Inhibition of Staphylococcus aureus by antimicrobial biofilms formed by competitive exclusion microorganisms on stainless steel

    Inhibition of Staphylococcus aureus by antimicrobial biofilms formed by competitive exclusion microorganisms on stainless steel Hyeri Son, Sunhyung Park, Larry R. Beuchat, Hoikyung Kim, Jee-Hoon Ryu PII: DOI: Reference:

S0168-1605(16)30467-6 doi: 10.1016/j.ijfoodmicro.2016.09.007 FOOD 7376

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

20 June 2016 31 August 2016 11 September 2016

Please cite this article as: Son, Hyeri, Park, Sunhyung, Beuchat, Larry R., Kim, Hoikyung, Ryu, Jee-Hoon, Inhibition of Staphylococcus aureus by antimicrobial biofilms formed by competitive exclusion microorganisms on stainless steel, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.09.007

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ACCEPTED MANUSCRIPT Inhibition of Staphylococcus aureus by Antimicrobial Biofilms Formed by Competitive

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Exclusion Microorganisms on Stainless Steel

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea

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a

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Hyeri Sona, Sunhyung Parka, Larry R. Beuchatb, Hoikyung Kimc, *, and Jee-Hoon Ryua, *

University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea Center for Food Safety and Department of Food Science and Technology, University of

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b

c

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Georgia, 1109 Experiment Street, Griffin, Georgia 30223-1797, USA Department of Food and Nutrition, Wonkwang University, 460 Iksandae-ro, Iksan, Jeonbuk

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54538, Republic of Korea

* Corresponding author Tel.: + 82 2 3290 3409; fax: + 82 2 3290 3918. E-mail address: [email protected] (Jee-Hoon Ryu). Tel.: + 82 63 850 6894; fax: + 82 63 850 7301. E-mail address: [email protected] (Hoikyung Kim).

ACCEPTED MANUSCRIPT Abstract The goal of this study was to develop a desiccation resistant antimicrobial surface

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using biofilm of competitive exclusion (CE) microorganism inhibitory to Staphylococcus

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aureus. We isolated 161 microorganisms from soils, foods, and food-contact surfaces that are

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inhibitory to S. aureus. Among them, three CE microorganisms (Streptomyces spororaveus strain Gaeunsan-18, Bacillus safensis strain Chamnamu-sup 5-25, and Pseudomonas azotoformans strain Lettuce-9) exhibiting strong antibacterial activity and high growth rates

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were selected for evaluation. These isolates formed biofilms within 24 h on stainless steel

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coupons (SSCs) immersed in Bennet’s broth and tryptic soy broth (TSB) at 25°C. Cells in these biofilms showed significantly (P  0.05) enhanced resistance to a desiccation (43%

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relative humidity [RH]) compared to those attached to SSCs but not in biofilms. The

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antimicrobial activities of biofilms formed by these isolates on SSCs against S. aureus at 25°C and 43% RH were determined. Compared to SSCs lacking biofilms formed by CE

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microorganisms, populations of S. aureus on SSCs harboring CE biofilms were significantly lower (P  0.05). Results indicate that persistent antimicrobial activity against S. aureus on

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stainless steel surfaces can be achieved by the presence of biofilms of CE microorganisms. This information will be useful when developing strategies to improve the microbiological safety of foods during storage, processing, and distribution by facilitating the development of effective antimicrobial food-contact surfaces.

Keywords: Competitive-exclusion microorganisms, Biofilm, Staphylococcus aureus, Stainless steel, Antimicrobial surface

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ACCEPTED MANUSCRIPT 1. Introduction Staphylococcal intoxication is one of the most common foodborne diseases

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worldwide. It results from ingestion of foods containing staphylococcal enterotoxin produced

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mainly by Staphylococcus aureus (Lawley, 2008; Seo and Bohac, 2013). The most common

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symptoms of staphylococcal foodborne intoxication are nausea, vomiting, and abdominal cramping (Forsythe, 2010). In the United States, it is estimated that 60 outbreaks, 1,502 illness, 67 hospitalization and 1 death occurred during 2011-2014 due to Staphylococcal food

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poisoning (CDC, 2014). S. aureus is highly resistant to desiccation and is not uncommonly

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found on food-processing equipment and surrounding environmental surfaces, becoming a source of food contamination (Bennett and Monday, 2003; Chaibenjawong and Foster, 2011;

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Hennekinne et al., 2012; Landgraf and Destro, 2013). To prevent S. aureus intoxication, it is

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important to inhibit the growth of S. aureus on food-contact surfaces as well as on surfaces in the surrounding environment.

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To inhibit the growth of hazardous microorganisms on food-contact surfaces and in food processing environments, biological methods which do not leave sanitizer residues,

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affect sensorial properties of foods, or cause corrosion of metals, have been examined. These methods typically involve the use of competitive exclusion (CE) microorganisms which have antagonistic activity against foodborne pathogens. The mechanisms of CE microorganisms to inactivate or prevent growth of pathogens are based on competition for attachment sites or nutrients, production of antimicrobial substances, or more rapid growth (Gálvez et al., 2012; Ukuku et al., 2015). Several studies focused on inactivation of Listeria monocytogenes, Salmonella enterica, and enteropathogenic Escherichia coli using CE microorganisms have been reported (Belák and Maráz, 2015; Leverentz et al., 2006; Liao, 2009). However, the inhibition of S. aureus by CE microorganisms on abiotic surfaces has not been reported. Biofilms have been defined as sessile bacterial communities attached to a biological 3

ACCEPTED MANUSCRIPT or abiotic surface, usually embedded in extracellular polymeric substances (EPSs) (Cloete et al., 2009; Costerton et al., 1999). Because EPSs act as physical barriers against environmental

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stresses, biofilm-embedded foodborne pathogens exhibit increased resistance to detrimental

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effects of stresses such as desiccation, antibiotics, and sanitizers (Kim et al., 2007; Olson et

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al., 2002; Robbins et al., 2005; Ryu and Beuchat, 2005a, 2005b). The increased resistance of hazardous microorganisms in biofilms to environmental stresses has therefore been a focus of research, with the aim of developing effective decontamination measures. In the study we

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report here, it was hypothesized that biofilm formation by CE microorganisms that inhibit S.

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aureus on an abiotic surface would be enhanced by their resistance to desiccation stress. If this hypothesis is true, antimicrobial biofilms formed by CE microorganisms on abiotic

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surfaces may have application to control the growth of S. aureus. For examples, the biofilms

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of CE microorganisms could be applied to the surfaces encountered during production of agricultural commodities, storage of raw and processed foods, production of processed foods,

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transportation and distribution of foods, etc. The objective of this study was to develop a desiccation resistant antimicrobial

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surface using biofilm of CE microorganism inhibitory to Staphylococcus aureus. To achieve this goal, we isolated and identified CE microorganisms from soils, foods, and food-contact surfaces that were inhibitory to S. aureus and evaluated their ability to form biofilms. The desiccation resistance profiles of CE microorganisms in biofilms formed on stainless steel were examined and their antimicrobial activities against S. aureus were confirmed.

2. Materials and methods 2.1. Strains of S.aureus used Five strains of S. aureus were used: ATCC 25923 (clinical isolate), KCTC 1928 (clinical isolate), ATCC 13565 (isolated from an outbreak associated with ham), ATCC 4

ACCEPTED MANUSCRIPT 23235 (isolated from turkey salad), and ATCC 27664 (isolated from chicken tetrazzini). Cryopreserved S. aureus cells were activated in 10 mL of tryptic soy broth (TSB; BBL/Difco,

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Sparks, MD, USA) at 37C for 24 h. Each of the five activated strains was transferred to 10

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mL of TSB at 37C at three consecutive 24 h intervals. A S. aureus cocktail (10 mL) was

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prepared by combining 2 mL of each of the five strains. The cocktail was centrifuged at 2,002  g for 15 min at room temperature (22±2C), supernatants were decanted, and the cells were

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resuspended in 10 mL of sterile 0.1% peptone water (PW). This procedure was repeated, and the suspensions were serially diluted in 0.1% PW to prepare S. aureus inocula (ca. 7 or 5 log

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CFU/mL).

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2.2. Isolation of microorganisms from soil, foods, and food-contact surfaces

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To screen for microorganisms with antimicrobial activity against S. aureus, isolates

contact surfaces.

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with different colony colors and morphologies were collected from soil, foods, and food-

Soil samples were collected from 30 diverse locations, including mountains, lakes,

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and dams, in the Republic of Korea. Samples were collected as described by Kim et al. (2011). Briefly, samples were collected from a depth of 10 cm in the ground. Subsamples (1 g) were suspended in 10 mL of sterile distilled water (DW) and incubated at 25C for 1 to 3 h with shaking at 150 rpm. The supernatant (0.1 mL) was spread-plated on humic acid vitamin agar medium and incubated at 28C for 3 to 14 days. Cells from selected colonies were transferred using a loop to 10 mL of 20% glycerol in DW and stored at -80C. Fresh produce (lettuce [Lactuca savita var. crispa], iceberg lettuce [Lactuca savita var. capitata], cabbage [Brassica oleracea var. capitata], red cabbage [Brassica oleracea var. capitata], Chinese cabbage [Brassica rapa], perilla leaf [Perilla frutescens var. japonica], spring greens [Brassica campestris], chicory [Cichorium intybus], and mustard greens 5

ACCEPTED MANUSCRIPT [Brassica juncea]) and fishery products (fish [mackerel and yellow croaker], crustaceans [shrimp], and shellfish [mussel and ark shell]) were purchased from eight stores in traditional

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wholesale markets (Cheongnyang wholesale market and Noryangjin fisheries wholesale

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market, Seoul, Republic of Korea). Samples (10 g) were combined with 100 mL of sterile

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0.1% PW and pummeled for 2 min using a stomacher (Interscience BagMixer® 400W; Interscience, Saint Nom, France). The homogenate was spread-plated on tryptic soy agar (TSA; BBL/Difco) supplemented with 0.1% (w/v) sodium pyruvate (Kanto Chemical Co,

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Inc., Tokyo, Japan), and incubated at 25C for 48 h. Colonies with different colors and

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morphologies were streaked on TSA, followed by incubation at 25C for 24 h and storage at 4C.

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To isolate microorganisms from food-contact surfaces, cutting boards and knives that

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had been used in two modern supermarkets and four stores in a traditional fisheries wholesale market located in Seoul were swabbed 100 times using a swab slightly moistened with sterile

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DW. The swabs were placed separately in 50 mL conical tubes containing 10 mL of sterile 0.1% PW, followed by vortexing for 2 min. Microorganisms were isolated using the methods

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described above for foods.

To evaluate isolates for antimicrobial activity against S. aureus, isolates from soil were streaked on Bennet’s agar and incubated at 25C for 6 days. One to five colonies per plate were transferred to 50 mL conical tubes containing 10 mL of TSB and 0.5 g of sterile glass beads (1 mm, Glass beads 1, Glastechnique Mfg., Germany), followed by incubation at 25C for at least 3 days with shaking at 200 rpm. Isolates from foods or food-contact surfaces were inoculated in TSB at 25C and transferred three times at 24 h intervals before examining for CE activity against S. aureus.

2.3. Double layer assay to screen CE microorganisms for inhibitory activity against S. aureus 6

ACCEPTED MANUSCRIPT The ability of isolates from soil, foods, and food-contact surfaces to inhibit S. aureus was determined using a double-layer assay (Zhao et al., 2004). Suspensions (10 µL) of cells

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of isolates prepared as described above were spot-inoculated onto TSA plates (spot diameter

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ca. 7 mm; four isolates per plate). Plates were held in a laminar flow biosafety hood at room

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temperature (22±2C) for 30 min and then incubated at 25C for 24 h. Molten TSA (10 mL) containing S. aureus (ca. 5 log CFU/mL) was poured onto the surface of TSA and the plates were dried in a laminar flow biosafety hood for 30 min followed by incubation at 37C for up

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to 24 h. The antimicrobial activity of isolates against S. aureus was assessed by measuring

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the diameters of zones of inhibition surrounding colonies.

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2.4. Identification of CE microorganisms inhibitory to S. aureus

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To identify the genus and species of isolates with strong antimicrobial activity against S. aureus, 16S rRNA sequence analysis was performed (http://www.macrogen.co.kr, Seoul,

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Korea). Sequences were analyzed using BLAST software available on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/, USA).

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Phylogenetic analysis of the 16S rRNA gene sequences of isolates was performed using Mega software version 5.05 with the neighbor-joining method. Bootstrap values are expressed as percentages of 1,000 replications.

2.5. Biofilm formation by CE microorganisms on stainless steel Stainless steel coupons (SSCs; Type 304, 5  2 cm, no. 4 finished) were washed using 15% phosphoric acid and 15% alkaline detergent as described by Nam et al. (2014). The washed SSCs were boiled in DW for 15 min, dried for 24 h, and autoclaved at 121C for 15 min. Suspensions of CE microorganisms prepared as described above were centrifuged at 7

ACCEPTED MANUSCRIPT 2,002  g for 15 min at room temperature (22±2C), and pelleted cells were resuspended in 10 mL of phosphate-buffered saline (PBS, pH 7.4). The procedure was repeated and cell

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suspensions were diluted in PBS to a density of ca. 5 log CFU/mL and deposited in a sterile

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sprayer (30 mL, code 46322; Daiso, Seoul, Korea). To attach CE microorganisms on SSC

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surfaces, a sterile SSC was deposited in a polystyrene dish (60 mm diameter by 8 mm height) which had been placed in a Petri dish (90 mm diameter by 15 mm height; SPL, Seoul, Korea). Suspensions (ca. 1 mL) of CE microorganisms were sprayed onto the surface of SSCs at an

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angle of 90 from the vertical and a height of 5 cm, followed by drying at room temperature

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(22±2C) for 1 h in a laminar flow biosafety hood. SSCs with attached cells were transferred to a new Petri dish, and 4 mL of Bennet’s broth (S. spororaveus strain Gaeunsan-18 and B.

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safensis strain chamnamu-sup 5-25) or TSB (P. azotoformans strains Lettuce-9) were

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deposited onto the SSC surfaces. The Petri dishes were sealed using Parafilm (Bemis, Neenah, WI, USA) and incubated at 25C for 0, 24, 48, 72, 96, or 120 h. After incubation, SSCs were

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removed from the Petri dish and rinsed in sterile DW (400 mL) with gentle movement in a circular motion for 5 s, and then placed in conical tubes containing 30 mL of 0.1% PW and 3

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g of glass beads (425 to 600 m, Sigma-Aldrich, St. Louis, MO, USA). The mixtures were vortexed at maximum speed for 1 min and suspensions were serially diluted in 0.1% PW. Undiluted (0.1 mL) or serially diluted (0.1 mL) suspensions were surface plated on Bennet’s agar or TSA using a spiral plater (Easy Spiral®; Interscience) and incubated at 25C for up to 72 h before colonies were counted. The theoretical detection limit of the spiral-plating method was 2.5 log CFU/coupon.

2.6. Attachment of CE microorganisms to stainless steel coupons To prepare SSCs containing attached CE microorganisms, suspensions (ca. 6.2, 9.1, and 8.9 log CFU/mL) of S. spororaveus strain Gaeunsan-18, B. safensis strain Chamnamu8

ACCEPTED MANUSCRIPT sup 5-25, and P. azotoformans strain Lettuce-9 were sprayed onto SSCs as described above

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and dried in a laminar flow biosafety hood at room temperature (22±2C) for 1 h.

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2.7. Survival of CE microorganisms in biofilms on SSCs under desiccating conditions

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The resistance of CE microorganisms attached to or embedded in biofilms on SSCs against desiccating conditions (43% relative humidity [RH]) was determined. To create a 43% atmospheric RH environment, 4 mL of saturated potassium carbonate (aw 0.4300.005;

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Daejung) solution was deposited in a Petri dish. The dish was sealed with Parafilm and stored

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at 25C for at least 24 h before use.

SSCs containing attached CE microorganisms or cells in biofilms prepared as

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described above were placed in a polystyrene dish, which had been placed in a Petri dish

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adjusted to 43% RH. The Petri dish was sealed with Parafilm and incubated at 25C for up to 5 days (120 h). After incubating for 0, 24, 48, 72, 96, or 120 h, SSCs were transferred to 50

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mL conical centrifuge tubes containing 3 g of glass beads and 30 mL of TSB, and vortexed at maximum speed for 1 min. The suspension was serially diluted in 0.1% PW. To determine

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the number of CE microorganisms on SSCs, undiluted suspensions (0.25 mL in quadruplicate and 0.1 mL in duplicate) and diluted suspensions (0.1 mL in duplicate) were surface-plated on Bennet’s agar or TSA and incubated at 25C for 72 h. The remaining suspensions were enriched at 25C for 72 h. If no colonies formed on Bennet’s agar or TSA plates, the enriched suspensions were streaked on Bennet’s agar or TSA and incubated at 25C for 72 h to verify the presence or absence of CE microorganisms. The theoretical detection limits for direct plating and enrichment were 1.5 log and 0.0 log CFU/coupon, respectively.

2.8. Antimicrobial activities of CE microorganisms in biofilms on SSCs against S. aureus Biofilms of CE microorganisms were formed on SSCs as described above. A 0.1 mL 9

ACCEPTED MANUSCRIPT S. aureus suspension (ca. 5 log CFU/mL) was spot-inoculated on the surface of SSCs (18±2 drops/coupon) with or without biofilms. The inoculated SSCs were placed in a Petri dish in

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which the RH had been adjusted to 43% and incubated at 25C for up to 48 h. After

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incubation for 0, 3, 6, 12, 24 or 48 h, the SSCs were transferred to 50 mL conical centrifuge

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tubes containing glass beads (3 g) and TSB (30 mL) and vortexed at maximum speed for 1 min. Suspensions were serially diluted in 0.1% PW, surface-plated on mannitol salt agar

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(MSA; BBL/Difco), and incubated at 37C for 24 h before colonies were counted. The remaining suspensions were enriched at 37C for 24 h. If no presumptive S. aureus colonies

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formed on MSA plates, enriched suspensions were streaked on MSA followed by incubation at 37C for 24 h. The theoretical detection limits for direct plating and enrichment were 1.5

2.9. Statistical analysis

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and 0.0 log CFU/coupon, respectively.

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All experiments were performed at least three times and two SSCs were examined in each replication. Data were analyzed using a general linear model with Statistical Analysis

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System software (SAS 9.3; SAS Institute, Cary, NC). Results were compared using Fisher’s least significant difference (LSD) test or SAS proc t-test. Significant differences at a 95% confidence level (P ≤ 0.05) are presented.

3. Results 3.1. Isolation and identification of CE microorganisms inhibitory to S. aureus In total, 2,065 microorganisms were isolated from soil (1,648 isolates), foods (312 isolates), and food-contact surfaces (105 isolates); 161 isolates showed inhibitory activities against S. aureus in double-layer assays (Table 1). The 17 isolates causing largest zones of inhibition are listed in Table 2. Of the isolates from soil, Chamnamu-sup 5–25 showed the 10

ACCEPTED MANUSCRIPT largest zone (3.7 cm) followed by Sanrimdongmulwon 1–3 and Gaeunsan-18. Among the isolates from fresh produce, Lettuce-9 caused the largest zone of inhibition (3.9 cm),

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followed by Perilla leaf-7 (3.7 cm) and Iceberg lettuce-20 (3.6 cm). When were cultured in

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TSB at 25°C, populations of 11 of the isolates (including Gaeunsan-18, Chamnamu-sup 5–25

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and Lettuce-9) increased to >9 log CFU/mL within 72 h (data not shown). Based on these results, Gaeunsan-18, Chamnamu-sup 5–25 and Lettuce-9 were evaluated for antimicrobial activity against S. aureus in subsequent experiments.

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Gaeunsan-18, Chamnamu-sup 5–25 and Lettuce-9 isolates were identified by 16S

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rRNA analysis and NBI BLAST software to be Streptomyces spp., Bacillus spp. and Pseudomonas spp., respectively. Using a neighbor-joining phylogenetic tree, Gaeunsan-18,

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Chamnamu-sup 5–25 and Lettuce-9 isolates were designated as Streptomyces spororaveus

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strain Gaeunsan-18, Bacillus safensis strain Chamnamu-sup 5-25, and Pseudomonas

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azotoformans strain Lettuce-9 (Supplementary Figure 1).

3.2. Biofilm formation by CE microorganisms on SSCs

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Figure 1 shows populations of S. spororaveus strain Gaeunsan-18, B. safensis strain Chamnamu-sup 5-25, and P. azotoformans strain Lettuce-9 in biofilms formed on the surface of SSCs immersed in Bennet’s broth or TSB at 25°C for up to 120 h. The initial populations of CE microorganisms attached to SSCs were 2.6 to 3.3 log CFU/coupon. Populations increased significantly (P ≤ 0.05) within 24 h, reaching 7.9 to 8.5 log CFU/coupon within 48 h; however, populations did not increase during incubation for an additional 3 days. These results demonstrate that S. spororaveus strain Gaeunsan-18, B. safensis strain Chamnamu-sup 5-25, and P. azotoformans strain Lettuce-9 can form biofilms on SSCs immersed in Bennet’s broth or TSB at 25°C.

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ACCEPTED MANUSCRIPT 3.3. Survival of CE microorganisms in biofilms on SSCs under desiccating conditions Figure 2 shows numbers of CE microorganisms attached to SSCs or in biofilms on

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SSCs exposed to 43% RH at 25°C for 0, 24, 48, 72, 96, and 120 h. Populations of each strain

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decreased significantly (P ≤ 0.05) as incubation time progressed, regardless of whether cells

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were attached or in biofilms. However, at a given incubation time, the number of CE microorganisms in biofilms was significantly (P ≤ 0.05) higher than that of attached cells. Among the CE microorganisms tested, P. azotoformans strain Lettuce-9 in biofilms showed

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the highest tolerance to 43% RH. For example, the number of cells attached to SSCs

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decreased by >3.4 log CFU/coupon within 24 h of drying, whereas the reduction in biofilm

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was 0.8 log CFU/coupon.

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3.4. Antimicrobial activities of CE microorganisms in biofilms against S. aureus on SSCs Figure 3 shows populations of S. aureus inoculated on SSCs with or without biofilms

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of CE microorganisms and incubated at 25°C and 43% RH for 0, 3, 6, 12, 24, or 48 h. The initial S. aureus population on SSCs was ca. 4.2 log CFU/coupon. After incubating SSCs

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without biofilms for 48 h, populations were ca. 3.0 log CFU/coupon. In contrast, the population of S. aureus decreased more rapidly on SSCs containing biofilms of CE microorganisms. Populations of S. aureus exposed to biofilms of S. spororaveus strain Gaeunsan-18, B. safensis strain Chamnamu-sup 5-25, and P. azotoformans strain Lettuce-9 decreased significantly (P ≤ 0.05) within 48 h to 1.9, 1.8, and 1.9 log CFU/coupon, respectively.

4. Discussion The hypothesis preceding initiation of this study was that biofilms of CE microorganisms on stainless steel would exhibit enhanced resistance to desiccation and have 12

ACCEPTED MANUSCRIPT persistent antimicrobial activity against S. aureus. Initially, 161 of 2,065 isolates (7.8%) from various samples of soils, foods, and food-contact surfaces were found to inhibit the growth of

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S. aureus. Of these 161 isolates, three with considerable antibacterial activity and high

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growth rates were selected for further evaluation. They were identified as Streptomyces

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spororaveus (strain Gaeunsan-18), Bacillus safensis (strain Chamnamu-sup 5-25), and Pseudomonas azotoformans (strain Lettuce-9). The genus Streptomyces is frequently used for the production of antibiotics (Dastager et al., 2007) and is distributed in soil and aquatic

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environments (Saadoun et al., 2002). Although antimicrobial effects of various Streptomyces

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spp. have been reported, the lethality of S. spororaveus against foodborne pathogens has not been described to date. B. safensis can be found in desert soil (Raja and Omine et al., 2012),

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root tubers (Singh et al., 2013), and the rhizosphere (Kothari et al., 2013). A number of

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reports have described the lethal activity of the Bacillus spp. against foodborne pathogens (Földes et al., 2000; La Ragione et al., 2003). However, to the best of our knowledge, the

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antimicrobial activity of B. safensis against foodborne pathogens has not been documented. Pseudomonas spp. are present in soil and water, and on the surface of plant roots (Paulsen,

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2005). Production of various antibiotics by Pseudomonas spp., e.g., P. graminis and P. syringae, has been reported (Alegre et al., 2013; Janisiewicz et al., 1999), but the antimicrobial activity of P. azotoformans against foodborne pathogens has not been described. We evaluated the ability of the three CE isolates to form biofilms on the surface of SSCs. Isolates were able to form biofilms on SSCs immersed in Bennet’s broth or TSB at 25°C within 24 h. It has been reported that Streptomyces spp. and Bacillus spp. can form biofilms on abiotic surfaces, although biofilm formation by S. spororaveus and B. safensis on SSCs has not been documented. Biofilm formation by P. azotoformans on SSC surfaces has not been reported; however, biofilm formation on other abiotic surfaces has been described. Arkatkar et al. (2010) reported that P. azotoformans formed biofilm on polypropylene films 13

ACCEPTED MANUSCRIPT incubated at 35 to 37°C. The resistance of CE microorganisms in biofilms to desiccation (25°C and 43% RH)

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was determined. The survival of CE microorganisms in biofilms on SSCs was significantly

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higher (P ≤ 0.05) than that of cells attached to SSCs without biofilm formation. Enhanced

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resistance of CE microorganisms against desiccation was not unexpected. It has been reported that the formation of biofilms increases cell resistance to environmental stresses, including desiccation. Van de Mortel et al. (2005) inoculated Pseudomonas putida onto the

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filter membranes and formed biofilms, followed by exposure to 56%, 76%, 85%, 92%, and

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100% RH at room temperature for up to 120 h. After 120 h, the survival rate of the P. putida in biofilms was 4- to 90-fold higher than that of membrane-attached cells. Hansen and Vogel

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(2011) incubated SSCs containing attached L. monocytogenes cells or cells in biofilms at

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15°C and 43% RH for up to 23 days, and found that populations in the biofilms were significantly (P ≤ 0.05) higher (1.1 log CFU/cm2) than populations of attached cells. The

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authors suggested that the enhanced resistance of cells in biofilms against desiccation was due in part to the cell envelope and/or EPS composition.

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In the final phase of the study, we investigated the lethal effects of biofilms of CE microorganisms formed on SSCs against S. aureus at 43% RH. We used five strains-cocktail culture of S. aureus to avoid the strain-dependant phenomena and tried to simulate the situation that CE biofilm were exposed to dry condition (43% RH ) at normal warm temperature (25C). It was observed that S. aureus was more rapidly inactivated when inoculated on the SSCs containing CE biofilm compared to the SSCs lacking CE biofilm. This indicates that biofilm formed by CE microorganisms on SSCs had antimicrobial activity against S. aureus. It is possible that CE microorganisms in biofilms may have caused sublethal injury of a portion of S. aureus cells, resulting in an underestimation of the number of survivors. Control of S. aureus on abiotic surfaces on which biofilms of CE 14

ACCEPTED MANUSCRIPT microorganisms had formed has not been reported. However, inactivation of other foodborne pathogens by biofilms of CE microorganisms has been described. Kim et al. (2013) evaluated

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the antimicrobial effects Paenibacillus polymyxa biofilms. Strong antimicrobial activity

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against E. coli O157:H7 at 25°C and 43, 85, and 100% RH was observed. SSCs with biofilms

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of P. polymyxa significantly (P ≤ 0.05) reduced the number of E. coli O157:H7 within 48 h, and the lethal effect was enhanced as the RH was decreased. Mariani et al. (2011) reported that the formation of biofilms of lactic acid bacteria on wooden shelves used for cheese

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ripening suppresses the growth of L. monocytogenes at 15°C and 98% RH. The mechanisms

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of CE microorganisms in inhibiting S. aurues strains were not investigated in this study. If CE microorganisms produce antimicrobial substances, then their toxicity to human and

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possibilities in causing antibiotic resistant microorganisms should be evaluated. If CE

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biofilms are established on food-contact surfaces, some portions of CE microorganisms would be transferred into foods, possibly resulting in facilitating food spoilage. Therefore, the

investigated.

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influence of CE microorganisms on the early spoilage of food products should be

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In summary, we have demonstrated that biofilms formed by three CE microorganisms on stainless steel inhibit the growth of S. aureus. Two CE microorganisms evaluated in the study (Bacillus safensis strain Chamnamu-sup 5-25 and Pseudomonas azotoformans strain Lettuce-9) have been deposited in the Korean Agricultural Culture Collection (KACC). Designated strain numbers are KACC 92124P, and KACC 92125P, respectively. In future research, larger-scaled validation experiments using stainless steel surfaces in food production facilities should be performed. Biofilm formation of the CE microorganisms on other types of surfaces such as plastic, glass, concrete, tile, etc. should be investigated. The influence of environmental conditions such as the presence of soil and indigenous microorganisms including viable but not-culturable cells on the bioflm formation of CE 15

ACCEPTED MANUSCRIPT microorganisms should be further studied. In addition, the optimum environmental conditions should be determined for maximizing the lethality of CE biofilms on abiotic surfaces. Our

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findings provide useful information when developing strategies involving the use of CE

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biofilms to lower the microbiological safety risks in food storage, processing, and distribution

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environments.

Acknowledgements

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This study was supported by a National Research Foundation of Korea (NRF) grant funded

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by the Korean Government (MSIP) (NRF-2013R1A2A2A01068475) and by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries,

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Republic of Korea (No. 316011-05-1-HD060).

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stainless steel: effect of exopolysaccharide and curli production on its resistance to

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Saadoun, I., Gharaibeh, R., 2002. The Streptomyces flora of Jordan and its' potential as a source of antibiotics active against antibiotic-resistant Gram-negative bacteria. World J.

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Seo, S., Bohac, G. A., 2013. Staphylococcus aureus. In: Doyle, M. P., Buchanan, R. L. (Eds.),

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new endoinulinase producing bacterial strain of Bacillus safensis AS-08. Biologia 68, 1028-1033.

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ACCEPTED MANUSCRIPT Figure legends (Son et al.) Figure 1.

Maturation of biofilms formed by Streptomyces spororaveus strain Gaeunsan-18

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(○); Bacillus safensis strain Chamnamu-sup 5-25 (□); and Pseudomonas

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azotoformans strain Lettuce-9 (△) on SSCs. Cultures were incubated on a shaker

at 200 rpm and 25°C for 5 days in Bennet’s broth (○,□) or TSB (△), followed by

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harvesting cells, suspending cells in PBS, and attaching cells to SSCs at 25°C for

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1 h. SSCs were immersed in 4 mL of Bennet’s broth (○, □) or TSB (△), followed by incubation at 25°C. Points indicate mean values ± standard deviations of

Survival of (A) Streptomyces spororaveus strain Gaeunsan-18; (B) Bacillus

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Figure 2.

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replicate experiments.

safensis strain Chamnamu-sup 5-25; and (C) Pseudomonas azotoformans strain

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Lettuce-9 attached to (□) and in biofilms on (○) SSCs under desiccation conditions. SSCs harboring attached cells or biofilms were incubated at 25°C and

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43% RH for up to 5 days. The detection limits by direct plating (dotted line) and enrichment were 1.5 and 0.0 log CFU/coupon, respectively. Points indicate mean values ± standard deviations of replicate experiments.

Figure 3.

S. aureus populations on SSCs, with (○) or without (□) biofilms of (A) Streptomyces spororaveus strain Gaeumsan-18, (B) Bacillus safensis strain Chamnamu-sup 5-25, and (C) Pseudomonas azotoformans strain Lettuce-9, incubated at 43% RH and 25°C for up to 48 h. The detection limits by direct plating (dotted line) and enrichment were 1.5 and 0.0 log CFU/coupon, respectively. Points are mean values ± standard deviations of replicate experiments. 21

ACCEPTED MANUSCRIPT

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Supplementary Figure 1. Neighbor-joining phylogenetic tree based for 16S rRNA gene

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sequences of (A) Streptomyces spororaveus strain Gaeunsan-18

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and related Streptomyces spp., (B) Bacillus safensis strain Chamnamu-sup 5-25 and related Bacillus spp., and (C) Pseudomonas azotoformans strain Lettuce-9 and related

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Pseudomonas spp. Bootstrap values of 1,000 replications are

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noted at the branch points. Scale bar, 0.01 substitutions per nucleotide position. PCR primers used in the study were 27F

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( AGA GTT TGA TCM TGG CTCA G) and 1492R (TAC GGY

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TAC CTT GTT ACG ACT T).

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ACCEPTED MANUSCRIPT Table 1. Numbers of microbial isolates collected from soil, foods and food contact surfaces.

Number of isolates inhibitory to S. aureus

Soil

1,648

142

Foods

312

19

3

Food contact surfaces

105

0

0

Total

2,065

161

6

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Number of isolates with large inhibition zone (>30 mm in diameter)a

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Total number of isolates

3

Antimicrobial activities of isolates against S. aureus cocktail were evaluated using a double-

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a

Source

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layer assay.

23

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Table 2. Diameters of zones of inhibition of a S. aureus (cocktail comprised of strains

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ATCC 25923, KCTC 1928, ATCC 13565, ATCC 23235, and ATCC 27664) caused by

Isolate

Diameter of inhibition zone (cm)a (mean±SD)

1

Chamnamu-sup 5–25

3.7±0.3

2

Sanrimdongmulwon 1–3

3

Gaeunsan-18

4

Daeseosandaem 3–239

2.8±0.2

Geumsan-207

2.5±0.0

Chungsando-218

2.5±0.0

7

Suwoongyo 1–227

2.4±0.0

8

Ga-pyeong-41

2.3±0.2

Soil

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5

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Rank

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Source

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competitive-exclusion microorganisms as determined by a double-layer assay.

3.4±0.0 3.2±0.1

Chamnamu-sup 6–11

1.6±1.0

1

Lettuce-9

3.9±0.2

2

Perilla leaf-7

3.7±0.2

3

Iceberg lettuce-20

3.6±0.3

4

Iceberg lettuce-7

2.9±0.3

5

Lettuce-13

1.3±0.0

6

Iceberg lettuce-19

1.0±0.1

7

Iceberg lettuce-17

0.9±0.0

8

Kimchi cabbage-8

0.8±0.1

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9

Fresh produce

a

A total of 17 isolates were screened for antimicrobial activity against a five-strain

S. aureus cocktail using a double-layer assay. 24

ACCEPTED MANUSCRIPT

9

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8

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7

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6 5 4

3 2

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CE microorganisms populations (log CFU/coupon)

10

1 0

24

48

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0

72

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Fig. 1. (Son et al., 2016)

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Time (h)

25

96

120

ACCEPTED MANUSCRIPT 10 9 8 7 6 5 4 3 2 1 0

CECE microorganism populations (log CFU/coupon) PT ED MA NU SC RI PT

A)

0

10 9 8 7 6 5 4 3 2 1 0

48

72

96

120

24

48

72

96

120

24

48

B)

0

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24

10 9 8 7 6 5 4 3 2 1 0

C)

0

72

Time (h)

Fig. 2. (Son et al., 2016)

26

96

120

ACCEPTED MANUSCRIPT 5

A)

4

CES. aureus populations (log CFU/coupon) PT ED MA NU SC RI PT

3 2 1 0

0

5

12

24

36

48

12

24

36

48

B)

4 3 2 1 0

0

5

C)

4

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3 2 1 0

0

12

24

Time (h)

Fig. 3. (Son et al., 2016) 27

36

48

ACCEPTED MANUSCRIPT 71

Streptomyces subrutilus strain NBRC 13388T Streptomyces avidinii strain NBRC 13429T

47

89

Streptomyces vinaceus strain CSSP186T Streptomyces cirratus strain CSSP547T Streptomyces nojiriensis strain NBRC 13794T

10 42

Streptomyces xanthophaeus strain NBRC 12829T

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Streptomyces nojiriensis strain LMG 20094T 9

Streptomyces spororaveus strain NBRC 15456T 12

Gaeunsan-18

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72

T

33

Streptomyces lavendulae strain NBRC 12789T

60

Streptomyces colombiensis strain NRRL B-1990T

43

Streptomyces goshikiensis strain NRRL B-5428T

55

94

55

Streptomyces sporoverrucosus strain NBRC 15458T

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Streptomyces manipurensis strain MBRL 201T Streptomyces cinnamonensis strain NBRC 15873T Streptomyces yokosukanensis strain NRRL B-3353T

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Actinomadura madurae ATCC 19425T

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Actinomadura madurae ATCC 19425

74

77

Bacillus axarquiensis strain CR-119

23

AC

93

T

T

Bacillus mojavensis strain NBRC 15718

92

76

Bacillus malacitensis strain LMG 22477

Bacillus subtilis strain 168

T

Bacillus vallismortis strain DSM 11031 Bacillus nematocida strain B-16

20 42

Bacillus amyloliquefaciens strain MPA 1034 T

Bacillus licheniformis strain DSM 13

31 86

T

Bacillus sonorensis strain NBRC 101234 Chamnamu-sup 5-25 Bacillus safensis strain FO-36b

77

T

Bacillus safensis strain NBRC 100820 Bacillus pumilus strain ATCC 7061

97

T

T

Bacillus stratosphericus strain 41KF2a Bacillus aerophilus strain 28K

66

28

Bacillus aerius_strain 24K

T

T

Actinomadura madurae ATCC 19425

28

T

T

Bacillus altitudinis strain 41KF2b

62

T

T

Bacillus atrophaeus 1942 strain 1942

100

T

T

T

T

ACCEPTED MANUSCRIPT

Pseudomonas proteolytica strain CMS 64

59 55

T T

T

Pseudomonas gessardii strain CIP 105469

Pseudomonas brenneri strain CFML 97-391

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64

Pseudomonas libanensis strain CIP 105460 28

Pseudomonas synxantha strain NBRC 3913

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23 55

T

T T

Pseudomonas mucidolens strain NBRC 103159

21

T

Lettuce-9 52

71

Pseudomonas azotoformans strain NBRC 12693 T

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Pseudomonas panacis strain CG20106

35

Pseudomonas cedrina strain CFML 96-198 Pseudomonas migulae strain CIP 105470

T

T

Pseudomonas chlororaphis strain ATCC 13985

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83

Pseudomonas chlororaphis strain NCIB 10068

56

Pseudomonas lini strain DLE411J

96

D

Pseudomonas brassicacearum strain NFM421

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29

T

T

T

Actinomadura madurae ATCC 19425

T

T

T

ACCEPTED MANUSCRIPT Highlights

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▶ Antimicrobial activity of CE biofilms on stainless steel to S. aureus was determined.

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▶ Three CE microorganisms inhibitory to S. aureus were isolated and identified.

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▶ CE microorganisms formed biofilms on stainless steel surfaces at 25°C within 24 h.

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▶ Resistance of CE microorganisms to desiccation was enhanced after biofilm formation.

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▶ Antimicrobial activity of CE microorganisms was maintained after biofilm formation.

30