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Title: Efficiency of the V3 region of 16S rDNA and the rpoB gene for bacterial 1

community detection in Thai traditional fermented shrimp (Kung-Som) using PCR-2

DGGE techniques 3

Running title: Bacterial community detection in fermented shrimp (Kung-Som) using 4

PCR-DGGE 5

Authors: Chatthaphisuth Sanchart1, Soottawat Benjakul

2, Onnicha Rattanaporn

3, 6

Dietmar Haltrich4, Suppasil Maneerat

1* 7

Affiliations:1Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince 8

of Songkla University, Hat Yai, 90112, Thailand; 2Department of Food Technology, 9

Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, 10

Thailand; 3Department of Biochemistry, Faculty of Science, Prince of Songkla 11

University, Hat Yai, Songkhla 90112 Thailand; 4Department of Food Sciences and 12

Technology, Food Biotechnology Laboratory, BOKU University of Natural Resources 13

and Life Sciences, Vienna, Austria. 14

*Corresponding author E-mail: [email protected]; Tel.: +66-74-286379; Fax: +66-15

74-558866 16

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Songklanakarin Journal of Science and Technology SJST-2014-0143.R1 Maneerat

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Abstract 25

Kung-Som is one of several Thai traditional fermented shrimp products, that is 26

especially in the southern part of Thailand. This is the first report to reveal the bacterial 27

communities in the finished product of Kung-Som. Ten Kung-Som samples were 28

evaluated using the polymerase chain reaction-denaturing gradient gel electrophoresis 29

(PCR-DGGE) methodology combined with appropriated primers to study the dynamics 30

of the bacterial population. Two primers sets (V3; 341f(GC)-518r and rpoB; 31

rpoB1698f(GC)-rpoB2014r primers) were considered as a possible tool for the 32

differentiation of bacteria and compared with respect to their efficiency of 16S rDNA 33

and rpoB gene amplification. PCR-DGGE analysis of both the V3-region and rpoB 34

amplicon was successfully applied to discriminate between lactic acid bacteria and 35

Gram positive strains in the bacterial communities of Kung-Som. In conclusion, the 36

application of these two primers sets using PCR-DGGE techniques is a useful tool for 37

analyzing the bacterial diversity in Kung-Som. Moreover, these preliminary results 38

provide useful information for further isolation of desired bacterial strains used as a 39

starter culture in order to improve the quality of Kung-Som. 40

Keywords: Kung-Som, rpoB gene, V3 region, PCR-DGGE, Bacterial community 41

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Introduction 49

Kung-Som is a traditional fermented shrimp product that is found widely 50

distributed in the south of Thailand. It is made from shrimp, sugar, salt and water and is 51

typically fermented with the natural, spontaneous microbial flora. The microbiology of 52

Kung-Som is diverse and complex. The principal microorganisms found in Kung-Som 53

are various lactic acid bacteria (LAB) (Tanasupawat et al., 1998; Hwanhlem et al., 54

2010). Species identification and population enumeration are critical in the study of 55

bacterial communities. Due to the limitations of conventional microbiological methods, 56

the identification of microorganisms that requires selective enrichment and subculturing 57

is problematic or impossible. Moreover, classical microbial techniques used have not 58

accurately analyzed the presence of the main bacterial species (Ben Omar and Ampe, 59

2000) and have not provided a completely accurate representation of these complex 60

communities. On the other hand, culture-independent molecular techniques have 61

provided better methods to give more information on the microbial diversity in complex 62

food samples (Cocolin et al., 2001; Ercolini, 2004). In addition, culture-independent 63

molecular techniques based on specific nucleotide sequences are widely used for 64

monitoring, detection, identification and classification of bacterial diversity. 65

In the recent decade, polymerase chain reaction-denaturing gradient gel 66

electrophoresis (PCR-DGGE) has been successfully applied to determine the 67

microbiology of fermented food such as fermented sausages (Fontana et al., 2005), 68

fermented grains (Chao et al., 2008), fermented meat (Hu et al., 2009), and fermented 69

dairy products (Liu et al., 2012), to name a few. This approach has provided new insight 70

into the microbial diversity and allowed a more rapid, high-resolution description of 71

microbial communities than did the traditional approaches since it allows the separation 72

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of DNA molecules that differ by single bases (Ercolini, 2004). The use of appropriate 73

consensus primers is also a critical point in determining the resolution of DGGE 74

analysis in mixed microbial systems, especially in LAB differentiation (Chen et al., 75

2008). From the evidence of several published papers, the 16S rDNA seems to be by far 76

the most widely used as a molecular marker for the determination of the phylogenetic 77

relationships of bacteria. The hypervariable V3-region on the 16S rDNA is the most 78

frequently used to start the study of an unknown and complex bacterial community. In 79

addition, the V3-region is considered to have a high grade of resolution and to be highly 80

variable, and it is regarded as a good choice when it comes to length and inter-species 81

heterogeneity (Coppola et al., 2001; Florez and Mayo, 2006; Hovda et al., 2007; Chen 82

et al., 2008). Unfortunately, one problem related to the use of 16S rDNA in DGGE 83

analysis is the complexity created by the existence of multiple heterogeneous copies 84

within a genome (Dahllof et al., 2000; Crosby and Criddle, 2003; Rantsiou et al., 2004). 85

Consequently, a solution to the problem of 16S rDNA heterogeneity is provided 86

by the analysis of a gene that exists in only a single copy (Fogel et al., 1999). Certain 87

protein-coding genes, such as the gene encoding the beta-subunit of DNA-directed 88

RNA polymerase, rpoB, have been proposed to fulfill this criterion. rpoB is used as a 89

potential biomarker to overcome identification problems because it is considered a 90

housekeeping gene. Targeting the rpoB gene allowed the reliable discrimination of 91

species. The use of this gene as a marker was able to avoid the intraspecies 92

heterogeneity problem caused by the use of the 16S rDNA, which appears to exist in 93

one copy only in bacteria (Dahllof et al., 2000; Ko et al., 2002). In addition, in some 94

strains of bacteria, an internal region of rpoB is a more suitable sequence than 16S RNA 95

because of its higher nucleotide polymorphism (Khamis et al., 2005). However, the use 96

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of rpoB presents a taxonomic disavadantage: the database of the sequence is less well 97

documented than that of the 16S rDNA (Rantsiou et al., 2004; Renouf et al., 2006). 98

There are no data using the PCR-DGGE technique to characterize the dominant 99

bacteria in Kung-Som product. Consequently, the aim of this present study was to focus 100

on the use of the hypervariable V3-region on the 16S rDNA and rpoB gene by using 101

PCR-DGGE techniques as a tool to reveal the bacteria that commonly develop in the 102

Kung-Som product and to compare the efficiency of the 16S rDNA and rpoB gene 103

sequences for species discrimination in such a complex food sample. The obtained 104

results provide preliminary information for study to apply in the microbial starter 105

cultures in Kung-Som fermentation. 106

Materials and Methods 107

Lactic acid determination in Kung-Som 108

Kung-Som samples were purchased from different local markets in Songkhla 109

Province, Thailand. The pH value was measured by a pH meter (420A ORION, USA). 110

Total acidity as lactic acid was determined according to the AOAC (AOAC, 1995). 111

Three independent measurements were made for each sample. Data presented are the 112

means and standard deviations calculated. 113

DNA extraction from Kung-Som 114

DNA was extracted from the juice sample of Kung-Som by the method Cocolin 115

et al. (2004), with slight modification. One millilitre of juice sample of each sample was 116

centrifuged at 14,000×g for 10 min at 4 °C to pellet the cells. The pellet was washed 117

twice with 1 ml of sterile 0.85% (w/v) NaCl. The pellet was resuspended in 50 µl of 20 118

mgml-1

lysozyme (Fluka, USA). After 30 min incubation at 37 °C, 30 µl of 25 mgml-1

119

proteinase K (AMRESCO®

, USA) and 150 µl proteinase K buffer were added. The 120

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tubes were incubated at 65 °C for 90 min before the addition of 400 µl breaking buffer 121

and incubated futher at 65 °C for 15 min. Then, 400 µl of phenol:chloroform:isoamyl 122

alcohol (25:24:1, pH 6.7) was added for extracting DNA, RNA and protein. The tubes 123

were centrifuged at 12,000×g at 4 °C for 10 min, the aqueous phase was collected and 124

the nucleic acid was precipitated with 1 ml of ice-cold absolute isopropanol. The DNA 125

was obtained by centrifugation at 14,000×g at 4 °C for 10 min, washed briefly with 70% 126

(v/v) ice-cold ethanol and centrifuged again. The DNA was dried at room temperature, 127

resuspended in 20 µl of RNase-DNase-free sterile water, and treated with 5 µl of 10 128

mgml-1

DNase-free Rnase (Vivantis, USA). After 5 min incubation at 37 °C, genomic 129

DNA was stored at -20 °C. 130

The V3 region of 16S rDNA and rpoB gene amplification 131

Primers 341f (5´-CCTACGGGAGGCAGCAG-3´) and 518r 132

(5´ATTACCGCGGCTGCTGG-3´) were used to amplify a region of approximately 200 133

bp of the V3 region of 16S rDNA (Muyzer et al., 1993). Primers rpoB1698f (5’-134

AACATCGGTTTGATCAAC-3’) and rpoB2014r (5’-CGTTGCATGTTGGTACCCAT 135

-3’) were used to amplify a region of approximately 350 bp of the rpoB gene (Dahllof et 136

al., 2000). Amplification reactions were carried out in volumes of 50 µl. In addition, a 137

GC clamp was added to the forward primer to improve the sensitivity in the detection of 138

mutations by DGGE (Sheffield et al., 1989). PCR products were examined by 2% (w/v) 139

agarose gel electrophoresis. These were used to check the quality and size of PCR 140

products before being subjected to DGGE analysis. 141

DGGE analysis 142

DGGE analysis was performed using the Dcode universal mutation detection 143

system apparatus (Cleaver Scientific, UK) according to Fontana et al. (2005) with slight 144

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modification. Thirty millilitres of PCR product was mixed with loading dye and applied 145

to 8% (w/v) polyacrylamide gels. A 28% to 55% denaturing gradient (100% of 146

denaturant corresponded to 7 mol l-1

urea and 40% formamide) were used for both the 147

341f(GC)-518r and rpoB1698f(GC)-rpoB2014r primer sets. Electrophoresis was run in 148

1X TAE buffer at constant temperature (60 ºC) for 10 min at 20 V and subsequently for 149

16 h at 85 V. After electrophoresis, the gel was stained for 30 min with 1X (final 150

concentration) SYBR Gold (Invitrogen, USA) in 1X TAE buffer, rinsed in water, and 151

then visualized and photographed under UV illumination with the Gel Documentation 152

(UVI-TECH, England). 153

After running the DGGE analysis, relevant bands were punched from the gel 154

with sterile pipette tips. Each piece was transferred into 20 µl of RNase-DNase-free 155

sterile water and incubated overnight at 4 ºC to allow the diffusion of the DNA. Then, 156

the eluted DNA was used as a template and re-amplification took place with primers 157

without the GC clamp. The PCR products were purified by using the HiYieldGel/PCR 158

DNA Fragments Extraction Kit (RBC, Taiwan), and sequenced by a DNA sequencer 159

(Ward Medic Ltd., Malaysia). 160

Construction of phylogenetic tree 161

Searches in GenBank with the BLAST program on the NCBI website were 162

performed to determine the closest known relatives of the determined 16S V3 region 163

and rpoB gene sequences. Multiple sequence alignments were created by using the 164

BioEdit version 3.3.19.0. The phylogenetic tree was obtained to compare similarities 165

among the sequences by the neighbor-joining method (Saitou and Nei, 1987) using the 166

MEGA software version 5. Kimura’s method was followed and 1,000 repetitions were 167

made for bootstrap (Tamura et al., 2011). 168

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Results and Discussion 169

Kung-Som is a one of the traditional fermented food products from southern 170

Thailand. The production process traditionally relies on a spontaneous fermentation 171

initiated by natural and fortuitous microorganisms, mainly various LAB and coagulase-172

negative cocci (CNC). Kung-Som is made from the main raw materials shrimp, sugar, 173

salt and water. These raw materials and personal hygiene can also be the possible 174

sources of pathogenic microorganisms or spoilage bacteria such as Escherichia coli, 175

Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus and Salmonella sp. In 176

all Kung-Som samples of this study, it was found that the pH and lactic acid 177

concentration ranged from 3.58 to 4.04 and 1.78% to 3.12%, respectively (Fig.1). The 178

pH of all samples was below 4.5 because LAB utilizes the carbohydrate substrates 179

available to produce organic acids, and especially lactic acid, as part of their 180

metabolites. These acids not only contribute to the taste, aroma and texture of the 181

product but also lower the pH of the product which is one of the important key factors 182

to ensure quality and safety (Visessanguan et al., 2006; Kopermsub and Yunchalard, 183

2010). Generally, a pH lower than 4.4 can inhibit growth of E. coli (Alvarado et al., 184

2006) and Salmonella sp. (Sorrells and Speck, 1970); a pH lower than 3.7 can inhibit S. 185

aureus (Alvarado et al., 2006); a pH lower than 4.0 inhibits B. cereus (Yang et al., 186

2008) and a pH of 4.5-5.0 has been demonstrated to inhibit V. parahaemolyticus 187

(Adams and Moss, 2008). Consequently, the pathogenic or spoilage bacteria were 188

inhibited by organic acids that affected the bacterial growth and extracted DNA 189

concentration in our samples. This finding is probably related to the PCR-DGGE 190

detection limit (104 cfuml

-1). In accordance with these results, no DNA bands 191

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corresponding to pathogenic and spoilage bacteria were detected in Kung-Som samples 192

of our study. 193

Renouf et al. (2006) reported that the first step for finding suitable primers is to 194

assume that the primers must be present in all the species and delimit variable 195

sequences to separate each species. The last step is to find the most suitable and 196

accurate gradient and the best DGGE conditions (temperature, time). Two sets of 197

primers (V3-region of 16S rDNA and rpoB gene) were considered suitable in this study 198

since it is a housekeeping gene. Consequently, the bacterial diversity of the Kung-Som 199

product was revealed by DGGE analysis. Figure 2 and 3 show the DGGE profiles 200

obtained by the DNA directly extracted from Kung-Som in different regions in 201

Songkhla Province, Thailand. Both the V3 region of 16S rDNA and the rpoB gene were 202

amplified from these DNA templates. The V3 region of 16S rDNA and rpoB gene 203

profiles displayed different patterns. The DGGE profile of the rpoB gene amplification 204

showed that the numbers of the bands were lower than those of the V3 region of 16S 205

rDNA amplification. For the DGGE profile and the phylogenetic relationship of the V3 206

region of 16S rDNA amplification (Fig. 2A and 2B), bands corresponding to 207

Tetragenococcus halophilus (band b), Lactobacillus farciminis (band d) and 208

L. plantarum (band l and k) were prominent in all Kung-Som samples, which is in 209

agreement with data published by Hwanhlem et al. (2010). Although band k was found 210

in all samples, it was faint. L. acetotolerans (band g) and L. rapi (band j, o, p) were 211

present in all samples except samples 3 and 6. Salinivibrio sharmensis (band a) and 212

Macrococcus sp. (band e) appeared only in sample 3 and L. crustorum (band h and i) 213

was only found in sample 6. Staphylococcus piscifermentans (band c), Weissella 214

thailandensis (band m) and W. cibaria (band n) were exhibited in some samples but with 215

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very weak intensity. Salinivibrio sharmensis (band a) was found only in sample 3. 216

Several bands originating from a single species were observed on the DGGE gels, and 217

these were from L. crustorum, L. rapi and L. plantarum. The reason for this is the 218

sequence heterogeneity as described by Crosby and Criddle (2003). 219

For the DGGE profile and the phylogenetic relationship of rpoB gene 220

amplification (Fig. 3A and 3B), bands 4 and 5, corresponding to W. thailandensis and 221

S. piscifermentans, were predominating in every sample. Furthermore, L. fermentum 222

(band 2) and L. reuteri (band 3) were present in all samples except sample 3 and 6. 223

Band 1 was only detected in sample 3. 224

Species-specific DGGE bands from two sets of primers for the main members of 225

LAB and CNC were exhibited. LAB (Lactobacillus, Tetragenococcus and Weissella), 226

including CNC species (Macrococcus and Staphylococcus), are the most commonly 227

isolated bacteria from fermented foods, especially meat and fish (Hu et al., 2008; 228

Kopermsub and Yunchalard, 2010; Hwanhlem et al., 2011). In addition, the genus 229

Salinivibrio was found to be the dominant species in sample 3 of the V3 region 230

amplification. These species are generally isolated from fermented fish samples 231

(Chamroensaksri et al., 2009). The differences in the results obtained, such as the 232

DGGE pattern from the rpoB gene amplification, indicated the occurrence of very low 233

bacterial diversity when compared with the V3-region amplification. These results 234

depended on the specificity of the primers (Endo and Okada, 2005; Renouf et al., 2006) 235

and the PCR conditions (Hongoh et al., 2003). 236

Rantsiou et al. (2004) and Renouf et al. (2006) reported that the use of rpoB 237

gene amplification combined with PCR-DGGE can only reveal the predominant species 238

in a sample. Moreover, Chen et al. (2008) suggested that the use of appropriate 239

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consensus primers is a critical point in influencing the resolution of DGGE analysis in 240

mixed microbial systems, especially in LAB differentiation. Endo and Okada (2005) 241

indicated that LAB could not be detected by universal bacterial PCR primer but were 242

detected when groups of LAB-specific primer were used. This is because the DGGE 243

profile can demonstrate only the diversity of bacteria present at more than 1% of the 244

target bacteria. Therefore, the detection of numerous different species present at low 245

concentrations appeared to be difficult using PCR-rpoB/DGGE. Thus, the rpoB gene 246

pattern exhibited the different species which did not appear in the V3-region pattern. 247

In both the rpoB gene and V3-region patterns, samples 3 and 6 showed different 248

DGGE patterns from those of compared to other samples. This is because these samples 249

were originated from different recipes or processes of preparation which could vary the 250

initial food matrix, fermentation process, personal hygiene, local tradition or local 251

geographic preferences. All of these are crucial factors in determining the growth of 252

specific microbial communities (Cocolin et al., 2004; Ercolini, 2004; Chen et al., 2008). 253

This outcome was related to the higher pH and lower lactic acid content of samples 3 254

and 6 (Fig.1). In accordance with this lower lactic acid concentration, a difference in the 255

LAB groups of these samples was detected (Fig. 2 and 3). 256

A number of faint bands could not be identified because of their low content 257

which might be related to the heterogeneous distribution of microorganism in the food 258

matrix (Florez and Mayo, 2006). In a detection limit analysis, an individual species 259

(Pediococcus pentosaceus DMST 18752) was identified by PCR-DGGE when its 260

number was higher than 104 cfuml

-1 (data not shown). The detection limit of PCR-261

DGGE depends on the species or perhaps even the strain considered. Furthermore, the 262

number and the concentration of the other members of the microbial community, along 263

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with the nature of the food matrix, all represent variables influencing the detection limit 264

of DGGE. These factors affect both the efficiency of DNA extraction and the PCR 265

amplification due to possible competition among templates (Ercolini, 2004; 266

Temmerman et al., 2004; De Vero et al., 2006). 267

Conclusion 268

The suitability of the primers used was based on the discriminatory efficiency of 269

the hypervariable V3-region and the rpoB gene that allowed species differentiation from 270

the dominant groups of bacteria in Kung-Som. Although the applications of PCR-DGGE 271

techniques combined with appropriate consensus primers to study complex microbial 272

communities originating from food samples have been shown to be an efficient tool for 273

detection of complex bacteria populations, we believe that our findings represent a 274

preliminary analysis. The data can not be considered sufficient to achieve a confident 275

identification at species level, but should suggest that the relevant genes together with 276

other target sequences such as other region of 16S rDNA, gyrA, gyrB, recA or rpoC, 277

should be used for unequivocal identification of individual species. Moreover, these 278

preliminary results provide useful information for improving product quality. An 279

improved understanding of the changing microflora in Kung-Som fermentations could 280

be used to develop a starter culture in the future. 281

Acknowledgements 282

This study was financially supported by the Thailand Research Fund through the 283

Royal Golden Jubilee Ph.D Program (Grant No. PHD/0060/2556), the Graduate School 284

and the Thailand Research Fund Senior Scholar Program Prince of Songkla University. 285

References 286

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415

Figure captions 416

Fig. 1 The pH value and titratable acidity of Kung-Som samples. 417

Fig. 2 DGGE profiles (A) and the phylogenetic tree based on V3-region on 16S rDNA 418

(B) of the bacteria community obtained from DNA directly extracted from Kung-Som 419

samples. The scale bar represents the number of inferred substitutions per site. 420

Fig. 3 DGGE profiles (A) and the phylogenetic tree based on rpoB gene (B) of the 421

bacteria community obtained from DNA directly extracted from Kung-Som samples. 422

The scale bar represents the number of inferred substitutions per site. 423

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Fig. 1

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a

e

h i

k

c

d

f g

j

l

n

o p

b

m

A

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

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Fig. 2

CS-d

Lactobacillus farciminis KCTC 3681T (AEOT01000034)

Lactobacillus crustorum LMG 23699T (AM285450)

CS-k, CS-l

Lactobacillus plantarum HK01 (JN671595)

CS-h, CS-i

Lactobacillus pentosus ATCC 8041T (D79211)

CS-j, CS-o, CS-p

Lactobacillus rapi YIT 11204T (AB366389)

Lactobacillus buchneri JCM 1115 (NR 041293)

Lactobacillus parabuchneri ATCC 49374 (AB205056)

CS-g

Lactobacillus acetotolerans JCM 3825T (AB303841)

Lactobacillus homohiochii NBRC 13120 (AB680370)

CS-b

Tetragenococcus halophilus NBRC 12172 (AP012046)

Tetragenococcus halophilus IAM 1676T (D88668)

Tetragenococcus koreensis JS T (AY690334)

Staphylococcus xylosus ATCC 29971T (D83374)

CS-c

Staphylococcus carnosus ATCC 51365T (AB009934)

Staphylococcus piscifermentans ATCC 51136T (AB009943)

CS-e

Macrococcus brunensis LMG 21712 (AY119686)

Macrococcus bovicus ATCC 51825 (Y15714)

Macrococcus hajekii CCM 4809 (AY119685)

Weissella confusa JCM 1093T (AB023241)

Weissella kimchii DSM 14295 T (AF312874)

CS-m

Weissella sp. NBRC 107204 (AB682503)

Weissella thailandensis FS61-1T (AB023838)

Weissella thailandensis 5-5 (HQ384297)

Weissella thailandensis fsh4-2 (HE575179)

CS-n

Weissella cibaria BJ31-3C (AY341572)

Weissella cibaria LMG 17699T (AJ295989)

Weissella paramesenteroides NRIC 1542 (AB023238)

Weissella paramesenteroides ATCC 33313T (ACKU01000017)

CS-a

Salinivibrio costicola ATCC 35508T (X74699)

Salinivibrio costicola SI3 (HQ641751)

Salinivibrio proteolyticus AF-2004 (DQ092443)

Salinivibrio sharmensis BAGTT (AM279734)

Salinivibrio budaii MML 1937 (JN848571)

Escherichia coli20-10-1-9 (JF919881)

100

98

71

95

84

93

59

89

89

88

68

87

86

69

61

58

63

50

55

78

0.1

B

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1 2

4

3

5

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

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Fig. 3

Lactobacillus fermentum CECT 5716 (CP002033)

Lactobacillus fermentum IFO 3956 (AP008937)

Lactobacillus fermentum CNRZ 246 (AF515648)

CS2-rpo

CS3-rpo

Lactobacillus reuteri BR11 (GU191835)

Lactobacillus reuteri SD2112 (CP002844)

Lactobacillus reuteri JCM 1112 (AP007281)

CS1-rpo

Lactobacillus pentosus IG1 (FR874854)

Lactobacillus pentosus MP-10 (FR871825)

Lactobacillus plantarum JDM1 (CP001617)

Lactobacillus plantarum DSM 20174 (AF515652)

CS4-rpo

Weissella thailandensis fsh4-2 (HE575158)

Weisella paramesenteroides (Y16471)

Weissella paramesenteroides DSM 20288 (AF531265)

Staphylococcus simulans DSM 20322 (JN190408)

Staphylococcus piscifermentans DSM 7373 (DQ120745)

Staphylococcus piscifermentans CCM 4789 (HM146318)

CS5-rpo

Staphylococcus condimenti DSM 11675 (DQ120734)

Staphylococcus carnosus subsp. utilisDSM 11677 (DQ120731)

Staphylococcus carnosus subsp. carnosus DSM 20501 (JN190401)

Escherichia coli CIP 54.8 (EU010107)

98

87

92

83

94

90

97

98

93

100

84

93

87

100

60

57 70

98

95

0.1

B

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