Inhibition of Cronobacter sakazakii Adhesion to Caco-2 Cells by

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Inhibition of Cronobacter sakazakii adhesion to Caco-2 cells by commercial dairy powders and raw buttermilk Daniel Ripollés, Saidou Harouna, José Antonio Parrón, Irene Arenales, Miguel Calvo, María Dolores Pérez, and Lourdes Sánchez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04971 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Journal of Agricultural and Food Chemistry

Running Head: Inhibition of C. sakazakii adhesion to Caco-2 cells

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Inhibition of Cronobacter sakazakii Adhesion to Caco-2 Cells

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by Commercial Dairy Powders and Raw Buttermilk

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Daniel Ripollésa, Saidou Harounaa, José A. Parróna, Irene Arenalesb,

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Miguel Calvoa, María D. Péreza, Lourdes Sáncheza *

10 a

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Departamento de Producción Animal y Ciencia de los Alimentos. Facultad de

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Veterinaria. Instituto Agroalimentario de Aragón (IA2) (Universidad de Zaragoza-

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CITA), Zaragoza, Spain

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b

Universidad Tecnológica de Tehuacán. San Pablo Tepetzingo, Tehuacán, Puebla, México

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ACS Paragon Plus Environment


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ABSTRACT

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Cronobacter sakazakii is a foodborne pathogen that has been associated with

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severe infections mainly in neonates. The binding of this bacterium to the host cell

27

surfaces represents the first step in the pathogenesis of the disease. An ELISA-

28

based assay has been developed using a polyclonal antiserum against C. sakazakii

29

to determine its adhesion to Caco-2 cells. The antiserum used recognised many of

30

the outer membrane proteins of C. sakazakii. A positive correlation was found

31

between the absorbance values obtained by ELISA and the number of bacteria

32

adhered to cells determined by plate counting. The inhibitory effect on bacterial

33

adhesion to cells observed with some dairy products was significant and

34

concentration-dependent.. Commercial buttermilk caused the maximal reduction of

35

the adhesion percentage (33.0 ± 5.07) at the highest concentration assayed (20

36

mg/mL), followed by butter serum (31.9 ± 5.36), skim milk (30.4 ± 5.07) and raw

37

buttermilk (25.6 ± 3.80)26.6 ± 1.58). In some cases, significant differences (p < 0.05)

38

were found on the inhibition exerted by the different products evaluated. The results

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obtained in this study demonstrate that dairy products contain some componentsa

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mixture of molecules with the ability to inhibit the adhesion of C. sakazakii to Caco-2

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cells.

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Keywords: Cronobacter sakazakii, adhesion to Caco-2 cells, ELISA-based assay,

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dairy products, milk proteins

45 46

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Con formato: Fuente: Cursiva

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1. INTRODUCTION

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Cronobacter sakazakii is a foodborne pathogen that affects mainly to

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newborns,neonates, infants and immunocompromised adults causing necrotizing

50

enterocolitis, meningitis and sepsis. with unusual resistance to dry, heat and acid

51

stress.1 The foodborne majority of the outbreaks associated with this pathogen have

52

been related with contaminated powdered infant formula, which may have been

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contaminated during the processing, or through utensils and equipment used in the

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its preparation and administration of formula.2 The ability of C. sakazakii to form

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biofilms on abiotic surfaces contributes to its resistance, increasing the risk of cross-

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contamination.2 Milk components, mainly proteins, have been proved to contribute to

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biofilm formation in by Cronobacter species.33 Despite the low incidence of disease

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caused by C. sakazakii, the mortality rates are high, ranging from 40 to 80%.2

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makes necessary to develop strategies directed to avoid infections with C. sakazakii,

60

especially in susceptible individuals.

2

Con formato: Superíndice

This

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The pathogenicity and virulence mechanisms of C. sakazakii are still under

62

investigation and its knowledge is probably far from complete. Recently, Singh et al.4


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described an array of C. sakazakii virulence factors responsible for tissue adhesion,


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invasion and host cell injury. These factors include some outer membrane proteins

65

(OMPs), lipopolysaccharides, a heat stable toxin and an iron acquisition system.

66

Several in vitro infection models have been used to elucidate its the mode of

Con formato: Fuente: Sin Cursiva

67

adherence of C. sakazakii to host cell surfaces.4,55,6

Con formato: Fuente: Cursiva Con formato: Superíndice

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Up to now, several many epithelial cell lines have been used to study the


69

mechanisms of bacterial adhesion such as Hep-2, Caco-2, HT-29 and INT-407.6–87-9


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One of the most widely used is Caco-2, a human colon adenocarcinoma cell line,

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which expresses, after differentiation, several morphological and functional 3



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characteristics of normal enterocytes. after differentiation.9

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pathogenic bacteria to intestinal epithelial cells is the first step in the intestinal

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colonisation and requires their binding to specific receptors on the cell surface.10,1111

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One of the most common means of interaction are lectin-like structures, located on

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the surface of bacteria, that bind complementary to carbohydrates attached to

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proteins (glycoproteins) or lipids (glycolipids)(glycoproteins o glycolipids) present on

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the cell membrane. However, the adherence can be mediated by other different

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mechanisms, in which bacterial polysaccharides carbohydrates and host lectins or

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protein-protein interactions are involved.1212

The attachment of


Con formato: Superíndice Con formato: Superíndice


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Some components of bovine milk, such as oligosaccharides (OS) (mainly acidic

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OS), glycolipids and glycoproteins (lactoferrin, immunoglobulins, mucin, etc) have

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been reported to inhibit the adhesion of pathogens to host cells by competition with

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bacterial receptors surface molecules or with binding sitesreceptors on intestinal

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epitheliuma.13,1413,14 Therefore, those molecules could constitute a new therapeutic

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strategy

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glycosylated components contained in dairy products could reduce the percentage of


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bacteria adhered to cells in susceptible individuals, which could mitigate the severity


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of disease. Therefore, those molecules could constitute a new therapeutic strategy to

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fight against antibiotic-resistant bacteria.13

to

fight

against

antibiotic-resistant

bacteria.13Furthermore,

these

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Different assays have been used to study the adherence of bacteria to

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susceptible cell lines. Among the most used techniques are direct microscopic

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counting after staining,8

counting of detection of radiolabelled bacteria15 and plate


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counting..16 An enzyme-linked immunosorbent assay (ELISA) was developed by Ofek


95

et al.et al.17 by immobilizing cells on the bottom of microtiter plates and after

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incubatingon of bacteria with cells with bacteria, their adhesion was determined by

9

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using specific antiserum following the standard guidelines for ELISA. This method is

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a reproducible and easy to perform assay and itthat can be a good choice when the

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number of samples is high.18

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The research aim of this preliminary study was to evaluate the effect of several

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dairy products on the adhesion of C. sakazakii to Caco-2 cells. To study bacterial

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adhesion, set up a method based on an enzyme-linked immunosorbent assay with

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specific antiserum obtained against C. sakazakii was set up. to determine its

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adhesion to Caco-2 cells This method allowed us to evaluate the effect of several

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dairy products on C. sakazakii adhesion to cells.

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2. MATERIALS AND METHODS

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2.1. Chemicals

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Precast polyacrylamide gels at 10% (m/v) were from Bio-Rad Laboratories

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(Hercules, CA, USA). Prestained protein ladder was obtained from Thermo Fisher

110

Scientific (Waltham, MA, USA). Polyacrylamide gels (8-25%) and low molecular

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weight marker were from GE-Healthcare (Uppsala, Sweden). Peroxidase substrate,

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3,3’,5,5’-tetramethylbenzidine(TMB) was supplied by ZEULAB (Zaragoza, Spain).

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Specific primary antibodies obtained in rabbit against mucin-1, lactadherin and

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butyrophilin were kindly donated by Dr. Jan Trige Rasmussen from the Protein

115

Chemistry Laboratory (University of Aarhus, Denmark). Sheep antibodies against

116

bovine immunoglobulin G (IgG) were obtained from ICN Biomedicals (Irvine, CA,

117

USA). Rabbit antibodies against bovine lactoferrin were previously obtained in our

118

laboratory.19

119 120

Dulbecco´s Modified Eagle Medium (DMEM), heat inactivated fetal bovine serum, non-essential

amino-acids,

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

5


acid




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(HEPES), L-glutamine, trypsin-ethylenediaminetetraacetic acid (EDTA), antibiotics

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(penicillin-streptomycin) and amphotericin B were obtained from Gibco,

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Technologies Corporation, (Paisley, UK). Tryptic soy broth (TSB), tryptic soy agar

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(TSA) and polyvinylidene difluoride (PVDF) membranes were purchased from Merck

125

Millipore (Darmstadt, Germany). Yeast extract (YE) was from Oxoid (Basingstoke,

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UK). Bovine pancreatic RNase and DNase, N-lauroyl sarcosinate, bovine serum

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albumin (BSA), peroxidase-conjugated goat anti-rabbit IgG and peroxidase

128

conjugated donkey anti-sheep IgG were purchased from Sigma-Aldrich (St Louis,

129

MO, USA). Glycine (purity > 99%) was obtained from Panreac Química SLU

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(Barcelona, Spain). Horse serum was kindly supplied by the Veterinary Hospital of

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Zaragoza University (Zaragoza, Spain). BCA protein assay kit was obtained from

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Pierce Biotechnology (Rockford, IL, USA).

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2.2. Source and analysis of commercial dairy powders and raw milk

(Life

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Three commercial dairy powders obtained from pasteurized milk (72 ºC for 15 s)

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were assayed in this study: skim milk, butter serum and buttermilk. Skim milk was

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obtained in the process of producing anhydrous milk fat, after fat concentration to

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75% by centrifugation. Butter serum was collected after phase inversion of

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concentrated fat and subsequent melting and centrifugation. Buttermilk was obtained

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during butter manufacture by churning cream. All products were supplied as

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powders.

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Raw buttermilk was prepared from bovine milk supplied by a local dairy farm

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(Villacorona, El Burgo de Ebro, Spain). Bovine milk was warmed to 50 °C and

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separated into cream and skim milk with a cream separator model ARR-DES 125

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(Suministros Químicos Arroyo, Santander, Spain). Then, butter was produced by

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cream churning and in this process, buttermilk was released. Buttermilk was filtrated 6


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through cheese cloth to remove butter granules and finally, it was freeze-dried and

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stored at -20 °C until further analysis.

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The compositional values of commercial dairy powders were provided by

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suppliers, and those of raw buttermilk were obtained by infrared spectroscopy using

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a MilkoScan 4000 (Foss Electric, Hilleroed, Denmark).

151

2.3. Detection of glycoproteins in commercial powders and raw buttermilk by

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

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Western-blotting

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Commercial dairy powders and raw buttermilk were prepared at 30 mg/mL in

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Mmilli-Q water, dissolved (1:1) in 126 mM Tris-HCl, pH 6.8, containing 20% glycerol,

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4%

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bromophenol blue and treated at 100 °C for 5 min. Afterwards, each sample were

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applied to 10% polyacrylamide gels and running conditions were 180 V for 30 min.

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Gels were stained with Periodic Acid Schiff reagent (PAS) or Coomassie Blue R

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according to standard procedures. Prestained protein ladder was used in the

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electrophoresis.

sodium

dodecyl

sulphate (SDS),

10%

β-mercaptoethanol

and

0.02%

162

The presence of the glycoproteins mucin-1, lactadherin, butyrophilin, lactoferrin

163

and IgG in commercial powders and raw buttermilk was detected by Western-blotting

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using PVDF membranes, according to the procedure described by Benfeldt et al.et

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al.20

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2.4. Caco-2 cell culture

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Human colon carcinoma Caco-2 cells (TC7 clone, which expresses biochemical

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and functional characteristics very similar to those of differentiated enterocytes) were

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kindly provided by the Physiology Department of the Veterinary Faculty of Zaragoza

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University (Zaragoza, Spain). Cells were grown in Dulbecco´s Modified Eagle

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Medium (DMEM) supplemented with 20% (v/v) heat inactivated fetal bovine serum, 2

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mM L-glutamine, 1% (v/v) non-essential amino-acids, 25 mM 4-(2-hydroxyethyl)-1-

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piperazineethanesulfonic acid (HEPES), 1% (v/v) antibiotics (100 U/mL penicillin, 100

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µg/mL streptomycin) and 2.5 µg/mL amphotericin B. Cells were grown in 25 cm2

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tissue culture flasks and incubated at 37 °C in 5% CO2 until confluence.

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For the adhesion assays, Caco-2 cells were seeded at a density of 1.4 x 104 cells

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per well onto a 96 well microplate (TPP Techno Plastic, Trasadingen, Switzerland)

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and incubated 20-21 days to obtain differentiated Caco-2 cells.9

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medium was replaced every other day and the day before the adhesion assay the

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culture was replaced by a medium without antibiotic and amphotericin B.

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2.5. Culture of C. sakazakii

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The culture

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A freeze-dried culture of C. sakazakii (CECT 858) was supplied by the Spanish

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Type Culture Collection (CECT, Valencia, Spain). After reviving the bacteria following

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supplier instructions for the culture, they were stored at -80 °C in sterile

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cryopreservation vials. Working cultures were obtained by transferring a porous bead

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of stock culture into 10 mL of TSB with 0.6% YE, and incubating it at 37 °C for 24 h.

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Afterwards, a loop of the culture was seeded onto TSA with 0.6% YE and incubated

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at 37 °C for 240 h to obtain isolated colonies of C. sakazakii.

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2.6. Obtaining antiserum against C. sakazakii

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The procedure used to obtain antibodies against C. sakazakii was previously

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described by Sun et al.et al.21 An isolated colony was cultured in 400 mL of TSB at

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37 °C for 24 h. After cultivation, bacteria were separated from nutrient broth by

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centrifugation at 5000g for 15 min at 4 °C and the pellet washed twice with 20 mL of

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0.85% NaCl with centrifugation between each wash at the same conditions 8


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mentioned above. The pellet was dissolved in 5 mL of 0.85% NaCl and heated at 120

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°C during 30 min. After this treatment, the bacterial suspension was aliquoted and

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stored at –20 ºC until use. Antiserum against heat inactivated C. sakazakii was

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obtained in rabbits by a procedure previously described.19 Antiserum was stored at

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-20 ºC until use.

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2.7. Extraction of the outer membrane proteins of C. sakazakii

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Outer membrane proteinsOMPs were extracted from C. sakazakii according to

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the method described by Jaradat et al.et al.22 An isolated colony of C. sakazakii was

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incubated during 20 h at 37 ºC in 10 mL of TSB medium and after that period the

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suspension was centrifuged at 3400g for 10 min at 4 °C. The cells were incubated

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with 0.1 µg/mL of bovine pancreatic RNase and DNase in 20 mM MgCl2 during 10

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min at 37 ºC and then sonicated in a water bath model (Ultrasons, (JP Selecta,

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Abrera, Spain) at 300 W at intervals of 1 min (10 cycles). After sonication, the

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suspension was centrifuged at 5000g for 30 min at 4 °C. The supernatant was re-

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centrifuged at 29000g for 2 h at 4 ºC and the pellet obtained was dissolved with in

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2% (w/v) N-lauroyl sarcosinate for 30 min. After this incubation, the solution was

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centrifuged again at 29000g for 2 h at 4 ºC and the precipitate was rinsed with 20 mM

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Tris-HCl containing 2% SDS and re-centrifuged at the last conditions mentioned. The

213

pellet from this centrifugation obtained was resuspended in distilled water and stored

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at -20 ºC until use. The proteins extracted from the C. sakazakii outer membrane

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were analyzed by SDS-PAGE in 8-25% polyacrylamide gels, including a low

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molecular weight marker. The main immunogenic proteins of C. sakazakii membrane

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were detected by Western-blotting using the polyclonal antiserum obtained in this

218

study, following the procedure according to Franco et al.et al.23

9



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The proteins contained in the most intense electrophoretic bands were analysed

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analyzed by matrix-assisted laser desorption/ ionisation -time- of- flight mass

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spectrometry (MALDI-TOF MS) at the Proteomic Platform of the Barcelona Scientific

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Park (Barcelona, Spain).

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2.8. Adhesion assay to Caco-2 cells

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The method used for determining the adhesion of C. sakazakii to Caco-2 cells

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was a modification of the procedure described by Le Blay et al.et al.18 Differentiated

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Caco-2 cells, after 20-21 days of culture in 96-wells plate, were washed with 150 µL

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of phosphate buffered saline (PBS, consisting of 0.14 mM NaCl, 2.6 mM KCl, 8.1 mM

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Na2HPO4, 1.4 mM KH2PO4, pH 7.4) per well and incubated with 150 µL of 5% (w/v)

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bovine serum albumin (BSA) containing 0.05% (v/v) Tween 20 (BSA-T) for 90 min to

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avoid non-specific binding of C. sakazakii to the plate. Afterwards, Caco-2 cell

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monolayers were washed twice with PBS.

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To prepare bacterial suspension for the assays, an isolated colony of C.

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sakazakii was transferred to 10 mL of TSB-YE and incubated at 37 ºC for 20 h.

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Afterwards, the medium was centrifuged at 3400g for 10 min at 10 °C and the pellet

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washed twice in PBS. Bacteria were suspended and diluted with supplemented

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DMEM without fetal bovine serum, antibiotics and amphotericin B, to obtain

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suspensions of 104-108 CFU/mL (determined by plate counting). A volume of 50 µL of

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bacterial dilutions previously prepared was added to each well of the plate with Caco-

239

2 cells and incubated 1 h at 37 °C. Finally, Caco-2 cells were washed twice with PBS

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(150 µL/well).

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2.9. Determination of C. sakazakii adhesion by ELISA-based method

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After incubating Caco-2 monolayers with bacteria and washing twice with PBS to

243

remove non-adhered bacterial, cell monolayers were fixed with 4% (w/v)

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paraformaldehyde in PBS for 10 min to prevent detachment of the cells or adhered

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bacteria. Then, cells were incubated with 80 µL/well of 0.1 M glycine during 5 min

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and washed twice with PBS. A volume of 100 µL of rabbit antiserum against C.

247

sakazakii, diluted 1/500 in PBS with 10% (v/v) horse serum, was added to each well

248

and incubated at 37 ºC for 1 h. After three washes with 150 µL per well of PBS with

249

0.05% Tween 20 (PBS-T), 100 µL of peroxidase-labelled goat anti-rabbit

250

immunoglobulin G, diluted 1/1,000 in PBS with 10% (v/v) horse serum, were added

251

and incubated at 37 ºC for 1 h. Afterwards, the wells were washed three times with

252

PBS-T and incubated with 100 µL of TMB for 20 min. After this incubation, 50 µL of 2

253

M H2SO4 was added to each well to stop the enzymatic reaction. The absorbance

254

was measured at 450 nm using a plate reader (Multiskan MS, (Labsystem, Helsinki,

255

Finland).

256

2.10. Determination of C. sakazakii adhesion by plate count

257

In order to correlate the absorbance values obtained by ELISA, with the counts of

258

bacteria, after the adhesion assay, adhered bacteria after removing the non-adhered

259

bacteria, the cells were released from Caco-2 cells using 50 µL per well of trypsin-

260

EDTA (0.25% trypsin, 0.91 mM EDTA). After incubation at 37 ºC forduring 15 min, 50

261

µL of DMEM with 20% inactivated fetal calf serum was added to the cells to inhibit

262

trypsin activity. Serial 10-fold dilutions of the suspension with recovered bacteria

263

were seeded onto TSA-YE plates and incubated at 37 ºC for 24 h. In the same cell

264

culture plate, for each bacterial dilution, two wells were employed to determine the

265

number of bacteria adhered to Caco-2 cells by plate counting and another two wells

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were used for the ELISA-based procedure above described. 11



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2.11. Effect of commercial powders and raw buttermilk on C. sakazakii

268

adhesion to Caco-2 cells

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All dairy products were prepared by dissolving powders at concentrations of 2, 10

270

and 20 mg/mL in PBS. Afterwards, solutions were centrifuged at 16000g for 15 min

271

and the supernatants obtained were filtered through 0.22 µm. The protein

272

concentration in the filtered samples (those prepared at 20 mg/mL) was determined

273

by the BCA protein assay kit.

274

The effect of dairy products on C. sakazakii adhesion was evaluated by the

275

procedure described in section 2.8 modified as follows. After blocking the plate with

276

BSA-T to avoid non-specific binding of C. sakazakii to the plastic, cell monolayers

277

were incubated with 50 µL of dairy samples previously prepared, as described above.

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After 1 h of incubation at 37 ºC, 50 µL of bacterial suspension (107 CFU/mL) were

279

added to the cells. The rest of the procedure was completed following the ELISA-

280

based, assteps described in section 2.9. The percentage of inhibition of bacterial

281

adhesion by each dairy product was calculated as follows:

282

% Inhibition = 100 - [(Abs450 value of sample/ Abs450 value of PBS) x 100].

283

Specific activity of dairy powders, initially prepared at 20 mg/mL, was calculated

284

by dividing the percentage of inhibition of bacterial adhesion by the protein content in

285

the final samples added to the wells.

286

2.12. Statistical analysis

287

Mean and standard deviations were calculated from data of at least nine

288

replicates from three independent experiments, using Graph Pad Prism 5.0 software.

289

Data were analyzsed for statistical significance using the same programIBM SPSS

290

Software 22.0.. Levene’s test was employed to check homoscedasticity of the data.

12


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Con formato: Sangría: Primera línea: 0.75 cm

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291

Kolmogorov-Smirnov and Shapiro-Wilk normality test was applied to check Gaussian

292

distribution prior to analyze data by two-way analysis of variance (ANOVA)one-way

293

analysis of variance (ANOVA) with Tukey’s multiple comparison test. Tukey’s post

294

hoc test was used to assess differences between mean values. Significance was

295

defined as p < 0.05.

296

3. RESULTS

297

3.1. Composition analysis and protein characterization of commercial dairy

298

powders and raw buttermilk

299

The content (w/w) of protein, lipid and lactose in the dairy samples products

300

evaluated in this study is shown in Figure 1. Compositional data of commercial

301

samples were provided by suppliers and those of raw buttermilk were obtained by

302

infrared spectroscopy. The percentage of protein, lipid and lactose ranged between

303

27.1-38.9%, 5-22.2% and 40.7-50%, respectively.

304

The SDS-PAGE profiles obtained for commercial samples and raw buttermilk

305

showed that the majority of electrophoretic bands corresponded to caseins and whey

306

proteins, though some milk fat globule membrane (MFGM) proteins were also

307

present in all products (Figure 2). Some milk glycoproteins reported with bacterial

308

antiadhesive activity, such as lactoferrin, immunoglobulin G, lactadherin, butyrophilin

309

and mucin-1, have been previewedwere observed on the electrophoretic profile of gel

310

stained with PAS and confirmed by Western-blotting (Figure 3). The two bands

311

observed in the Western-blotting performed to detect bovine IgG correspond to its

312

light and heavy chains, due to their disruption caused by treatment with β-

313

mercaptoethanol.

314

3.2. Identification of C. sakazakii outer membrane proteins

13



315

The SDS-PAGE profile of the proteins extracted from C. sakazakii outer

316

membrane revealed several bands between 10 and 50 kDa. The two major bands,

317

identified as A and B bands in Figure 4a were scinded and analyzed by MALDI-TOF

318

MS.

319

Three main proteins were identified in band A: outer membrane protein F, outer

320

membrane protein A and outer membrane porin protein C. Another three major

321

proteins were identified in band B: maltoporin, elongation factor Tu and long-chain

322

fatty acid outer membrane transporter. Data of sequence coverage, molecular weight

323

and isoelectric point of the proteins identified in electrophoretic bands A and B are

324

shown in Table 1. After analysing analyzing the outer membrane proteins OMPs by

325

Western-blotting with polyclonal specific antiserum, we observed that antibodies

326

recognised all the main proteins present in the electrophoresis profile (Figure 4b).

327

Therefore, it can be concluded that the majority of the outer membrane

328

proteinsOMPs of C. sakazakii are immunogenic and that the antiserum was adequate

329

to be used in the ELISA-based assay..

330

3.3. Adhesion of C. sakazakii to Caco-2 cells

331

In order to quantify the extent of C. sakazakii adhesion to Caco-2 cells,

332

increasing bacterial concentrations were added to the wells of the culture plate. After

333

adhesion of bacteria to cells, these were released with trypsin-EDTA and plated on

334

TSA-YE agar or detected by ELISA in order to compare the estimated counts of

335

bacteria by both techniques. A linear relationship was obtained within the range 3-6

336

Log CFU/well, with a correlation coefficient (r2) of 0.939 (Figure 5). The results

337

obtained in these experiments led us to consider this ELISA-based assay as

338

adequate to study the effect of different dairy products on C. sakazakii adhesion.

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339

The bacterial suspension used for testing the anti-adhesive properties of dairy

340

products was of 107 CFU/mL that resulted in around 45% of adhesion efficiency

341

(adhered bacteria to cells respect to the total added bacteria). To establish this value,

342

several bacterial suspensions were assayed previously, from 104 to 108 CFU/mL and

343

it was observed that the percentage of bacteria adhered to cells decreased when the

344

number of added bacteria was above 107 CFU/mL, probably due to a saturation

345

effect of the cell monolayer (data not shownTable 2). Therefore, this value was

346

considered as optimal for bacterial enumeration and also for evaluating the inhibitory

347

activity of dairy products.

348

3.4. Effect of commercial dairy powders and raw buttermilk on C. sakazakii

349

adhesion

350

The inhibitory effect of commercial dairy powders and raw buttermilk on C.

351

sakazakii adhesion to Caco-2 cells was evaluated at concentrations of 2, 10 and 20

352

mg/mL (Table 32). PBS was used as control (considered as 0% of inhibition).The

353

inhibitory effect obtained with increasing concentrations of products may be

354

explained by the additive effect of components such as s OSoligosaccharides,

355

glycosphingolipids and glycoproteins. Differences in the inhibitory activity between

356

the products and concentrations were found statistically significant in all cases

357

respect to the control (PBS), considered as 0% of inhibition. Commercial products

358

(skim milk, butter serum and buttermilk) showed higher inhibitory activity than raw

359

buttermilk at all concentrations assayed. The highest inhibitory effect on adhesion

360

was produced by commercial buttermilk at 20 mg/mL. The differences in the

361

inhibitory activity were not statistically significant (p < 0.05) among between all

362

commercial dairy products at the same concentration tested. However, significant the

363

differences (p < 0.05) were found between raw buttermilk and commercial dairy 15



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buttermilk powders were statistically significant (p

Inhibition of Cronobacter sakazakii Adhesion to Caco-2 Cells by

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