Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts on four food-contact surfaces at 22 °C and 10 °C

Accepted Manuscript Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts on four food-contact surfaces at 22 °C and 10 °C Piumi De Abrew Abeysundara, Nitin Dhowlaghar, Ramakrishna Nannapaneni, Mark W. Schilling, Sam Chang, Barakat Mahmoud, Chander S. Sharma, Din-Pow Ma PII:

S0956-7135(17)30237-2

DOI:

10.1016/j.foodcont.2017.04.043

Reference:

JFCO 5600

To appear in:

Food Control

Received Date: 24 January 2017 Revised Date:

7 April 2017

Accepted Date: 28 April 2017

Please cite this article as: Abeysundara P.D.A., Dhowlaghar N., Nannapaneni R., Schilling M.W., Chang S., Mahmoud B., Sharma C.S. & Ma D.-P., Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts on four food-contact surfaces at 22 °C and 10 °C, Food Control (2017), doi: 10.1016/j.foodcont.2017.04.043. 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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Running title: Growth and biofilm formation by L. monocytogenes in cantaloupe extracts.

2 Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts

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on four food-contact surfaces at 22°C and 10°C

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Piumi De Abrew Abeysundara1, Nitin Dhowlaghar1, Ramakrishna Nannapaneni1*, Mark W.

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Schilling1, Sam Chang1, Barakat Mahmoud1, Chander S. Sharma2, and Din-Pow Ma3

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Mississippi State, MS 39762, USA, 2Poultry Science Department, Mississippi State University,

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Mississippi State, MS 39762, USA and 3Department of Biochemistry, Molecular Biology,

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Department of Food Science, Nutrition and Health Promotion, Mississippi State University,

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Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762,

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USA

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Listeria monocytogenes, Growth, Biofilm, Cantaloupe

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[email protected]

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Author for correspondence: Phone: 662-325-7697, Fax: 662-325-8728, E-mail:

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ABSTRACT The nationwide listeriosis outbreak that occurred in the United States during 2011

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highlighted the importance of preventing cantaloupe contamination with Listeria monocytogenes

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(Lm) within farm and processing environments. The objectives of this study were to determine

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the effects of strain and temperature on growth and biofilm formation of Lm in cantaloupe flesh

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and peel extracts on different food-contact surfaces. Growth of Lm strains was markedly greater

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at high concentration of cantaloupe extracts and temperature in comparison to low concentration

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and temperature. For 50 mg/ml of cantaloupe extract inoculated with 3 log CFU/ml, the growth

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of Lm was 8.5 log CFU/ml in 32 h at 22°C and 6-7 log CFU/ml in 72 h at 10°C. For 2 mg/ml of

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cantaloupe extract that was inoculated with Lm, the growth was 7-7.5 log CFU/ml in 72 h at

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22°C and 3.5 log CFU/ml in 72 h at 10°C. There were no differences (P ˃ 0.05) among Lm

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strains for biofilm formation in cantaloupe extracts, but biofilm formation was greater at high

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temperature and high concentration. For 50 mg/ml cantaloupe extracts inoculated with 3 log

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CFU/ml, the biofilm formation of Lm on stainless steel surface was approximately 7 log

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CFU/coupon at 22°C in 4-7 days and 5-6 log CFU/coupon at 10°C in 7 days. For 2 mg/ml

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cantaloupe extracts, the biofilm formation of Lm on the stainless-steel surface was approximately

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5-6 log CFU/coupon at 22°C and 4-4.5 log CFU/coupon at 10°C in 7 days. The biofilm

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formation by cantaloupe outbreak strain Lm 2011L-2625 in cantaloupe extracts was least on

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buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces (P <

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0.05). These findings show that a very low concentration of nutrients from cantaloupe flesh or

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peel can induce Lm growth and subsequent biofilm formation on different food-contact

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processing surfaces.

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1. Introduction Listeria monocytogenes is a Gram-positive rod shape, non-spore forming, facultative

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anaerobic, bacterium which is responsible for listeriosis in humans and animals (Gandhi &

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Chikindas, 2007). Among all the foodborne pathogens, L. monocytogenes is the most dangerous

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due to its high mortality rate of 20-25% (Todd & Notermans, 2011). One of the deadliest

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listeriosis outbreaks (33 deaths and one miscarriage) reported in the world history which

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occurred in the USA in 2011 was associated with whole cantaloupe (McCollum, et al., 2013).

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Among the 13 known serotypes of L. monocytogenes, strains that belong to serotypes 1/2a, 1/2b

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and 4b are mostly associated with foodborne listeriosis outbreaks (Borucki, Peppin, White, Loge,

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& Call, 2003; Nelson, et al., 2004) .

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L. monocytogenes is commonly found in soil, sewage and fecal matter (Locatelli, Spor,

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Jolivet, Piveteau, & Hartmann, 2013). Cantaloupe can often be contaminated with pathogens that

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are associated with soil and animals since it is grown close to the ground. The outer rind of

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cantaloupe fruit provides an excellent surface for pathogenic microorganisms to attach and hide

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(Webb, Erickson, Davey, & Doyle, 2015a, 2015b). L. monocytogenes cells that are attached to

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the rind can contaminate cantaloupe-processing environments and promote growth and

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subsequent biofilm formation on food-contact and non-food contact surfaces when appropriate

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environmental conditions exist. The investigations conducted by U.S. Food

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Administration during the multi-state cantaloupe outbreak in 2011 have found the presence of L.

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monocytogenes on the food-contact and non-food contact surfaces of the cantaloupe processing

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facility of Jensen farm, Colorado (McCollum, et al., 2013).

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and

Drug

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A biofilm is a collection of microbial cell communities that are attached to a surface and

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embedded in a self-produced matrix that consists of polysaccharides (Vestby, Møretrø, 3

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Langsrud, Heir, & Nesse, 2009; Whitehead & Verran, 2015). Biofilms are the most predominant

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form of bacteria in food processing environments since microbial cells have extra protection

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from desiccation, UV light, antimicrobials and sanitizing agents (Bernbom, Vogel, & Gram,

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2011; Vestby, et al., 2009). Biofilms formed on the food-contact surfaces can cause cross-

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contamination when improperly cleaned, thus creating a serious food safety issue (Wilks,

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Michels, & Keevil, 2006).

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L. monocytogenes is capable of adhering to various types of food-contact surfaces that are

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found in the food processing environments (Di Bonaventura, et al., 2008). Attachment and

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biofilm formation of L. monocytogenes is influenced by the physiochemical properties of

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environment (nutrients, temperature, pH, salt concentration) as well as the cell surface

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(hydrophobicity, flagellation and motility) (Di Bonaventura, et al., 2008; Kalmokoff, et al.,

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2001). Survival, growth and biofilm formation in standard microbiology growth media may not

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reflect the actual behavior of L. monocytogenes in food processing environments. This is because

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the media contain optimum levels of all the nutrients that are necessary for microbial growth. For

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example, Beuchat, Brackett, Hao, and Conner (1986) studied the growth of L. monocytogenes in

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cabbage juice at 5°C and observed that L. monocytogenes was capable of growing in sterile

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cabbage juice containing <5% NaCl. In contrast, L. monocytogenes growth was observed when

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≤10% NaCl was present in tryptic phosphate broth. Kim, Ryu, and Beuchat (2006) studied the

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biofilm formation of Enterobacter sakazakii in different growth media at 25°C and found that E.

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sakazakii did not form biofilms on the stainless steel containing lettuce juice broth but produced

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biofilms on the same surfaces in infant formula broth. These findings suggest that the growth and

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biofilm formation of foodborne bacterial pathogens can drastically differ depending on the

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growth media.

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In cantaloupe processing environments, L. monocytogenes can be exposed to nutrients that

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are leaked from cantaloupe fruits. Both cantaloupe flesh and peel contain nutrients such as sugar,

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proteins, carotenoids, vitamin C and various minerals that are essential for microbial growth

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(Koubala, Bassang’na, Yapo, & Raihanatou, 2016). There are no published data on the growth

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and biofilm formation of L. monocytogenes in cantaloupe residue that persists on the food

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processing surfaces. Penteado and Leitao (2004a) studied the growth of L. monocytogenes in

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low-acid fruit pulps (melon, watermelon and papaya) at 10°C, 20°C and 30°C. According to their

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study, the growth of L. monocytogenes varied depending on the fruit type.

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Even though food-soils are removed from the cantaloupe processing environments routinely,

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residues may persist on the food-contact surfaces when they are improperly cleaned (Fryer &

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Asteriadou, 2009). There is a need for critical understanding of the survival, growth and biofilm

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formation that reflects the actual behavior of L. monocytogenes in cantaloupe residue when it is

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present on food processing surfaces. Therefore, the objectives of the present study were to

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determine the effects of strain, temperature, nutrient level, and food-contact surface on the

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growth and biofilm formation of L. monocytogenes in cantaloupe flesh and peel extracts.

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2. Materials and methods

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2.1. Bacterial strains and culture conditions

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L. monocytogenes strains that were used in this study are described in Table 1. The working

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stock cultures of these strains were prepared by inoculating a colony grown on PALCAM agar

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onto tryptic soy agar that contained 0.6% yeast extract (TSAYE) slants and incubated at 37°C for

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24 h. These working stocks were stored at 4°C for 4-5 weeks. The overnight culture of each

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strain was prepared by inoculation of 10 ml of tryptic soy broth that contained 0.6% yeast extract

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(TSBYE) from the working stocks and incubated at 37°C for 18 to 20 h with shaking at 150 rpm

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(Imperial ІІІ incubator, Lab-Line Instrument Inc., IL, USA).

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2.2. Food-contact surface preparation

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Four types of food-contact surfaces that are commonly used in the cantaloupe processing

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industry were obtained (Table 2) and were cut into 1 cm×1.5 cm size coupons. Only stainless

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steel coupons were reused during experiments. These coupons were soaked in an alkali detergent

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for 1 h and washed thoroughly with deionized water. Washed stainless steel coupons were

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autoclaved at 121°C for 15 min. Buna-n rubber, ultra-high molecular weight polyethylene and

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thermoplastic polyurethane coupons were surface sterilized by submerging in 100% alcohol for

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15 min prior to washing with sterile water to eliminate alcohol residue. Sterile coupons were

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surface dried inside a biosafety cabinet prior to use. Sterility of these coupons was confirmed by

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incubating coupons in fresh TSBYE at 37°C for 48 h.

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2.3. Preparation of cantaloupe extracts

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Cantaloupe (Cucumis melo L. var. reticulatus) fruits were obtained from a local supermarket

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and washed under running tap water for 1 min. The peel was then removed and the flesh was

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deseeded. To prepare cantaloupe flesh and peel extracts, 100 g of cut cantaloupe flesh or peel

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was blended with 400 ml of deionized water for 30 seconds using a juice extractor (Hamilton

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beach, Model: 53514). The blended flesh and peel extracts were then filtered using a filter bag

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(BA6141/STR) and centrifuged (2500 × g) (Beckman, Model TJ-6 centrifuge) for 5 min to

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eliminate coarse particles. Cantaloupe extracts were autoclaved at 121°C for 15 min and stored at

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room temperature until needed. These cantaloupe extracts (200 mg/ml) were further diluted in

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physiological saline to obtain 50 mg/ml and 2 mg/ml of concentrations, respectively, based on

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the fresh weight of the cantaloupe.

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2.4. Determination of physical/chemical properties of cantaloupe extracts

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The pH of sterile cantaloupe flesh and peel extracts was determined using a calibrated pH

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meter (Accumet Basic, AB15 pH meter, Fisher Scientific). Brix values were determined using a

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refractometer (RHB-18ATC, GxPro). In order to determine the dry weight of cantaloupe flesh or

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peel extract, 25 ml of each extract was transferred to a pre-weighed 50 ml centrifuge tube. These

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extracts were freeze dried until a constant weight was obtained. The dry weight was calculated

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using the following equation.

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Dry weight (mg/ml) = (Post dry weight of the tube - Weight of the empty tube) mg

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Volume of extract (ml)

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2.5. Evaluation of growth of L. monocytogenes in cantaloupe extracts at different temperatures Growth of L. monocytogenes EGD (Bug600), 2011-2624 and G1091 in 50 and 2 mg/ml of

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cantaloupe flesh and peel extracts at 22°C and 10°C was studied. The overnight cultures of these

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strains were prepared as described previously. One ml volumes of overnight cultures were

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centrifuged for 5 min (9000 ×g, MARATHON 21000R, Fisher Scientific). The resulting cell

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pellets were resuspended in 1 ml of physiological saline to obtain 9 log CFU/ml of stationary

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phase cells.

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Cantaloupe flesh or peel extracts (50 and 2 mg/ml) were inoculated with the L.

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monocytogenes stationary phase cells to yield an initial cell concentration of 3 log CFU/ml. The

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inoculated cantaloupe extracts were incubated at 10°C (Low temperature, VWR, Scientific

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Products, Sheldon manufacturing, INC., OR, 97113) and at room temperature (22°C). Cell

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numbers were enumerated for 72 h at 8 h intervals by serially diluting an aliquot from the cell

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suspensions in physiological saline and plating on tryptic soy agar that was enriched with 0.6%

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yeast extract, Escullin and Ferric ammonium citrate (TSA-YEEF). These plates were incubated

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at 37°C (Imperial III Incubator, Lab line instrument Inc., IL, USA) for 48 h to obtain colony

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forming unit (CFU) counts.

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2.6. Calculation of generation time

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The generation time (g) was calculated from the slope of the line of the semi logarithmic plot

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of exponential growth using the mean of 4 replications. The equation of g = 0.301/slope of the

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semi logarithmic plot of exponential growth was used to calculate the generation time as

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described previously (Penteado & Leitao, 2004a, 2004b).

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2.7. Evaluation of biofilm formation by different strains of L. monocytogenes in cantaloupe

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extracts at different temperatures

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One ml from each overnight culture (Table 1) was centrifuged, and the resulting cell pellets

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were resuspended in 1 ml of physiological saline. Cantaloupe flesh or peel extracts (50 and 2

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mg/ml) were inoculated with stationary phase cells to obtain a 3 log CFU/ml initial cell

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concentration. Sterile stainless steel coupons were placed in polystyrene 24-well plates with one

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coupon per well. Each well was filled with 2 ml of cell suspension that was prepared in either

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cantaloupe flesh or peel extract. These coupons were incubated at 22°C or 10°C for 1, 4 or 7

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days.

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2.8. Enumeration of attached cells in the biofilm To enumerate attached cells in biofilms at the end of 1, 4 or 7 days, used growth media was

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removed from the wells and coupons were washed twice by adding 2.75 ml of physiological

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saline to remove loosely bound cells. Washed coupons were transferred into plastic tubes (15 ml-

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TronadoTM) each containing 5 ml of 0.1% peptone water with 0.02% Tween 80 and five sterile

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glass beads. Tubes were vortexed for 1 min (Vortex mixer, Labnet International, Inc, Edison, NJ,

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USA) and aliquots of detached cell suspensions were serially diluted in physiological saline.

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Each dilution was plated on TSA-YEEF and incubated at 37°C for 48 h.

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2.9. Effect of the nature of the food-contact surface on biofilm formation by L. monocytogenes

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2011-2625 in cantaloupe extracts at different temperatures

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Stainless steel, buna-n rubber, ultra-high molecular weight polyethylene and thermoplastic

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polyurethane coupons were placed in 24-well plates with one coupon per well. L. monocytogenes

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2011L-2625 cell suspension of 2 ml at 3 log CFU/ml was prepared in cantaloupe flesh or peel

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extract (50 and 2 mg/ml) and added to each well. These coupons were incubated at 22°C and

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10°C for 1, 4 or 7 days. At the end of each incubation period, biofilm formation on the four

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different surfaces was enumerated as described previously.

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2.10. Statistical analysis

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A completely randomized design with a 2-way factorial structure and 4 replications was

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used. Mean values (log CFU/ ml or log CFU/coupon) were calculated using four values obtained

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and analysis of variance with Tukey’s honestly significant difference test (P < 0.05) was

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performed to separate means where significant differences occur. For the comparison study of

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biofilm formation among 6 strains, the two factors included strain and temperature. For the

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comparison of average biofilm formation of L. monocytogenes in 1, 4 and 7 days, the days were

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compared with combination of concentration and temperature combination. For the comparison

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of biofilm formation of L. monocytogenes 2011-2625 on different food contact surfaces, the two

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factors included surfaces and temperature (P < 0.05).

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3. Results

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3.1. Physical and chemical properties of cantaloupe extract

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The pH of cantaloupe flesh and peel extracts was around 6.5. Cantaloupe flesh extract had

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around 1.6°B of total soluble solids while cantaloupe peel extract showed a 0.7 °B of total

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soluble solids. The fresh weight for both cantaloupe flesh and peel extracts was 200 mg/ml. The

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dry weight of cantaloupe flesh extract was 1.7 mg/ml and peel extract was 0.7 mg/ml.

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3.2. Growth curves of L. monocytogenes in cantaloupe extracts

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The growth rate of L. monocytogenes in cantaloupe flesh and peel extracts was greater at

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higher cantaloupe extract concentration and higher temperature. The growth curves of L.

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monocytogenes in cantaloupe flesh at 50 and 2 mg/ml at 22°C and 10°C are shown in Figure 1.

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In 50 mg/ml cantaloupe flesh, the cell numbers of L. monocytogenes Bug600, 2011L-2625 and

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G1091 increased by 5.5 log CFU/ml in 32-40 h at 22°C and remained stable at that level

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afterwards, while L. monocytogenes increased by 3.5 log CFU/ml in 72 h at 10°C (Fig. 1ACE).

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In 2 mg/ml cantaloupe flesh extract, the cell numbers of L. monocytogenes strains Bug600,

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2011L-2625 and G1091 were increased by 4.5 log CFU/ml in 56 h at 22°C and 0.5 log CFU/ml

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in 72 h at 10°C (Fig. 1BDF). In 50 and 2 mg/ml cantaloupe flesh extract, the cell number of L.

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monocytogenes from 8 – 72 h was greater (P < 0.05) at 22°C when compared to 10°C.

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A similar pattern was also observed for the growth of L. monocytogenes in cantaloupe peel

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extract at 50 and 2 mg/ml concentrations at 22°C and 10°C (Fig. 2). In 50 mg/ml cantaloupe peel

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extract, the cell numbers of L. monocytogenes Bug600, 2011L-2625 and G1091 were increased

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by 5.5 log CFU/ml at 22°C in 24-40 h and remained stable at that level afterwards, while L.

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monocytogenes strains were increased by 3-4 log CFU/ml at 10°C in 72 h (Fig. 2ACE). In 2

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mg/ml cantaloupe peel extract, the cell numbers of L. monocytogenes strains Bug600, 2011L-

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2625 and G1091 were increased by 4-4.5 log CFU/ml in 48-56 h at 22°C and 0.5 log CFU/ml at

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10°C in 72 h (Fig. 2BDF). For 50 and 2 mg/ml cantaloupe peel extract, the cell number of L.

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monocytogenes from 8 – 72 h was greater (P < 0.05) at 22°C when compared to 10°C.

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The lag phase of L. monocytogenes strains was less than 8 h in 50 and 2 mg/ml cantaloupe

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flesh or peel extracts at 22°C while it was extended to 16-18 h in 50 mg/ml or 72 h in 2 mg/ml

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cantaloupe extracts at 10°C.

In 50 mg/ml cantaloupe extracts, the generation time was markedly increased by 2-4 h by

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shift of temperature from 22°C to 10°C for all L. monocytogenes strains tested (Table 3). In 2

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mg/ml cantaloupe extract, the generation time of L. monocytogenes extended by about 2-4 h

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beyond the corresponding generation time in 50 mg/ml cantaloupe extract at 22°C. Since no

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exponential growth was observed during the duration of 72 h, the generation time was not

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calculated for L. monocytogenes in 2 mg/ml at 10°C.

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3.3. Influence of strain and temperature on biofilm formation by L. monocytogenes on stainless

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steel surface containing cantaloupe extracts

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Biofilm formation by L. monocytogenes strains was greater in 50 mg/ml cantaloupe extract at

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22°C (P < 0.05) as compared to 2 mg/ml at 10°C. In 50 mg/ml cantaloupe flesh extract, biofilm

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formation of L. monocytogenes strains was 5-6 log CFU/coupon in 1 d and 7 log CFU/coupon in 11

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4 or 7 d at 22°C. At 10°C, biofilm formation was 1.5 to 3.5 logs less compared to 22°C (P <

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0.05) (Fig. 3ACE). In 2 mg/ml cantaloupe flesh extract, biofilm formation by L. monocytogenes

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was 4.5 log CFU/coupon in 1 d or 5-6 log CFU/coupon in 4 or 7 d at 22°C. At 10°C biofilm

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formation was 1 to 2.5 logs less (P < 0.05) than that at 22°C (Fig. 3BDF). With one exception,

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there were no differences (P < 0.05) in biofilm formation among the six strains of L.

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monocytogenes in cantaloupe flesh extract under all environmental condition tested. For 50

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mg/ml cantaloupe flesh extract, L. monocytogenes Bug600 had 1 log less biofilm formation in 1

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day as compared to other strains at 22°C.

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A similar pattern was also observed for the biofilm formation by six strains of L.

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monocytogenes in cantaloupe peel extract at 50 and 2 mg/ml concentrations at 22°C and 10°C

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(Fig. 4). In 50 mg/ml cantaloupe peel extract, biofilm formation of L. monocytogenes strains was

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approximately 6 log CFU/coupon in 1 d and 7 log CFU/ coupon in 4 or 7 d at 22°C. At 10°C

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biofilm formation was 1.5 to 4 logs less (P < 0.05) than that at 22°C (Fig. 4ACE). In 2 mg/ml

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cantaloupe peel extract, biofilm formation by L. monocytogenes strains were approximately 4-5

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log CFU/coupon in 1 d or 5-6 log CFU/coupon in 4 or 7 d at 22°C while their corresponding

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biofilm formation at 10°C was 1.5 to 3 logs less (P < 0.05) than that observed at 22°C (Fig.

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4BDF). No significant differences in biofilm formation were observed among the six strains of L.

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monocytogenes in cantaloupe peel extract under all environmental condition tested with the

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exception of 50 mg/ml cantaloupe flesh extract in 1 day at 10°C, where L. monocytogenes G1091

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had 1 log greater biofilm formation as compared to other strains.

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In 50 mg/ml cantaloupe flesh or peel extract, there was no increase (P < 0.05) in average

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biofilm formation by L. monocytogenes observed from 4 to 7 d at 22°C, but there was 1.5 - 2.0

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log increase observed at 10°C (Table 4). In 2 mg/ml cantaloupe flesh or peel extract, there was

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no increase (P < 0.05) in average biofilm formation by L. monocytogenes strains observed from

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4- 7 d at both 22°C and 10°C.

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3.4. Biofilm formation by L. monocytogenes 2011L-2625 on different food-contact surfaces

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containing cantaloupe extracts

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In 50 mg/ml cantaloupe flesh extract inoculated with 3 log CFU/ml, L. monocytogenes

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2011L-2625 biofilm formation was approximately 6.5 log CFU/coupon in 1 d or 7 log

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CFU/coupon in 4 or 7 d on stainless steel, polyethylene and polyurethane surfaces 22°C. The

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corresponding biofilm formation on buna-n rubber was 2.0-3.5 logs less (P < 0.05) than that

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observed on the other three surfaces at 22°C (Fig. 5ACE). In 50 mg/ml cantaloupe flesh extract,

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L. monocytogenes 2011L-2625 formed 1-2 log CFU/coupon biofilm on all the four surfaces in 1

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d at 10°C. However, L. monocytogenes 2011L-2625 formed 5-6 log CFU/coupon biofilms on

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stainless steel, polyethylene and polyurethane surfaces in 4 or 7 d at 10°C while its

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corresponding biofilm formation on buna- n rubber was 2 logs less (P < 0.05) than that observed

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on other surfaces at 10°C.

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In 2 mg/ml cantaloupe flesh extract, L. monocytogenes 2011L-2625 biofilm formation was

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approximately 4 log CFU/coupon in 1 d or 6 log CFU/coupon in 4 or 7 d on the stainless steel,

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polyethylene and polyurethane surfaces. The corresponding biofilm formation on buna-n rubber

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was approximately 1.5 logs less (P < 0.05) than that observed on the other three surfaces at 22°C

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(Fig. 5BDF). In 2 mg/ml cantaloupe flesh extract, biofilm formation by L. monocytogenes

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2011L-2625 was approximately 2 log CFU/coupon on all the four surfaces in 1 d at 10°C.

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However, L. monocytogenes 2011L-2625 formed 5.5-6 log CFU/coupon of biofilm on the

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stainless steel, polyethylene and polyurethane surfaces in 4 or 7 d while its corresponding

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biofilm formation on buna-n rubber was 1-1.5 logs less (P < 0.05) than that observed on the

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other surfaces at 10°C. In 50 mg/ml cantaloupe peel extract, L. monocytogenes 2011L-2625 biofilm formation was

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approximately 6.5-7.0 log CFU/coupon in 1, 4 or 7 d on stainless steel, polyethylene and

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polyurethane surfaces while its corresponding biofilm formation on buna-n rubber was 2.5-3.5

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logs less (P < 0.05) than that observed on the other three surfaces at 22°C (Fig. 6ACE). In 50

291

mg/ml cantaloupe peel extract, L. monocytogenes 2011L-2625 formed 1-2 log CFU/coupon

292

biofilm on all four surfaces in 1 d at 10°C. However, L. monocytogenes 2011L-2625 formed 4.5-

293

6.0 log CFU/coupon biofilms on stainless steel, polyethylene and polyurethane surfaces in 4 or 7

294

d while its corresponding biofilm formation on buna-n rubber was 1-2 logs less (P < 0.05) than

295

that observed on other surfaces at 10°C.

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In 2 mg/ml cantaloupe peel extract, L. monocytogenes 2011L-2625 biofilm formation was 4-

297

5 log CFU/coupon in 1 d or 6 log CFU/coupon in 4 or 7 d on stainless steel, polyethylene and

298

polyurethane surfaces. The corresponding biofilm formation on buna-n rubber was

299

approximately 1-2 logs less (P < 0.05) than that observed on the other surfaces at 22°C (Fig.

300

6BDF). In 2 mg/ml cantaloupe flesh extract, biofilm formation by L. monocytogenes 2011L-

301

2625 was approximately 2 log CFU/coupon on all the four surfaces in 1 d at 10°C. However, L.

302

monocytogenes 2011L-2625 formed 6 log CFU/coupon biofilms on stainless steel, polyethylene

303

and polyurethane surfaces in 4 or 7 d while the corresponding biofilm formation on buna-n

304

rubber was 1-2 logs less (P < 0.05) than that observed on other surfaces at 10°C.

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Overall, biofilm formation of L. monocytogenes 2011L-2625 on buna-n rubber was

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significantly lower than the biofilm formation on stainless steel, polyethylene and polyurethane

307

surfaces in 50 and 2 mg/ml cantaloupe flesh and peel extract concentrations at 22°C and 10°C.

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4. Discussion In the cantaloupe processing environments, nutrients from cantaloupe fruits can leak and

310

accumulate on food contact surfaces when improperly cleaned. Such residues may promote the

311

growth and subsequent biofilm formation by L. monocytogenes on the food processing surfaces.

312

This study focused on the critical understanding of the survival, growth and biofilm formation

313

that reflected the behavior of L. monocytogenes in cantaloupe residue if present at two

314

concentrations on different processing surfaces. Cantaloupe extracts were autoclaved at 121°C

315

for 15 min to eliminate any interferences from background microflora and spores against L.

316

monocytogenes. Autoclaved food extracts were frequently used for studying the behavior of L.

317

monocytogenes, Escherichia coli, Salmonella Enteritidis in fruit or vegetable juices. For

318

example, Raybaudi-Massilia, Mosqueda-Melgar, and Martín-Belloso (2009) studied the

319

antimicrobial activity of malic acid against Listeria monocytogenes, Salmonella Enteritidis and

320

Escherichia coli O157:H7 in apple, pear and melon juices using autoclaved fruit juices at 121°C

321

for 15 min. Mosqueda-Melgar, Raybaudi-Massilia, and Martín-Belloso (2007) sterilized melon

322

and water melon juice at 121°C for 15 min to study the combined effects of high-intensity pulsed

323

electric fields and natural antimicrobials to inactivate pathogenic microorganisms in fruit juices.

324

Oliveira, et al. (2014) sterilized fruit juices at 115°C for 10 min to study the effectiveness of a

325

bacteriophage in reducing Listeria monocytogenes in apple, pear and melon juices. Beuchat, et

326

al. (1986) studied the growth of L. monocytogenes in cabbage juice after autoclaving at 121°C

327

for 15 min. Preliminary studies revealed that growth of L. monocytogenes was similar in

328

autoclaved or filter sterilized cantaloupe flesh or peel extracts with respect to the length of the

329

lag phase or maximum growth in cell population. Autoclaving did not change the pH or the brix

330

value of cantaloupe flesh and peel extracts significantly. The pH of both cantaloupe flesh and

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peel extracts were around 6.4-6.5 and was optimum for L. monocytogenes growth. The brix value

332

indicated that cantaloupe flesh extract contained more soluble solids than the peel extract which

333

was also reflected in the dry weight that was calculated for each extract. This was probably due

334

to the high sugar content in cantaloupe flesh when compared to peel.

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This study was conducted at two temperature conditions. The 22°C condition was chosen as

336

cantaloupe processing operations are mostly done at room temperature. The 10°C condition was

337

chosen to study how lowering temperature can affect the growth and biofilm production of L.

338

monocytogenes. While whole or cut cantaloupe products are recommended to be stored at 2-5°C,

339

in real world, temperature fluctuations can happen during cold storage environments and in

340

transportation, where temperature may raise up to 10°C (BettGarber, Greene, Lamikanra,

341

Ingram, & Watson, 2011; Koseki & Isobe, 2005). Therefore, observations taken at 10°C will be

342

useful to understand growth and biofilm formation of L. monocytogenes at low temperature

343

conditions in the presence of cantaloupe residue.

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Results indicate that concentration as low as 2 mg/ml of cantaloupe flesh and peel extract is

345

an adequate substrate for L. monocytogenes to induce growth. The influence of temperature on

346

the growth rate of L. monocytogenes was evident in 50 mg/ml cantaloupe flesh extract where all

347

L. monocytogenes strains yielded a doubling time of approximately 2 h at 22°C and 4-6 h at

348

10°C. Similarly, Penteado and Leitao (2004a) and de Modelos (2007) observed that the

349

generation time of L. monocytogenes ScottA in cantaloupe pulp increased from 0.84 h to 1.74 h

350

or from 1.74 h to 7.12 h, respectively, when the growth temperature was decreased from 30°C to

351

22°C or from 22°C to 10°C.

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The concentration of cantaloupe extract in the growth media also affected the generation time

353

of L. monocytogenes. For example, for 50 mg/ml cantaloupe extracts, L. monocytogenes strains

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yielded a generation time of approximately 2 h as compared to 4.5-5.5 h for 2 mg/ml cantaloupe

355

extract at 22°C. Overall, the lesser temperature and decreasing nutrient concentration in the

356

growth media, there was an increase in the doubling time that inversely decreased the growth

357

rate of L. monocytogenes.

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Results from the current study indicate that low temperature (10°C) is not a barrier for L.

359

monocytogenes growth in cantaloupe processing environments. For example, all L.

360

monocytogenes strains showed rapid growth at 10°C in 50 mg/ml cantaloupe extracts. However,

361

there was no significant growth until 72 h in 2 mg/ml cantaloupe extracts suggesting that the

362

growth of L. monocytogenes was inhibited at 10°C when the available nutrients were limited.

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A stainless steel surface was used for the comparison study of biofilm formation by L.

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monocytogenes strains belonging to serotypes 1/2a, 1/2b and 4b since it is the most commonly

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found surface in food processing environments (Hilbert, Bagge-Ravn, Kold, & Gram, 2003).

366

With few exceptions, there were no differences (P < 0.05) in biofilm formation among the six L.

367

monocytogenes strains that were tested in cantaloupe flesh or peel extract under different

368

environmental conditions. Previously, it was reported that L. monocytogenes strains belonging to

369

serotype 1/2a formed biofilms with greater bacterial counts than 1/2b and 4b (Borucki, et al.,

370

2003). Di Bonaventura, et al. (2008) also reported that L. monocytogenes strains belonging to

371

serotype 1/2c formed biofilms with greater bacterial counts on stainless steel and glass when

372

compared to strains belonging to serotype 1/2a, 1/2b or 4b at 37°C. However, it was recently

373

reported that the ability of L. monocytogenes to form biofilms depends on the individual strains

374

while the correlation between biofilm formation and L. monocytogenes serotype remained

375

inconclusive (Doijad, et al., 2015).

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L. monocytogenes biofilm formation was greater (P < 0.05) at 22°C when compared to 10°C

377

in 1, 4 or 7 days in both 50 and 2 mg/ml cantaloupe flesh concentrations. Similarly, Di

378

Bonaventura, et al. (2008) reported that L. monocytogenes biofilm formation was greater at 37°C

379

or 22°C when compared to 12°C or 4°C. This may be due to the increase in cell hydrophobicity

380

with increasing temperature, which enhances the subsequent cell attachment to hard surfaces

381

(Chavant, Martinie, Meylheuc, Bellon-Fontaine, & Hebraud, 2002; Di Bonaventura, et al., 2008).

382

Flagella induced cell motility is required for cell attachment and the initiation of biofilm

383

formation (Todhanakasem & Young, 2008; Vatanyoopaisarn, Nazli, Dodd, Rees, & Waites,

384

2000). Di Bonaventura, et al. (2008) found that the motility of L. monocytogenes was drastically

385

reduced at lower temperature, where among 44 L. monocytogenes strains, 30 were motile at 22°C

386

while only 4 strains were motile at 12°C. This suggests that at 10°C, the decreased motility of L.

387

monocytogenes could result in lower cell attachment and subsequently less biofilm formation on

388

stainless steel as compared to 22°C in cantaloupe extract. Ochiai, et al. (2014) reported that the

389

persistent L. monocytogenes strains that were obtained from a chicken processing plant had

390

enhanced ability to alter biofilm formation in response to changing the temperature from 30°C to

391

37°C when compared to non-persistent L. monocytogenes strains. These findings suggest that

392

regulation of biofilm formation through temperature control in L. monocytogenes may be

393

significant for its survival and growth in food processing environments.

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Biofilm formation of foodborne pathogens under very low concentrations of nutrients has

395

been studied by many researchers (Solomon, Niemira, Sapers, & Annous, 2005; Stepanović,

396

Ćirković, & Ranin, 2004; Yang, Khoo, Zheng, Chung, & Yuk, 2014). Foodborne pathogens may

397

have access to different levels of nutrients depending on where they persist in food processing

398

facilities. In the present study, it was observed that the decreasing cantaloupe extract

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concentration from 50 to 2 mg/ml decreased L. monocytogenes biofilm formation at 22°C and

400

10°C in 7 days. Stanley and Lazazzera (2004) reported that starvation could induce more biofilm

401

formation in Bacillus subtilis. The starvation condition of Bacillus subtilis promoted higher

402

activation of transcription factors such as RpoS and Spo0A that are needed for biofilm

403

formation. Yang, et al. (2014) reported that S. Enteridis produced more cellulose in the

404

extracellular polymeric substances matrix of biofilm when grown in 1/20 TSB as compared to an

405

un diluted TSB. However, the effects of nutrient density of the growth medium on biofilm

406

formation may depend on the nature of the microorganism. Stepanović, et al. (2004) observed

407

that L. monocytogenes produced a greater amount of biofilm in TSB when compared to 1/20

408

TSB, while Salmonella spp. showed the opposite behavior. Similarly, Solomon, et al. (2005)

409

reported that Salmonella spp. from clinical samples promoted greater biofilm formation in 1/20

410

TSB when compared to TSB while the meat isolate Salmonella spp. from meat samples did not

411

differ in biofilm formation as the nutrient density in the growth media varied.

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Various materials such as stainless steel, glass, polyurethane, buna-n rubber and polyethylene

413

are used in the food processing and retail environments as food-contact surfaces. L.

414

monocytogenes cells adhere to a wide variety of surfaces found in the food processing and retail

415

environments (Beresford, Andrew, & Shama, 2001). It was observed that L. monocytogenes

416

formed biofilms on stainless steel, buna-n rubber, polyethylene, polyurethane surfaces in

417

cantaloupe extracts at 50 and 2 mg/ml concentrations at both 22°C or 10°C. However, regardless

418

of the growth temperature or the concentration of cantaloupe extract, L. monocytogenes 2011L-

419

2526 formed 1-3.5 logs less biofilm on buna-n rubber as compared to stainless steel,

420

polyethylene or polyurethane.

421

monocytogenes and Salmonella formed less biofilm on buna-n rubber than stainless steel surface.

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Previously, Ronner and Wong (1993) also reported that L.

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422

Surfaces such as glass and stainless steel are hydrophilic while rubber and plastic surfaces are

423

more hydrophobic (Stepanović, et al., 2004).

424

monocytogenes formed more biofilm on hydrophilic materials as compared to hydrophobic

425

materials regardless of the growth temperature while Chavant, et al. (2002) reported that L.

426

monocytogenes biofilm formation was more rapid on hydrophilic surfaces as compared to

427

hydrophobic materials. Even though polyethylene and polyurethane are hydrophobic, biofilm

428

formation on those surfaces was as great as stainless steel in this study.

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Bonsaglia, et al. (2014) reported that L.

In summary, results indicate that L. monocytogenes can induce significant growth and

430

biofilm formation on different food-contact surfaces in very low concentrations of cantaloupe

431

flesh and peel extracts. The growth and biofilm formation of L. monocytogenes was greater in 50

432

mg/ml cantaloupe flesh or peel extract concentration at 22°C as compared to 2 mg/ml cantaloupe

433

flesh or peel extract at 10°C. No major differences were found among the L. monocytogenes

434

strains tested for biofilm formation in cantaloupe extracts. L. monocytogenes formed less biofilm

435

on buna-n rubber than stainless steel, polyurethane and polyethylene containing cantaloupe

436

extract. These findings illustrate the critical importance of appropriate cleaning and sanitizing of

437

cantaloupe processing surfaces because even the presence of small sediments of cantaloupe

438

residue on the food-contact surfaces can promote growth and subsequent biofilm formation in

439

the processing environments, which creates serious food safety risks.

440

Acknowledgements

441

This research was supported in part by the Strategic Research Initiative and Food Safety

442

Initiative awards to RN by the Mississippi Agricultural and Forestry Experiment Station

443

(MAFES) under project MIS-401160. The authors would like to thank Dr. Cathy C. Webb,

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Center for Food Safety, Department of Food Science and Technology, University of Georgia for

445

providing L. monocytogenes outbreak strains used in this study.

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Table 1

448

L. monocytogenes strains tested in this study.

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L. monocytogenes

Serotype

Isolation source

Obtained from

2011L-2625

1/2a

Cantaloupe outbreak 2011

UGA

EGD (Bug600)

1/2a

Clinical

2011-2624

1/2b

Cantaloupe outbreak 2011

F8385

1/2b

Carrot

ScottA

4b

Clinical

G1091

4b

Coleslaw

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UGA: University of Georgia

459

FDA: Food and Drug Administration, USA

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460

451 Institute Pasteur, 452 France 453 UGA 454 UGA 455 FDA 456 UGA 457

22

461

Table 2

462

Description of food-contact surfaces tested in this study. Material

Thickness Manufacturer

Used for

(inch) Stainless steel (304 #4) 0.018

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Thyssen Krup Table tops, Utensil &

0.031

Warco Nitrite

Conveyer belt

Thermoplastic

0.01

Habasit

Conveyer belt

0.125

Sibe

Cutting boards, Conveyer belt,

Automation

Table tops

Ultrahigh molecular weight polyethylene

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Nitrile buna rubber

polyurethane

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equipment surface

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465

Table 3

466

Generation time of L. monocytogenes in cantaloupe extracts at 10°C and 22°C

strain

pe extract

Generation time of L. monocytogenes (h) 50 mg/ml 10°C

EGD (Bug600)

Flesh

5.9 (0.93)

2.0 (0.95)

2011L-2624

Flesh

4.4 (0.95)

2.0 (0.91)

G1091

Flesh

4.1 (0.89)

2.2 (0.99)

EGD (Bug600)

Peel

5.6 (0.98)

2011L-2624

Peel

G1091

Peel

2 mg/ml

10°C NA

NA

5.2 (0.83)

1.5 (0.91)

NA

5.2 (0.87)

4.3 (0.94)

2.3 (0.85)

NA

4.1 (0.84)

3.2 (0.92)

1.8 (0.88)

NA

5.7 (0.70)

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NA: Not available

5.4 (0.83)

4.6 (0.86)

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22°C

NA

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469

22°C

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L. monocytogenes

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Table 4

471

Average biofilm formation of six strains of L. monocytogenes in cantaloupe extracts under

472

different conditions.

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Age of

Cantaloupe

Average biofilm formation of L. monocytogenes

biofilm

extract

(log CFU/coupon)

(days)

2 mg/ml 22°C

Flesh

2.2 ±0.1aA

4

Flesh

3.8 ± 0.1bA

7

Flesh

5.2 ± 0.1cB

1

Peel

2.1 ± 0.18aA

4

Peel

7

Peel

22°C

5.5 ± 0.2aC

2.0 ± 0.1aA

4.1 ± 0.1aB

7.0 ± 0.1bC

3.5 ± 0.1bA

5.5 ± 0.2bB

7.0 ± 0.1bC

4.0 ± 0.2bA

5.6 ± 0.1bB

6.0 ± 0.10aC

2.1 ± 0.04aA

4.3 ±0.12aB

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50 mg/ml

4.1 ± 0.07bA

6.9 ± 0.09bC

3.5 ± 0.02bA

5.3 ± 0.07bB

5.7 ± 0.08cB

7.1 ± 0.06bC

4.2 ± 0.13bA

5.3 ± 0.09bB

Numbers in the same column of the same extract sharing different lowercase letters or numbers

474

in the same raw sharing different upper case letters are significantly different.

475

Fig. 1. Growth of L. monocytogenes Bug600 (A, B), 2011-2625 (C, D) and G1091 (E, F) in 50

476

mg/ml (A, C, E) and 2 mg/ml (B, D, F) of cantaloupe flesh extract at 10°C (□) and 22°C (■).

477

Error bars indicate standard error.

478

Fig. 2. Growth of L. monocytogenes Bug600 (A, B), 2011-2625 (C, D) and G1091 (E, F) in 50

479

mg/ml (A, C, E) and 2 mg /ml (B, D, F) of cantaloupe peel extract at 10°C (□) and 22°C (■).

480

Error bars indicate standard error.

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Fig. 3. Biofilm formation by six strains of L. monocytogenes on the stainless steel surface in 50

482

mg/ml (A, C, E) and 2 mg /ml (B, D, F) of cantaloupe flesh extract at 10°C (□) and 22°C ( ) in

483

1 day (A, B),4 days (C, D) and 7 days (E, F). Note: Bars with different lowercase letters indicate

484

significant differences between biofilms formed at the same temperature and * indicate

485

significant difference between biofilms belong to the same strain formed at different temperature

486

based on Tukey’s Honestly Significant Difference test ANOVA test (P < 0.05). Error bars

487

indicate standard error.

488

Fig. 4. Biofilm formation by six strains of L. monocytogenes on the stainless steel surface in 50

489

mg/ml (A, C, E) and 2 mg /ml (B, D, F) of cantaloupe peel extract at 10°C (□) and 22°C ( ) in

490

1 day (A, B),4 days (C, D) and 7 days (E, F). : Bars with different lowercase letters indicate

491

significant differences between biofilms formed at the same temperature and * indicate

492

significant difference between biofilms belong to the same strain formed at different temperature

493

based on Tukey’s Honestly Significant Difference test ANOVA test (P < 0.05). Error bars

494

indicate standard error.

495

Fig. 5. Biofilm formation by L. monocytogenes 2011L-2625 on four food-contact processing

496

surfaces in 50 mg/ml (A, C, E) and 2 mg /ml (B, D, F) of cantaloupe flesh extract at 10°C (□)

497

and 22°C ( ) in 1 day (A, B),4 days (C, D) and 7 days (E, F). Bars with different lowercase

498

letters indicate significant differences between biofilms formed at the same temperature.

499

Fig. 6. Biofilm formation by L. monocytogenes 2011L-2625 on four food-contact processing

500

surfaces in 50 mg/ml (A, C, E) and 2 mg /ml (B, D, F) of cantaloupe peel extract at 10°C (□) and

501

22°C ( ) in 1 day (A, B),4 days (C, D) and 7 days (E, F). Bars with different lowercase letters

502

indicate significant differences between biofilms formed at the same temperature.

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Highlights Low concentrations cantaloupe extracts can induce Lm growth and biofilm formation.

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Lm 2011L-2625 biofilms was least on buna-n rubber when compared to other surfaces.

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No differences in biofilm formation for six Lm strains tested in cantaloupe extracts.

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Lm biofilms increased with higher conc. of cantaloupe extracts at 20°C and 10°C.

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