Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection

Accepted Manuscript Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection Pattanan Yatip, D. Nitin Chandra Teja, Timothy W. Flegel, Chumporn Soowannayan PII:

S1050-4648(17)30685-X

DOI:

10.1016/j.fsi.2017.11.008

Reference:

YFSIM 4942

To appear in:

Fish and Shellfish Immunology

Received Date: 1 August 2017 Revised Date:

28 September 2017

Accepted Date: 6 November 2017

Please cite this article as: Yatip P, Nitin Chandra Teja D, Flegel TW, Soowannayan C, Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.11.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Extract from the fermented soybean product Natto

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inhibits Vibrio biofilm formation and reduces shrimp

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mortality from Vibrio harveyi infection

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Pattanan Yatip1, D. Nitin Chandra Teja1, Timothy W. Flegel1, 2 and Chumporn

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Soowannayan1, 2

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Center of Excellence for Shrimp Molecular Biology and Biotechnology, Mahidol University,

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Bangkok 10400, Thailand 2

National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Nueng, Klong Luang, Pathumtani, Thailand,

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Author for Correspondence: [email protected] or [email protected]

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ABSTRACT

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Many bacteria, including Vibrio pathogens of shrimp, need to colonize and/or form biofilms

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in hosts or the environment to cause disease. Thus, one possible control strategy for shrimp

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Vibriosis is biofilm inhibition. With this objective, an extract from the Japanese fermented

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soybean product, Natto was tested with the luminescent shrimp pathogen Vibrio harveyi

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(VH) for its ability to inhibit or degrade biofilm and to interfere with cell growth in broth.

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Natto is a traditional fermentation product of Bacillus subtilis var Natto (BSN1). Using 96

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well microtiter plates coated with 0.4% chitosan, we found that biofilm formation by VH was

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inhibited, while growth in parallel broth cultures was not. When an extract from Natto

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prepared using BSN1 was mixed with feed for the whiteleg shrimp Penaeus vannamei before

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ACCEPTED MANUSCRIPT immersion challenge with V. harveyi at 106 cfu/ml, survival was significantly higher (p≤0.05)

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than for control shrimp given feed without these additives. Further work done to test whether

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D-amino acids were involved in biofilm formation as previously reported for B. subtilis,

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Staphylococus aureus and Pseudomonas aeruginosa gave negative results. In conclusion, we

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discovered that Natto extract can inhibit Vibrio biofilm formation and that it or BSN1 alone

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added to shrimp feed can significantly reduce shrimp mortality in immersion challenges with

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pathogenic VH. This shows some promise for possible application against Vibriosis in

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shrimp since Natto is generally regarded as safe (GRAS) for human consumption.

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Keywords: Vibrio harveyi, biofilm, Natto, Bacillus subtilis, shrimp, vibriosis

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INTRODUCTION

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Shrimp bacterial diseases including luminescent shrimp disease caused by Vibrio species

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have long been problematic for the shrimp culture industry and have dramatically increased

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in recent years with the emergence of acute hepatopancreatic necrosis disease (AHPND) [1,

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2]. Prior to the advent of AHPND, it had been estimated that losses due to shrimp bacterial

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diseases accounted for approximately 20% of global disease losses in cultivated shrimp [3].

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AHPND is responsible for a major part of the mortality in the current epidemic of early

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mortality syndrome (EMS) that has caused a dramatic decrease in production of cultivated

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shrimp. It is now known that AHPND may be caused by several species of Vibrio that are

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capable of producing Pirvp toxins A and B [4-7]. In some countries production was reduced

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by 75% within two years [8, 9]. Although viral and parasitic disease outbreaks occur together

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with EMS, bacterial diseases still persist and are still problematic [10, 11].

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ACCEPTED MANUSCRIPT For most bacterial diseases of human and animals, it has been estimated that more than 80%

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are associated with the presence of stable bacterial communities enclosed by self-produced

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extracellular matrices or biofilms [12]. With shrimp pathogenic bacteria, such biofilms may

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be formed within the shrimp or in the environment where shrimp reside. In fact, bacterial

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biofilms have been found in the gastrointestinal (GI) tract, the stomach chambers and the

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hepatopancreas of shrimp infected with V. harveyi (e.g., tea brown gill syndrome and

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luminescent shrimp) and with V. parahaemolyticus (AHPND) [13].

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One strategy to control pathogenic bacteria is to control their ability to form biofilms using

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substances to either inhibit biofilm formation or cause biofilm degradation. Such substances

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are produced by plants, animals and microbes [14]. In this study we tested an extract from

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“Natto”, a traditional Japanese fermented soybean product. We considered this to be a good

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resource because Natto is generally regarded as safe (GRAS), so that any potential

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application that might arise from our work would not require extensive safety testing. The

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preparation of Natto involves fermentation of cooked soybeans with the bacterium Bacillus

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subtilis var natto. Natto is known to be rich in nutrients and to have relatively high

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antioxidant activities [15, 16]. Proteins derived from Natto consist of at least seventeen

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different amino acids including glutamic acid, glutamine, aspartic acid, leucine, proline,

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serine, lysine, methionine, threonine, glycine, isoleucine, tyrosine, phenylalanine, histidine,

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arginine, alanine and valine [17, 18]. A portion of these amino acids are found in free form,

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especially in the gooey or slimy polymer matrix characteristic of Natto where D forms of

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many amino acids are present [18]. D-amino acids are produced by cultures of many bacteria

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during their stationary phase of growth [19]. These D-amino acids are more common than

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originally thought; some are found incorporated in microbial proteins [20] and some have

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been found to modulate bacterial cell wall function [19]. The D-amino acids produced by B.

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ACCEPTED MANUSCRIPT subtilis have been shown to prevent biofilm formation and to degrade biofilms formed by

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bacteria including Staphylococus aureus and Pseudomonas aeruginosa [21, 22]. These

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findings led the authors to suggest that the D-amino acids constituted a widespread signal for

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biofilm disassembly [21]. In this study, we investigated whether Natto extract could inhibit V.

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harveyi growth and/or biofilm formation and whether the extract or B. subtilis var natto itself

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could protect shrimp against V. harveyi when mixed with shrimp feed.

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MATERIALS AND METHODS

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Vibrio isolates

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Bacterial cultures used in this study consisted of a natural isolate Vibrio harveyi (1114GL)

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(referred to here as VH0) that was obtained from shrimp affected by tea brown gill syndrome

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[23, 24] and a lysogenic clone derived from it and infected with the bacteriophage VHS1,

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(referred to here as VH1). These two bacterial cultures were used as representatives of shrimp

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pathogenic Vibrio bacteria that have been found in the shrimp stomach and hepatopancreas

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[13]. The bacteria were stored at -80 °C in Mueller Hinton broth (MHB) supplemented with

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3% NaCl containing 20% glycerol. These Vibrio isolates were cultured and tested in Mueller

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Hinton broth supplemented with 3% NaCl (MHBS) or on 1.5% agar plates of the same

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medium (MHAS).

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Bacillus subtilis var Natto isolates

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Bacillus subtilis var natto isolates were obtained by isolation from commercial Natto

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purchased at local supermarkets by streaking onto tryptic soy agar (TSA) (Difco, USA)

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followed by incubation overnight at 37°C. From these products, only one type of colony of a

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light yellow color with a rough surface and undulating margin arose on the TSA plates. The

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isolates obtained were named B subtillus-natto (BSN) and they were stored at -80 °C in

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ACCEPTED MANUSCRIPT tryptic soy broth (TSB) (Difco) containing 20% glycerol. One of these isolates called BSN1,

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was arbitrarily selected for the work described in this paper. For experiments, stored BSN1

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was revived by overnight culture in TSB (100 µl stock in 5 ml) at 37°C before streaking a

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portion of the broth on TSA using a sterile inoculation loop. A single colony was

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subsequently picked and inoculated into 5 ml TSB. The culture was incubated overnight at

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37°C with 250 rpm shaking. The cells were spun down by centrifugation at 9,000 g for 10

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min using a refrigerated centrifuge (Kubota 3700, Japan) and the cell pellet was collected.

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Genomic DNA was extracted using the phenol chloroform extraction method [25]. To

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identify the bacteria, the 16s ribosomal RNA gene was amplified using gene-specific primers

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40F:

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GACTACCAGGGTATCTAATCC-3′ [26] with the method described earlier [7]. The PCR

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products obtained were cloned into pGEM-T EASY vector (Promega) and transformed into

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E. coli JM109. The transformed cells were selected on LB agar containing 50 µg/ml

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ampicillin. Clones (2) with inserts were confirmed by colony PCR and sent for sequencing

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(Macrogen, Korea). The nucleotide sequence data were subjected to BLAST sequence

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analysis against NCBI databases for bacterial identification.

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Growth of BSN1 and Vibrio on various agar media

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To prepare for growth inhibition assays on agar plates a suitable medium had to be chosen

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that would accommodate growth of both BSN1 and the Vibrio isolates VH0 and VH1. Thus,

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BSN1 and Vibrio isolates VH0 and VH1 were streaked on TSA plates and MHAS plates and

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incubated overnight at either 30oC, 33.5oC or 37oC. Then their growth was assessed and the

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data obtained were used to design a medium that would be suitable for the inhibition assays.

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ACCEPTED MANUSCRIPT Effect of BSN1 on VH0 and VH1 growth on agar

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To determine whether or not the BSN1 had any effect on growth of V. harveyi VH0 and

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VH1, overnight cultures of the three bacteria were cross streaked on a medium optimized for

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dual growth (see previous section) and the plates were incubated overnight at 33.5ºC before

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the cultures were examined for any evidence of growth inhibition.

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Preparation of Natto using BSN1

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Laboratory Natto was prepared following a previously published method [27]. Briefly, the

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soybeans were autocalved for 40 min at 121oC before cooling and inoculated with an

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overnight pure culture of BSNI in TSB to achieve a final concentration of 104 cells per 100 g

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cooked beans. Then the mixture was incubated at 42oC for 2 days before use in preparing

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Natto extract.

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Natto extract preparation

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To prepare Natto extract from the viscous, gooey or slimy material present in Natto, 10 ml of

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sterile distilled water was added to 1 g of Natto followed by shaking. The viscous-material-

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water mixture was then centrifuged at 8,000 g for 10 min at 25°C. The supernatant was

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collected and filtered through a 0.22 µm membrane filter. Carbohydrates in the extract were

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quantified using a carbohydrate quantification assay described by Masaku and colleagues

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[28] with glucose as the standard and protein was quantified using the Bradford protein

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quantification assay [29] with bovine serum albumin (BSA) as the standard. The quantities of

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carbohydrate and protein in the extracts were 5.74 mg/ml and 421.69 ng/ml respectively. The

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Natto extract was stored at 4°C.

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ACCEPTED MANUSCRIPT Effect of Natto extract on Vibrio growth in broth

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To determine whether the Natto extract had any effect on growth of VH0 and VH1, a

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microtiter plate growth assay was used. Revived VH0 and VH1 stocks grown overnight in 3

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ml MHBS at 30°C with 250 rpm shaking and then diluted with sterile medium to an optical

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density (OD600) of 0.1 in preparation for further dilution to 1:10 in new medium before

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transfer to uncoated 96-well microtiter plates at.180 µl per well. Then, 20 µl of appropriately

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diluted Natto extract was added to obtain 3 different final protein concentrations of the

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extract in the cultures at 4.2 ng/ml, 42.2 ng/ml and 421.7 ng/ml protein, with an additional

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control well to which 20 µl distilled water was added. For each treatment and control there

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were 8 replicates. The cultures and extracts were mixed by pipetting before the microtiter

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plates were incubated at 30°C for 16 h with agitation at 250 rpm. Growth of each culture was

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measured by light absorbance at 600 nm using a microplate reader.

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Effect of Natto extract on biofilm formation

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To determine the effect of Natto extract on biofilm formation by VH0 and VH1, a biofilm

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assay was carried out in microtiter plates using the same protocol as the growth assays above

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except that the chitosan-coated polystyrene microtiter plates were used [30]. Natto extracts

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were added in 20 µl at the same concentrations and the control wells were the same. The

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plates were incubated at 30°C for 24 h without agitation. Eight replicates were done for each

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treatment and control.

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To quantify biofilms, culture medium was removed from the plates by overturning on

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absorbent paper. The wells were carefully washed twice with water to remove non-adherent

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cells before they were stained by addition of 220 µl of 0.3% crystal violet for 15 min. The

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excess dye was removed by three times washing with water. The plates were turned up-side

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ACCEPTED MANUSCRIPT down and the stained biofilms were allowed to dry overnight in room temperature. To

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quantitatively measure the thickness of the formed biofilms, 220 µl of 33% acetic acid was

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added to each well of the microtiter plates to dissolve the stained biofilms. To assure that the

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biofilms were completely dissolved, the plates were left standing at RT for 15 min before

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they were quantitated using a microplate reader set at absorbance at 600 nm. Statistical

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analysis was done using SPSS software (one-way ANOVA) with differences being

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considered statistically significant at p ≤ 0.05.

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The effect of amino acids on VH0 and VH1 biofilm formation

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Experiments were carried out to determine whether D-amino acids present in Natto and

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stationary phase cultures of B. subtilis [D-phenylalanine (D-Phe), D- Tyrosine (D-Tyr), D-

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Valine (D-Val)] [19] actively inhibit V. harveyi biofilm formation and/or degrade formed

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biofilms, as has been previously reported [21, 22]. The experimental protocol was similar to

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that used above for the effect of Natto extract on biofilm formation except that the following

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amino acids were added instead of Natto extract: D-Alanine (D-Ala), D-Histidine (D-His), D-

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Methionine (D-Met), D-Lysine (D-Lys), D-Aspartic acid (D-Asp) and D-Glutamic acid (D-

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Glu) (All from Sigma Chemicals, USA). All amino acids were tested at three different

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concentrations of 0.1 mM, 0.5 mM and 1 mM.

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Preparation of feed supplemented with Natto extract or BSN1

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To determine whether Natto extract and bacteria isolated from Natto (i.e. B. subtilis natto =

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BSN1) are effective/protective in V. harveyi infected shrimp, experiments with appropriately

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supplemented feeds were carried out. For Natto-extract supplemented feed, 1 g of

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commercial shrimp feed pellets (Charoen Pokphand shrimp feed number 1) was placed in a

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50 ml sterile beaker and 1 ml of Natto extract (421.7 ng protein/ml) was added followed by

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vigorous stirring using a sterile, stainless-steel spatula for approximately one minute until the

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mixture was uniform. At first there was free liquid present and this was gradually absorbed

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by the feed pellets. The mixture was then allowed to dry at room temperature overnight

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before storage in clean plastic bags at 4°C (no longer than two weeks) until used.

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BSN1-supplemented feed was prepared using a protocol similar to that reported by Rengpipat

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et al [30, 31]. Briefly, overnight cultures (̴ 18 h) of the bacteria were prepared in 25 ml of

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TSB and incubated at 37°C with 250 rpm agitation. Optical density (O.D.) at 600nm of the

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culture fluid was determined (O.D. 5.0 or 8×107 CFU/ml) before the cell pellet was collected

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by centrifugation at 3,124g for 15 min using a refrigerated centrifuge set at 4°C and the

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supernatant medium was poured off. The wet weight of the soft pellet was determined (0.18 g

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or 2×109 CFU) before 0.54 g of feed (number 1) was added to the tube followed by rapid and

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thorough mixing using a sterile, stainless steel spatula for approximately 1 minute until a

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uniform mixture was obtained. Then the mixture was transferred to a sterile petri dish to dry

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at RT for 3 hours. The final calculated density of bacteria in the feed was (2.8 x 109 cfu/g air

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dried feed). It was stored in clean plastic bags at 4°C (no longer than one week).

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Shrimp challenge experiments

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White leg shrimp (Penaeus vannamei) PL (210, approx. 7 mg body weight each) that

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originated from specific pathogen free (SPF) broodstock were purchased from Sibsaen

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hatchery, Thailand. Upon arrival they were tested for freedom of white spot syndrome virus

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(WSSV) and yellow head virus (YHV) infections by PCR and RT-PCR respectively using the

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methods described previously [31, 32]. The PLs were divided into 7 groups, one control and

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6 bacterial-challenge groups. In each group there were 3 replicates of 10 PLs. These shrimp

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were placed in 21 wide mouth bottles (10 PLs/bottle) containing 300 ml of artificial sea water

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ACCEPTED MANUSCRIPT (Marina, Bangkok, Thailand) at 15 ppt salinity and provided with an air stone. The bottles

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were placed in a fresh water bath equipped with heating rods set at 30°C to ensure stable

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temperature throughout the experiment. The shrimp in the control group were fed with

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normal feed twice a day at 10% body weight/meal for the entire experiment. Excess feed was

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removed daily. Among the 6 bacterial-challenge groups, two groups were fed with normal

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un-supplemented feed. After 7 days of feeding, one of the 2 groups was challenged with VH0

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while the other was challenged with VH1. The remaining 4 bacterial-challenge groups

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consisted of 2 groups fed with Natto-extract supplemented feed and 2 groups fed with BSN1-

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supplemented feed (see the diagram in Fig. 1). After 7 days of feeding, the shrimp were

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challenged with either VH0 or VH1.

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Bacterial challenges were carried out by immersion exposure. Bacteria were obtained from

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overnight cultures with optical densities (OD) in the range of 1.5-2.5 and calibrated for OD

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1.0 = 107 cfu/ml. Broth was added to the PL test bottles to achieve a final concentration 106

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cfu/ml. In the negative control bottles, an equal volume of sterile broth was added. The

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shrimp were exposed to the bacteria for 24 h before 50% of the water in each bottle was

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replaced with clean water of the same salinity. Shrimp mortalities were observed for 5 days

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after beginning the immersion challenge during which time moribund shrimp were fixed with

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Davidson’s fixative and processed for paraffin embedding and sectioning as described by

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Bell and Lightner [33]. The cut sections were stained with hemotoxylin and eosin before they

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were studied under a light microscope.

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Figure 1. Schematic diagram shows Natto experimental procedures

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RESULTS

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The bacterium isolated from commercial Natto was Bacillus subtilis var Natto

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Sequences obtained from cloned amplicons of PCR reactions targeting the rRNA gene of the

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bacterium isolated from Natto were identical to one another, and they were found to match

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the 16s rRNA gene of many isolates/strains of Bacillus subtilis including B. subtilis var.

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Natto with identities between 99% and 100%.

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BSN1 and Vibrio growth on various agar media

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When BSN1 and standard Vibrio isolates VH0 and VH1 were streaked on TSA plates

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incubated overnight at 30oC, BSN1 grew poorly and both Vibrio isolates gave clearly visible

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growth but not as good growth as on MHAS. In contrast, BSN1 grew very well on TSA at 11

ACCEPTED MANUSCRIPT 37oC while the two Vibrio isolates grew even more poorly than they did at on TSA 30oC.

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These results were as expected, given the nature of the two species. With MHAS, growth of

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the two Vibrio isolates was very good at 30oC but less so on the same medium at 37oC while

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BSN1 grew very poorly on MHAS at both temperatures. These results were used to design

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media that might allow the Vibrio isolates and BSN1 to be grown together on agar medium

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for cross-growth-inhibition assays, and it was found that TSA with a total NaCl content of

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1.5% and 33.5 oC was suitable for obtaining reasonable growth with the two Vibrio isolates

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together with BSN1.

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BSN1 does not inhibit growth of VH0 and VH1 in agar cultures

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Using the medium for dual culture of BSN1 with VH0 or VH1 (described above), it was

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revealed that there were no indications of growth inhibition between BSN1 and the two

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Vibrio isolates (Fig. 2 C).

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Natto extract does not inhibit growth of Vibrio harveyi (VH0 and VH1)

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When anti-Vibrio growth activities of Natto extract were evaluated using microtiter plate

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growth assays, there was no growth inhibition for both types of V. harveyi (VH0 and VH1) at

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extract protein concentrations between 4.2 ng/ml and 421.7 ng/ml (Fig. 2A). Indeed, Natto

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extract at all protein concentration significantly promoted the growth of both VH0 and VH1

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(p<0.05) (Fig. 2A).

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Natto extract inhibits Vibrio biofilm formation by VH0 and VH1

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The effect of the Natto extract on biofilm biomass of V. harveyi as determined using the

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crystal violet assay revealed that all concentrations of Natto extract significantly (p<0.05)

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inhibited biofilm formation by both VH0 and VH1 (Fig. 2B) and the degree of biofilm

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ACCEPTED MANUSCRIPT inhibition was found to be directly correlated with the concentration of the extract used. The

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highest protein concentration tested (421.7 ng/ml) gave the greatest inhibition in terms of

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biofilm biomass (70.6% and 68.9% for VH0 and VH1, respectively). At lower concentrations

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of the extract, biofilm formation reduction was more pronounced with VH0 than with VH1

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(Fig. 2B).

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Figure 2. The effect of Natto extract on growth and biofilm formation by V. harveyi GL1114

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(VH0) and its lysogen VH1 and Bacillus subtilis isolated from Natto on growth of the two

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bacteria. (A) Results for mean absorbance (OD600) of VH0 and VH1 grown with Natto

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extracts containing protein at 4.2, 42.2 and 421.7 ng/ml and carbohydrate at 0.057, 0.57 and

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5.7 mg/ml and showing no interference with growth for either VH0 or VH1. (B) Results of

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crystal violet assays showing significantly (p<0.05) reduced biofilm formation by both VH0

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and VH1 at the same concentrations of Natto extract as in (A). (C) Example of VH0 and VH1

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grown on TSA supplemented with 1.5% NaCl that was cross streaked with BSN1 and the two

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Vibrio isolates. No growth inhibition was observed.

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D-Amino acids do not inhibit Vibrio growth and biofilm formation

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To determine whether D-forms of amino acids found in Natto and amino acids found in B.

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subtilis cultures, inhibit growth and biofilm formation by VH0 and VH1, both planktonic 14

ACCEPTED MANUSCRIPT cells and biofilms of the VH0 and VH1 were grown with and without different synthetic

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amino acid supplementation. Amino acids tested were 6 major D-amino acids previously

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reported from Natto, including D-Ala, D-His, D-Met, D-Lys, D-Asp and D-Glu and 3 of the

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most abundant D-amino acids found in B. subtilis culture, i.e., D-Phe, D-Tyr, D-Val. These

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amino acids were tested individually and in some combinations. The results obtained from

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these studies showed that the amino acids tested neither inhibited biofilm formation nor

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growth of the bacteria VH0 and VH1 with few exceptions. Detailed results are shown in

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supplementary information.

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Natto extract and BSN1 protect shrimp against Vibrio harveyi

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From these experiments supplementing feed with either Natto extract or the BSN1 was found

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to protect PL of the whiteleg shrimp (P. vannamei) against challenge with V. harveyi.

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Challenged shrimp that were fed with either of the supplemented feeds had significantly

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higher (p<0.05) survival rates than those that were fed normal, un-supplemented feed (Fig.

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3). Specifically, survivals of shrimp fed normal un-supplemented feed prior to challenge with

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VH0 or VH1 were approximately 29% and 35%, respectively while survivals of shrimp fed

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Natto-extract supplement were 76% and 58%, respectively, and survivals of shrimp fed with

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B. subtilis supplement were 86% and 94%, respectively (Fig. 3A and 3B).

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Histopathology of moribund shrimp challenged with Vibrio

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Hematoxylin and eosin stained tissues of infected and moribund shrimp collected from the

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experiment revealed typical vibriosis pathologies at varying degrees, especially in the

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hepatopancreas (HP), regardless of the feed supplementation. Since there were more

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moribund shrimp in the challenged, un-supplemented feed groups, more shrimp with HP

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pathologies were observed in these groups. These pathologies included hepatopancreatic

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ACCEPTED MANUSCRIPT necrosis, sloughing of hepatopancreatic epithelial cells, hemocytic infiltration and flattening

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of HP epithelial cells in HP tubules. Examples of HP pathologies are shown in Fig. 4C-F.

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Shrimp in the unchallenged control group had normal histology as expected (Fig. 4A and

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4B). HP tissue of surviving, grossly normal shrimp specimens fed with Natto-extract

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supplement or BSN1-supplement and then challenged with VH0 or VH1 showed normal HP

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histology, similar to that in the unchallenged control group (Fig.4A and 4B).

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ACCEPTED MANUSCRIPT Figure 3. Effect of feed supplementation with Natto-extract (+Natto) or BSN1 (+BSN) on

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survival of shrimp challenged with V. harveyi by bath immersion at 106cfu/ml. (A) Mean

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percent survivals ± SD for challenge with V. harveyi VH0. (B) Mean percent survivals ± SD

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for challenge with V. harveyi VH1. The mean percent survivals were higher in both VH0 and

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VH1 infected shrimp groups that were fed with supplemented feed when compared to those

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of the infected shrimp that were fed with un-supplemented feed.

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ACCEPTED MANUSCRIPT Figure

4

Histopathology

of

shrimp

challenged

with

V.

harveryi.

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(A-B) Hepatopancreatic (HP) tissue of uninfected control shrimp stained with H&E and

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showing normal HP histology. (C-D) HP tissue of a moribund shrimp specimen fed with

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normal, un-supplemented feed and collected at day 3 after challenged with VH0 and showing

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detachment of epithelial cells from the basement membrane of affected hepatopancreatic

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tubules and some hemocyte infiltration. (E-F) Same as in the previous panel except

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challenged with VH1 and showing pathology similar to that in panel (C-D). (D and F) Rod-

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shaped bacterial cells were found in HP of both VH-challenged shrimp (black arrows).

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4. Discussion

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We have shown that Natto extract and BSN1 can inhibit biofilm formation by shrimp

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pathogenic V. harveyi without inhibiting growth in broth culture and on agar plates. We also

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showed that D-amino acids were unlikely to have been the active compounds that degraded

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and/or inhibited biofilm formation by VH0 and VH1. Our results are in agreement with the

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results reported by Sarkar and Pires [34] but somewhat contradict the results obtained from

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Kolodkin-Gal and colleagues [21] and Hochbaum and colleagues [22]. The latter two

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publications proposed that D-amino acids constitute a widespread signal for biofilm

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disassembly [21]. Detailed discussion regarding D-amino acids is provided in the

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supplementary information. We also showed that the Natto extract as well as BSN1 itself

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added as a supplement to shrimp feed could greatly reduce shrimp mortality in challenge tests

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using 2 related V. harveyi isolates (VH0 and VH1). In this study the survival rate of the VH1-

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challenged P. vannamei PLs that were fed with BSN1-supplemented feed was higher than that of

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shrimp fed with non-supplemented feed. It was also higher than that of P. vannamei PLs fed with

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BSN1- supplemented feed and challenged with VH0. We have no explanation for this difference, but

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it is possible that VH1 biofilm is more sensitive to B. subtilis cells or its product. In our study the

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biofilm produced by VH0 was found to be significantly thicker than that of VH1 in the presence of

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Natto extract, especially at higher protein concentrations (e.g., 42.169 ng/ml) (see Fig. 2B). Although

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at the highest concentration of Natto extract tested (protein concentration 421.69 ng/ml) the

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thicknesses of both bacterial biofilms did not differ significantly. VH1 has been reported to be more

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virulent than VH0 for the giant tiger shrimp P. monodon, but this difference has not been observed in

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the whiteleg shrimp P. vannamei

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feed, VH1 was induced into lytic cycles that would result in fewer bacterial cells. If so, it would

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explain the fewer Vibrio cells in the VH1 infected group (higher survival) when compared to the same

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shrimp infected with VH0 that has no phage. However, this phenomenon was not observed in growth

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inhibition assays that showed no Vibrio growth inhibition in the presence of BSN1 or Natto extract.

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More detailed research is needed to examine the mechanism by which BSN1 or its products protect

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shrimp against vibriosis.

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[35, 36]. It is also possible that in the presence of B. subtilis in

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A number of isolates of Bacillus and especially B. subtilis have been reported to act as

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protective probiotics in shrimp culture, and it has been proposed that they can enhance both

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shrimp immunity and growth [37-41]. They have also been shown to produce many digestive

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enzymes such as proteases, carbohydrolases and lipases that may be useful for shrimp in

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helping to release nutrients from feed [39-41]. It has also been proposed by some researchers

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that such probiotic bacteria compete with shrimp pathogenic bacteria for gut colonization and

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thus reduce the chance of colonization by harmful bacteria [42-45]. In one report, B. subtilis

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probiotic was shown to reduce Vibrio spp. counts in the shrimp intestine by 2 logs after 1

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month culture with probiotic treatment when compared to counts in shrimp from ponds that

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were not treated [37]. However, the results from the study by Ziaei-Nejad and colleagues [40]

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showed that although Bacillus bacteria were detected in the digestive tract of probiotic-

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treated-shrimp and not in the digestive tract of non-treated shrimp, the Bacillus colonization

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in the digestive tract was at a very low level (0.07–0.30% of the total bacterial population).

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The results suggested that colonization competition may not be the reason for reduced Vibrio

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protease and lipase were found to be significantly higher (P < 0.05) in the probiotic-treated

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shrimp suggesting that such activities might be the reason for better survival (11–17% higher)

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and higher wet weight (8–22% higher) of the probiotic-treated shrimp when compared to the

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untreated controls [40]. Our results in tests with Natto extracts that contain no Bacillus cells

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was also in agreement with these results in that similar higher survival rates of shrimp were

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obtained in both Natto extract (without cells, 76% and 86% for VH0- and VH1-challenged

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shrimp, respectively) and Bacillus subtilis treated shrimp (58% and 96% for VH0- and VH1-

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challenged shrimp, respectively) when compared to the untreated control shrimp (approx.

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29% and 35% for VH0- and VH1- challenged shrimp, respectively). In conclusion, from this

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study we can confirm that the biofilm formation inhibition compounds in Natto extract were

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unlikely to be D-forms amino acids. However, BSN1 Natto extract supplementation or BSN1

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supplementation in shrimp feed can reduce the shrimp mortality caused by Vibrio infection,

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so they could be used as shrimp feed additives to prevent or reduce the effect of the bacteria

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infections.

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ACKNOWLEDGEMENTS

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This project was supported by The National Research Council of Thailand (grant number

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264498), Mahidol University (grant number 165996), the Office of the Higher Education

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Commission, Thailand and Mahidol University under the National Research Universities

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Initiative.

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ACCEPTED MANUSCRIPT 6. REFERENCES

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Highlights -Extract of fermented soybean, Natto inhibits biofilm formation the bacterial growth.

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of the shrimp pathogen Vibrio harveyi without interfering with

- These inhibitions are not caused by the D-amino acids known bacteria used in Natto fermentation.

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to constitute Natto or supernatant of Bacillus subtilis culture, the

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- Shrimp feed supplemented with Natto extract or B. subtilis cells were found to greatly reduce Penaeus vannamei shrimp post larvae mortality from V. harveyi infection. -Less severe pathologies were observed in V. harveyi infected

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shrimp that were fed with Natto extract as well as B. subtilis cells supplemented feed.

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-This shows some promise for possible application against Vibriosis in shrimp since Natto is generally regarded as safe

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(GRAS) for human consumption.