared to be capable of inhibiting clot formation

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in both MRSA and MSSA (at 25 and 12.5 mg/mL) while leaf extracts were only

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capable of inhibiting coagulase activity in MRSA and at 6.25 mg/mL (½ of the MIC).

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It is interesting to note that, contrary to the antimicrobial activity the extracts with

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higher phenolic content didn’t necessarily exhibit the highest inhibition of activity, thus

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biding to the enzymes active site or modifying its activity) or that the difference in

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phenolic profiles between fruits and leaves may result in different interaction (Riihinen

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et al., 2008).

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Previous reports regarding the effect of plant extracts upon extracellular enzymes and

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virulence factors of S. aureus showed that garlic extracts (1 mg/mL) inhibited coagulase

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and Nepeta cataria L. extracts completely inhibited DNAse activity of both MRSA and

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MSSA.

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concentrations, completely inhibited S. aureus DNAse and were capable of reducing

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coagulase activity (Nostro, Angela Cannatelli, et al., 2001; Nostro, Bisignano, et al.,

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2001). Furthermore, Tranter, Tassou, and Nychas (1993) showed that phenolic

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compounds are capable of interfering with various S. aureus extracellular enzymes. As

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such, the results reported in the present work are in sync with those reported by the

355

overall community for Vaccinium extracts and other plants.

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Conclusions

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Analyzing the results obtained for the aqueous blueberry extracts, it can be seen that the

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major phenolic compound present is chlorogenic acid, that all extracts were capable of

359

effectively inhibiting MRSA and MSSA growth, biofilm formation and enzymatic

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activity, with the latter two being inhibited at sub-MIC concentrations. As blueberry is

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rich in phenolic compounds and these compounds are known to interact with bacterial

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membranes (leading to their disruption) we hypothesize that this interaction will be

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responsible for most of the activity attributed to the studied extracts. As such, blueberry

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extracts show potential for usage by the food industry due to their capability to inhibit S.

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aureus (MRSA and MSSA) and its virulence factors.

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Acknowledgments

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The present work was supported by National Funds from FCT through project Pest-

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PE/EQB/LA0016/2013 and by funds of the QREN-ADI project 13736 “Myrtillus –

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Mirtilo com inovação”. Additionally, the author S. Silva would like to acknowledge

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FCT for her Ph.D. grant SFRH/BD/90867/2012.

Helichrysum

italicum

alcoholic

extracts,

at

sub-MIC

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Tables

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Table 1. Flow conditions for HPLC analysis. Time (min) Flow (mL/min) 0 - 60 0.65 60 – 65 0.5 65 – 70 0.5

% Solvent Aa 100 – 40% 40 – 90% 90 – 100%

% Solvent Bb 0 – 60% 60 – 10% 10 – 0%

a

Solvent A - formic acid, water and methanol (92.5:5:2.5); b Solvent B – methanol, water and formic acid (25:50:25) - under the following conditions: linear gradient starting at 0 to 60% solvent B in 60 min at 0.65 mL/min, 60 to 10% in 5 min at 0.5 mL/min and from 5 to 0% in 5 min

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Table 2. Concentration (µg/mg of freeze dried powder) of the main identified compounds present in the extracts.. Concentration (µg/mL) Fruit Leaf decoction infusion 1.27 ± 0.10 15.47 ±0.01 nd 1.06 ± 0.01 nd 12.18 ± 0.10 nd 6.01 ± 0.73 nd 0.29 ± 0.01 nd < 0.14 nd < 0.14 0.03 ± 0.0 nd 0.03 ± 0.0 nd

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Fruit infusions 1.05 ± 0.01 nd 0.43 ± 0.02 nd nd nd <0.02 0.04 ± 0.0 0.04 ± 0.0

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Leaf decoction 13.82 ± 0.04 1.00 ± 0.02 13.19 ± 0.20 5.13 ± 0.09 0.12 ± 0.0 < 0.12 < 0.12 nd nd

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Table 3. Minnimum inhibitory concentration for each of the extracts tested.

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Fruit infusions MRSA 50a MSSA 50a a, no MBC; b, MBC = 25 mg/mL

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Chlorogenic acid Neochlorogenic acid Caffeic acid Quercetin-3-glucoside Cyanidin-3-galactoside Cyanidin-3-arabinoside Peonidin-3-glucoside Delphinidin-3-galactoside Malvidin-3-glucoside nd, not detected

MIC (mg/mL) Fruit Leaf infusion Leaf decoction decoction 50a 12.5b 12.5b 50a 12.5b 12.5b

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Leaf decoction

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MSSA + -

14.1±0.5

-

16.3±1.0

14.3±1.0

-

16.5±0.6

21±1.4

+

14.3±1.0

14.5±0.3

-

14.3±0.5

16.6±0.5

-

16.5±0.5

15.9±0.6

+

16.5±0.4

14.2±0.8

-

+

17.5±0.6

18.4±0.9

+

+

17.0±0.3

22.5±1.9

+

+

15.3±0.5

16.5±0.6

-

+

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15.3±0.5

-

+

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-

+

17.6±0.8

17.5±0.6

+

+

16.7±0.6

21.4±1.0

+

+

not detected; +, with clot; -, without clot

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Coagulase

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Positive Control 25 mg/mL 6.25 mg/mL 3.125 mg/mL 25 mg/mL 6.25 mg/mL 3.125 mg/mL 6.25 mg/mL 3.125 mg/mL 1.56 mg/mL 6.25 mg/mL 3.125 mg/mL 1.56 mg/mL

DNase (halos in mm) MRSA MSSA 16.5±0.6 21±0.82

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Figure Captions

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Figure 1 – Inhibition of MRSA (■) and MSSA (■) growth as induced by the different

543

extracts (halo diameter given in mm).

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Figure 2 – Impact of the different extracts upon the total viable counts of MRSA and

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MSSA over a 24 h growth period.

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Figure 3. Viable counts values (A), values in log of CFU/ml, and biofilm formation

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inhibition (B), values in reduction of log of CFU/ml, for the different conditions of

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biofilm formation. 1/2 MIC – 12.5 mg/mL of fruit extracts and 6.25 mg/mL for leaf

551

extracts; 1/4 MIC – 6.25 mg/mL for fruit extracts and 3.125 and for leaf extracts; 1/8

552

MIC – 3.125 mg/mL for fruit extracts and 1.56 mg/mL for leaf extracts. Different letters

553

represent statistically significant values (p < 0.05).

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Figure 4. Biomass production inhibition in the presence of V. corymbosum fruit and

555

leaf extracts. All results are given in biofilm inhibition percentage. 1/2 MIC – 12.5 g L

556

of fruit extracts and 6.25 mg/mL for leaf extracts; 1/4 MIC – 6.25 mg/mL for fruit

557

extracts and 3.125 and for leaf extracts; 1/8 MIC – 3.125 mg/mL for fruit extracts and

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1.56 mg/mL for leaf extracts. Different letters represent statistically significant values (p

559

< 0.05).

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ACCEPTED MANUSCRIPT Fruit and leaf extracts are rich in phenolic compounds All extracts inhibit both MRSA and MSSA All extracts exhibit antibiofilm activity

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Enzymatic activity was affected by the presence of the extracts