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Characterization of Weissella confusa DD_A7 isolated from kimchi
LWT - Food Science and Technology 111 (2019) 663–672
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Characterization of Weissella confusa DD_A7 isolated from kimchi 1
1
Debasish Kumar Dey , Bon Gyo Koo , Chanchal Sharma, Sun Chul Kang
∗
T
Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, 38453, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Weissella confusa DD_A7 Non-pathogenic bacteria 16S-rRNA SNPs Antibacterial activityAbbreviations: NCBI National center for biotechnology information ML Maximum likelihood method MCL Maximum composite likelihood MRS De man Rogosa and sharpe medium SNP Single nucleotide polymorphism RMS Root mean square PBS Phosphate buffer saline BATH Bacteria adhesion to hydrocarbon CFCS Cell-free culture supernatant AO Acridine orange EtBr Ethidium bromide Ca++ Calcium SEM Scanning electron microscope SD Standard deviation NCCP National culture collection for pathogens ATCC American type culture collection
Foodborne pathogenic bacteria like, Escherichia coli 0157:H7, Bacillus cereus and Salmonella typhimurium are the biggest threat to food industries. Their growth in the environment and transmission of deadly diseases has become a challenge. The present study confirmed the genetic identity of DD_A7 is similar to the reported probiotic strains of Weissella confusa by analyzing the polymorphism in the conserved region of 16S-rRNA sequence. The Kimchi isolated, non-pathogenic W. confusa DD_A7 strain has shown the potential to control the growth of foodborne pathogens. Furthermore, the cell-free culture supernatant of DD_A7 shows the potential to rapture the membrane integrity of E. coli 0157:H7 by influencing the Ca++ ion channels. The safety concern associated with the usage of DD_A7 strain was also determined by testing its antibiotic susceptibility against different antibiotics and confirmed its non-toxic behavior on mammalian cells. Nevertheless, the strain has also encompassed several applications such as isolation of amylolytic and proteolytic enzymes. Collectively, the study will help to control the growth of foodborne pathogenic bacteria using the studied probiotic strain. In addition, the study will also help to construct a species-specific probe in future for the identification of the non-pathogenic strain of W. confusa for its application in food industries.
∗ Corresponding author. Department of Biotechnology, College of Engineering, Daegu University Jillyang, Naeri-ri, Gyeongsan, Gyeongbuk, 38453, Republic of Korea. E-mail addresses: [email protected] (D.K. Dey), [email protected] (B.G. Koo), [email protected] (C. Sharma), [email protected] (S.C. Kang). 1 Authors contributed equally.
https://doi.org/10.1016/j.lwt.2019.05.089 Received 23 February 2019; Received in revised form 9 May 2019; Accepted 18 May 2019 Available online 20 May 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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1. Introduction
this study. The pathogenic bacteria were grown in Luria-Bertani Broth (LB) media at 37 °C.
Food is a necessity for the survival and presences of pathogens in it have been the biggest culprit for food industries. Numerous government agencies and educational institutions are working to bring advancement in the field of food science and technology (Kim, Parka, & Kang, 2018). The field of study importantly deals with the quality of food and safety aspects. However, despite of numerous advancements in the field, food safety is still a concern. As these pathogens are associated with several virulence factors which cause severe disease and illness such as, listeriosis, diarrhea, hemolytic-uremic syndrome, and so on in human. Currently, the demands of functional foods are increasing tremendously because of their beneficial properties (Montoro et al., 2016; Shangpliang, Sharma, Rai, & Tamang, 2017). Among the several other functional foods, Korean cuisine, kimchi, has gained importance due to its health benefits (Khan & Kang, 2016). Researchers have shown that microbes isolated from Kimchi possess an inherent ability to modulate the host immune response by producing a large number of secretory metabolites such as proteins, enzymes, antimicrobial compounds, and vitamins (Dey, Khan, & Kang, 2019; Lee et al., 2014). Additionally, several pieces of evidence have underscored the importance of Kimchi and the non-pathogenic microbes isolated from Kimchi regulating apoptosis in cancer cell (Choi et al., 2002), healing infections (RestaLenert & Barrett, 2003), having anti-aging potential (Kim et al., 2002), and alleviating inflammatory associated diseases by inhibiting the growth of pathogens (Dey et al., 2019; Lomax & Calder, 2009; Yadav, Jain, & Sinha, 2007). Thus, functional foods and the microbes present in it have demonstrated positive health effects on the host and are thereby considered beneficial (Domingos-Lopes, Stanton, Ross, Dapkevicius, & Silva, 2017). A Gram-positive bacterium, the genus Weissella is closely related to lactic acid bacteria (Lee et al., 2012). Several reports highlight the presence of Weissella confusa in fermented foods such as sugar cane, moth bean, meat, rice wine, and sausage, and discuss its beneficial contributions (Fairfax, Lephart, & Salimnia, 2014; Sharma, Kandasamy, Kavitake, Prathap, & Shetty, 2018). As per the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), W. confusa has traditionally and to date been used for food fermentation processes (Jeong et al., 2018; Sturino, 2018; Vogel et al., 2011). However, some species of Weissella are also reported as an opportunistic pathogen (Fairfax & Salimnia, 2012; Lee, Huang, Liao, Lai, & Lee, 2011), although the criteria for the identification of pathogenic strain is not yet identified (Fairfax et al., 2014). Thus, identification and isolation of a potent probiotic strain of W. confusa are imperative to use further in the food industry and its co-cultures may improve the technological properties of the other fermented food to confer the human health in several other ways. Hence, the present study was undertaken to identify a potent starter strain of Weissella from Kimchi by analyzing the 16S-rRNA sequence. We identified the single nucleotide polymorphisms (SNPs) in the conserved region of 16S-rRNA sequence. The study has shown the potential of DD_A7 inhibiting the growth of foodborne pathogenic bacteria which further justified the prophylactic potential of the strain. In addition, we have shown its future industrial application to extract amylolytic and proteolytic enzymes.
2.2. Isolation, purification, and culture condition of kimchi isolated probiotic A total of 20 Kimchi samples were obtained from 15 different local restaurants of Daegu, South Korea. The samples were serially diluted in saline, spread on de Man, Rogosa and Sharpe (MRS) agar (Sigma), and incubated aerobically at 37 °C for 24 h. 2.2.1. Identification of isolates In the study, 18 pure colonies were isolated from the Kimchi sample and further subjected to Gram staining, according to the method suggested by Kozaki, Uchimura, and Okada (1992). Once the strain was identified as Gram-positive bacteria, we processed the sample for 16S ribosomal RNA sequencing provided by Genotech Crop (Pohang, South Korea) using the Sanger dideoxy sequencing (Sequencing kit, Applied Biosystems, Foster City), according to the method of Hellberg et al. (2013). Accession numbers were obtained by depositing the 16S-rRNA sequence of the strains at the National Center for Biotechnology Information (NCBI), GenBank database. The isolates were stored at −80 °C, supplemented with 50% glycerol for further use. In total, we identified three different bacteria, Lactobacillus Plantarum (2 strain) and W. confusa (1 strain) which was reported in our previous study (Khan, I., and Kang, S.C., 2016; Dey et al., 2019). 2.2.2. Estimation of genetic diversity To compare possible similarity or diversity, nBLAST (NCBI) was performed using the 16S-rRNA sequences of Kimchi isolated W. confusa DD_A7 against the published database sequences. The top 19 strains selected and multiple sequence alignment was performed with reference to DD_A7 using CLUSTALW version 1.6 (Kyoto University Bioinformatics Center, Japan), and incorporated in MEGA version 7.026 (Center for Evolutionary Medicine and Informatics, Biodesign Institute, McAllister Ave, USA). The nucleotide substitution matrix and transition-transversion bias (R) were estimated for the best model by applying the maximum likelihood method. The pattern of nucleotide substitution and transition-transversion bias was also estimated using the Maximum Composite Likelihood algorithm with the Tamura-Nei model. The evolutionary history was inferred using the Maximum likelihood method (ML); additionally, positions with a gap and missing data were eliminated. Phylogenetic analyses were conducted using the MEGA version 7.026 (Center for Evolutionary Medicine and Informatics, Biodesign Institute, McAllister Ave, USA) by applying the UPGMA method. 2.2.3. Single nucleotide polymorphism (SNP) analysis Next, we performed SNP analysis to identify the species-specific mutation and similarly of DD_A7 with the pre-reported probiotic strain of W. confusa. To this aim, we analyzed the 16S-rRNA conserved sequences among the 19 different strains of W. confusa and compared with DD_A7. Multiple sequence alignment was critically visualized for detecting any polymorphism present in both the forward and reverse strand, which was further confirmed by plotting the substitution matrix in the study. The substitution matrix characterized the exchange rate of one character to another character over time.
2. Material and methods 2.3. Preparation of cell-free culture supernatant 2.1. The bacterial strain used and culture condition The cell-free culture supernatant (CFCS) of DD_A7 was prepared as described by Chhabriaa and Desai (2018), with some minor modifications. Briefly, the overnight grown culture of DD_A7 in MRS broth at 37 °C was centrifuged at 6000 rpm for 15 min at 4 °C, the supernatant was collected and further filter-sterilized. The filtered supernatant was then freeze-dried to collect the dry mass and stored in −80 °C until
The pathogenic bacteria Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889) and Extended-spectrum beta-lactamase (ESBL)-E. coli 4 (NCCP, KBN10P03347), and ESBL-E. coli 5 (NCCP, KBN10P03452) were used in 664
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bacterial cells were treated with or without CFCS (as described above). After treatment, cells were washed three times using 50 mM PBS (pH 7.3), followed by centrifugation at 4000 rpm for 5 min. The centrifuged cells were resuspended in PBS and thinly smeared on a glass slide, followed by fixation with glutaraldehyde (2.5 g/100 ml; Electron Microscopy Science, USA) for 2 h. Fixed cells were subsequently dehydrated using graded ethanol (ranging from 50% to 100%), followed by CO2 drying. The dried cells were coated with gold in a sputter coater and observed under a scanning electron microscope (Hitachi-S4300, Japan).
used. 2.4. Influence of CFCS on the pathogenic bacteria CFCS of DD_A7 was tested against seven different pathogenic bacterial strains after neutralizing the pH. The pathogenic bacteria used in the study include Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889), ESBL-E. coli 4 (NCCP, KBN10P03347), and ESBL-E. coli 5 (NCCP, KBN10P03452). To investigate the antagonistic activity, we applied the agar well dilution assay as described by Fatima, Ahmada, and Chaudhrya (2017), with some minor changes. The well-grown bacterial culture (107 CFU/ml) was spread on the specific agar plate with 50% (v/v) of CFCS in the center well; plates were incubated at 37 °C for 24 h, and the clear zone of inhibition around the well was measured to evaluate the antibacterial activity.
2.6. DNase activity of CFCS The DNase activity of the CFCS was evaluated using the E. coli 0157:H7 plasmid. The protocol was followed as described by Jakhar et al. (2014), with some modifications. The plasmid was incubated with different concentrations of CFCS (2%, 4%, 8%, and 16%) to evaluate its effect on DNA, and resolved on 1% agarose gel to visualize and observe its effect. Where the plasmid subjected to 2 mM H2O2 treatment followed by exposure to UV light for 1 min was considered as a positive control in the experiment.
2.5. Effect of CFCS on the foodborne pathogenic Escherichia coli 0157:H7 2.5.1. Determination of bacterial cell viability The bacterial cell viability was evaluated for the 24 h CFCS (50%) pre-treated E. coli 0157:H7 culture in the presence of acridine orange (AO)/ethidium bromide (EtBr) (50 μg/ml; vol/vol) dual stain under the fluorescence microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification, as described by Hameed et al. (2016). AO stains both the live and dead cell population, whereas EtBr stains only the cells with disintegrated membrane and permits EtBr to enter the cell. Hence, live cells appear green and dead cells appear red in color. The fluorescence intensity was measured and analyzed using ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).
2.7. Safety assessment of DD_A7 2.7.1. Antibiotic susceptibility test Resistance towards antibiotics such as gentamycin (10 μg/ml), spectinomycin (100 μg/ml), ampicillin (10 μg/ml) and kanamycin (50 μg/ml) was evaluated by the disc overlay diffusion method as described by Odeyemi and Ahmad (2017), with some minor modification. Paper discs overlaid with antibiotics were placed on the agar surface seeded with the micro-organism (107 CFU/ml) and incubated for 24 h at 37 °C. The clear zone of inhibition was measured around the discs to analyze the resistivity.
2.5.2. Measurement of membrane integrity Rhodamine 123 (Rh 123, 10 μg/ml) staining was used to analyze the effect on the membrane integrity, as described by Warnes, Caves, and Keevil (2012). Pathogenic E. coli 0157:H7 were pre-treated for 24 h with 50% CFCS, followed by staining with Rh 123, and observed under a microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification to measure the intensity of green fluorescence. In the experiment, untreated cells were considered as a control. The green fluorescence indicates accumulation of the dye on the inner surface of the intact cell membrane. Images were further analyzed under the 3D surface plot using ImageJ 1.50i version (National Institute of Health, Bethesda, MD, USA).
2.7.2. Cytotoxic assessment on mammalian cells In order to estimate the toxic effect of CFCS, morphological changes of HCT-116 (ATCC® CCL-247™) and RAW 264.7 (ATCC® TIB-71™) cell were evaluated after exposure to varying concentrations of CFCS (0%, 10%, 30%, and 50%) for 24 h; morphological variations were assessed under the inverted microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at the magnification of 20×. Additionally, the effect on cell density after treatment was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Briefly, 1 × 105 cells/well were seeded in a 96-well tissue culture plate and treated with the above-mentioned concentrations of CFCS; to evaluate the toxic effect, cell viability was compared with untreated control cells.
2.5.3. Effect on intracellular Ca++ ions In order to estimate the intracellular Ca++ ion, Fura-2AM a cellpermeable fluorescent probe was used, as described by Werthén and Lundgren (2001), with some modification. Briefly, pathogenic E. coli 0157:H7 cells were treated with or without CFCS (50%) for 24 h. After 24 h incubation at 37 °C, the bacterial culture was centrifuged at 4000 rpm for 5 min. The bacterial pellets were then incubated with 5 μM of Fura 2-AM for 60 min at 37 °C in dark, followed by washing with 50 mM phosphate buffer saline (PBS, pH 7.3) and perfused in HBSS (Hank's balanced salt solution) buffer. The fluorescence intensity of cells was analyzed under a fluorescence microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification. Fluorescence intensity was quantified using ImageJ 1.50i version (National Institute of Health, Bethesda, MD, USA), after nullifying the background staining.
2.7.3. Affect of CFCS on zebrafish larvae The non-toxic behavior of CFCS was determined by morphological analysis in the zebrafish model system. Briefly, CFCS (10, 30, and 50%) and LPS (0.1%) treatment was given to 6 days post fertilization (dpf) zebrafish larva (n = 10) in a 6 well plate and were maintained in E3 medium for 24 h. To prepare a 60X stock E3 medium, take 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 and dissolve the ingredients in distilled water to a final volume of 2 L (adjust the pH to 7.2 with NaOH). To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L and add 0.00003% methylene blue (Sigma-Aldrich) to the solution. 2.8. Application of DD_A7
2.5.4. Scanning electron microscopy (SEM) To further support the membrane permeability findings, E. coli 0157:H7 were examined under the scanning electron microscope (SEM) and images obtained were compared with the untreated sample, as described by Nuño, Alberto, Arena, Zampini, and Isla (2018). Briefly,
2.8.1. Enzymatic activity of DD_A7 The amylolytic and proteolytic activities were evaluated using the streaking method, as reported by Sharma et al. (2018). Bacterial cultures were streaked on the starch (1% w/v) and skim milk (10% w/v) agar plates, respectively, followed by incubation at 37 °C for 24 h. 665
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Table 1 Various strains of W. confusa isolated from different food sources and human gastrointestinal tract, and reported from different geographical areas. Strain
NCBI accession number
Query cover
E value
Ident
Isolation matrix
Geographic origin
Genome size
DD_A7 BSR5 JCM 1093 NH 02 SM32 SM10 MMZ50B HYM105 HBUAS54173 HBUAS54147 IMAU50297 IMAU50289 IMAU50206 IMAU50203 HBUAS52116 HA12 HBUAS54202 HBUAS54166 HBUAS54121 HBUAS54114
MH341584.1 KY203672.1 LC063164.1 AB425970.1 KU060296.1 KU060300.1 EU157913.1 KT982252.1 MH701962.1 MH701936.1 MG547239.1 MG547233.1 MF623270.1 MF623233.1 MH392877.1 MF148168.1 MH701991.1 MH701955.1 MH701912.1 MH701906.1
98% 97% 97% 97% 97% 97% 97% 96% 96% 96% 96% 96% 96% 96% 97% 96% 96% 96% 96%
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%
Kimchi Fermented batter of Kodomillet and Moth bean Sugar cane Thai fermented sausage sugarcane stalk after cutting sugarcane stalk after cutting Gueddid an artisanal Tunisian dry fermented meat Huyumei broad bean paste Young peoples intestinal tract Young peoples intestinal tract human intestine human intestine rice wine rice wine Zha-Chili larvae and adults of sheep abomasal nematode parasite Young peoples intestinal tract Young peoples intestinal tract Young peoples intestinal tract Young peoples intestinal tract
South Korea India Japan Thailand South Africa South Africa Tunisia China China China China China China China China New Zealand China China China China
1558 1547 1538 1534 1537 1537 1548 1513 1499 1499 1499 1499 1499 1499 1499 1516 1499 1499 1499 1499
Bacterial cells were suspended at the MOI of 1:100 ratio to HCT116 cells and allowed the bacterial cell to adhered on the colon cells for 2 h. After 2 h of incubation, cells was fixed with the antifade solution (Sigma) and observed under the fluorescent microscope.
2.8.2. Bacterial adhesion to different solvents and auto-aggregation ability The Bacteria Adhesion To Hydrocarbon (BATH) test was used to evaluate the cell surface properties of strain DD_A7. Briefly, bacterial cells were incubated in different solvents (xylene, ethyl acetate, and chloroform) for 4 h, as proposed by Dias, Duarte, and Schwan (2013) and Mohanram, Jagtap, and Kumar (2016). The auto-aggregation ability of strain DD_A7 was also analyzed as described by Khan and Kang (2016). To calculate the auto-aggregation ability, OD was recorded for the next 24 h at intervals of every 4 h, using a spectrophotometer at 600 nm (UV-2120 Optizen, Mecasys, South Korea). The below-mentioned equation was then applied for calculations:
Auto − aggregation % =
bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp
2.8.5. Bile-salt tolerance of DD_A7 Tolerance of DD_A7 against bile-salt was examined by measuring the absorbance values of the culture at 600 nm in MRS broth with different concentrations of bile salts [0.05%, 0.1%, 0.3% (w/v)] during 24 h of incubation at 37 °C with the initial turbidity of 0.2 OD600 and compared with its control.
1−At x 100 A0
2.9. Statistical analysis
where, At indicates the reading after incubation of bacteria at different time points, and A0 indicates the readings at the initial point (0h) at 600 nm.
All results are expressed as the mean ± standard deviation (SD) and the statistical significance is calculated using Student's “t” tests.
2.8.3. Biofilm formation ability The biofilm formation ability of DD_A7 was analyzed in 30 mm flat bottom plates, as described by Al-Shabib et al. (2018), with little changes in the protocol. Briefly, 1% well-grown cultures of DD_A7 were inoculated at initial turbidity of 0.2 at OD600 nm, in freshly prepared MRS broth. The cultures were allowed to grow without shaking for the next 72 h at 37 °C. The plates were then washed twice with PBS to remove non-adherent cells from the surface. Plates with the adherent colonies were stained with crystal violet for 20 min, rinsed with PBS to remove the extra stain, and images were captured under an inverted microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at a resolution of 100X. Images were then analyzed under the 3D surface plot using ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).
3. Result and discussion 3.1. Concordant phylogeny clade of DD_A7 with pre-reported prebiotic W. confusa The 16S-rRNA sequence of DD_A7 was submitted to GenBank against the accession ID (MH341584.1), and FASTA sequence was retrieved for further analysis (https://www.ncbi.nlm.nih.gov/nuccore/ 1389426126). Next, we selected 19 sequences from the public database which exhibits 99% similarity with identical genotypic identity and covered the maximum query in the range between 96 and 98% with a minimum error value (0.00) in respect to DD_A7 sequence. The strains of 19 different sequences were either isolated from food or found in a healthy human gastrointestinal tract (Table 1). We analyzed the root centered phylogenic tree with maximum likelihood and built the gene cluster (Fig. 1) based on the 16S-rRNA sequencing among the chosen strains. This group of 19 strains is further referred to as the W. confusa group. The result of the evolutionary divergence reveals an overall mean distance of 0.001 (p distance) (Supplementary Table 1). The evolutionary history was inferred using the Maximum Likelihood method. Initial tree(s) for the heuristic search were obtained by Neighbor-Join algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. Further topology was selected with superior log-likelihood value. All positions containing gap (s) and missing data were eliminated. The Phylogenetic tree revealed
2.8.4. Assessment for adhesion property of W. confusa DD_A7 on colon cells The adhesive property of DD_A7 was evaluated using HCT-116 cell line model as described by Jayashree et al., 2018, with some little modification. Briefly, the overnight grown culture of DD_A7 was centrifuged at 5000 rpm for 3 min. After centrifuge, cells were washed with PBS for three times and suspended in 1 ml of 0.1 M sodium carbonate buffer containing 2 μg/ml of Fluorescein isothiocyanate (FITC). The cells were incubated at 25 °C for 1 h. After incubation, cells were washed thrice with PBS to remove the excess stain. On the other hand, HCT-116 cells were stained with Mitotracker Deep Red (MTDR) (1 μg/ ml) and DAPI (1 μg/ml), incubated for 30 min at 37 °C in a 5% CO2. 666
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Table 3 Antibacterial potential of DD_A7 against pathogenic bacteria. Results are expressed as the diameter of the clear zone of inhibition (in mm). The zone diameter interpretive criterion was followed as described by CLSI M100-S20 (2010). Experiments were performed in triplicates and data are presented as mean ± SD. Strain name
ATCC number
Zone of inhibition (in mm)
Listeria monocytogenes Staphylococcus aureus Salmonella typhimurium Bacillus cereus Escherichia coli 0157:H7 ESBL-E. coli 4 ESBL-E. coli 5
ATCC 7644 ATCC 12600 ATCC 43174 ATCC 13061 ATCC 43889 NCCP, KBN10P03347 NCCP, KBN10P03452
22 9 17 20 28 17 24
alignment, as shown in Supplementary Fig. 1. Next, polymorphism was differentiated based on their types, i.e., transition and transversion (Table 2). Transition polymorphisms are the interchange either between two purines or two pyrimidines, whereas transversion polymorphisms are the interchanges of purine to pyrimidine or vice versa. In the current study, transversion was found to be more prevalent, where 40 sites were detected with a change in the nucleotide G to nucleotide T, and 30 sites were found for T to G transversion. On the other hand, only 36 sites were detected with a transition, where G was replaced by A. This result of the SNP analysis indicates that transversion is the commonly occurring polymorphism, especially the transversion of G/T or T/G. In addition, the site 1529(G/T) and 1530(T/G) were the predominant sites located in 18 sequences out of 19 when aligned with the query sequence. To further validate the results of evolution, nucleotide substitution pattern (Supplementary Table 2) and substitution matrix (Supplementary Table 3) were built using the MEGA software, version 7.026.
Fig. 1. The full-length 16S-rRNA sequence was aligned, and a phylogenetic tree was constructed by the UPGMA method using the MEGA software, version 7.026. The tree represents the relationship with other bacterial species. The data represents the similarity of DD_A7 with W. confusa.
that all native strains fit into an evolutionary cluster comprising the members of W. confusa strains, and other species are grouped into a separate cluster according to their relatedness found if any. A total of 201 nodes were thus identified (Fig. 1). The tree is drawn to scale of 0.005, with branch lengths measured in the number of substitutions per site. Further, we critically analyzed the sequence of the W. confusa group, and polymorphic sites were evaluated where two different nucleotide bases were detected at a particular position. The detected polymorphic sites in the sequences were analyzed on sequence
3.2. CFCS of DD_A7 as a potent antibacterial agent The isolated DD_A7 strain under evaluation was found to have
Table 2 Different types of polymorphisms observed in the multiple sequence alignment performed using CLUSTALW, version 1.6 (Kyoto University Bioinformatics Center, Japan). Accession no.
Transition
Transversion
KY203672.1
4(G/A), 5(G/A), 6(G/A), 15(T/C), 16(C/T), 1531(G/A), 1533(G/A), 1535(T/C) 4(G/A), 5(G/A), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/A) 14(T/C), 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/A) 5(A/G), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 111(G/A), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1547(G/A) 5(A/G), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 36(C/T), 1054(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/ A) 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1550(A/G), 1551(C/T), 1555(G/A) 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A) – – – – – – – 1145(C/T), 1168(C/T), 1207(A/G), 1531(G/A), 1533(A/G), 1535(T/ C), 1542(G/A), 1546(G/A) 1168(C/T) 331(A/G) 41(T/C) –
7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T)
LC063164.1 AB425970.1 KU060296.1 KU060300.1
EU157913.1 KT982252.1 MH701962.1 MH701936.1 MG547239.1 MG547233.1 MF623270.1 MF623233.1 MH392877.1 MF148168.1 MH701991.1 MH701955.1 MH701912.1 MH701906.1
7(T/G), 10(G/T), 17(T/G), 1529(G/T), 1530(G/T), 1534(G/T), 1356(G/C), 1545(G/T), 1548(G/C), 1549(A/C) 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(G/C), 1545(G/T), 1548(G/C) 7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1546(G/T), 1548(G/C), 1549(A/C) 7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1548(G/C), 1549(A/C) 17(T/G), 214(A/C), 1444(C/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/ T), 1548(G/C), 1549(A/C), 1552(C/A), 1553(C/G), 1554(C/G), 1556(T/G), 1559(A/C) 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1548(G/C), 1549(A/C) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 101(T/G), 1529(G/T), 1530(T/G)
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Fig. 2. Microscopic assessment of pathogenic E. coli 0157:H7 (ATCC 43889) after treatment with 50% CFCS. Bacterial cell death was examined by the AO/EtBr dual staining platform. The fluorescent intensity was recorded under the inverted microscope (Scale bar = 10 μm).
Fig. 3. Potential of 50% CFCS effecting the necessary biomolecules of pathogenic E. coli 0157:H7. (A) Effect on the membrane integrity was assessed by Rh 123 staining. Increase in bright green fluorescence intensity indicates the intact membrane of pathogenic bacteria due to the accumulation of dye on the cell surface. (B) Intracellular Ca++ accumulation was quantified using the ImageJ software after staining with Fura-2AM. (C) Scanning electron microscope analysis of E. coli O157: H7: untreated control cells show a regular and intact cell morphology, whereas CFCS treated cells show a destroyed cell membrane morphology. Scale bar = 2.5 μm. (D) DNase activity of CFCS was visualized on 1% agarose gel. From left to right → M, 1 Kb DNA marker; Lane 1, Control (E. coli plasmid); Lane 2, DNA damage induced by H2O2 treatment followed by 1 min UV radiation was exposed to the plasmid; Lane 3, CFCS (2%) treated plasmid; Lane 4, CFCS (4%) treated plasmid; Lane 5, CFCS (8%) treated plasmid; Lane 6, CFCS (16%) treated plasmid. The ratio of Nicked: supercoiled DNA was estimated. Statistical significance was determined with the help of Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
antagonism varied among different strains, with inhibition ranging from 9 to 28 mm, as presented in Table 3. The CFCS was observed to be most effective against the Escherichia coli 0157:H7 (ATCC 43889) with 28 mm zone of inhibition, whereas Staphylococcus aureus (ATCC 12600) showed the least susceptibility (9 mm of clear zone of inhibition). Since E. coli 0157:H7 was found to be the most susceptible strain, we decided to use it in all further experiments.
antimicrobial activity against pathogenic bacteria such as Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889), ESBL-E. coli 4 (NCCP, KBN10P03347) and ESBL-E. coli 5 (NCCP, KBN10P03452). We observed that 50% CFCS of DD_A7 has antagonistic effects against all the tested pathogens, but the results revealed that the degrees of 668
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3.3. Influence of CFCS on the DNA integrity
Table 4 (A) Antibiogram susceptibility test of DD_A7 against the commonly prescribed antibiotics, (B) Enzymatic activity of W. confusa DD_A7. Experiments were performed in triplicates and data are presented as mean ± SD. A. Antibiotic
Concentration Zone of inhibition
Resistant Intermediate Susceptible
Gentamycin Spectinomycin Ampicillin Kanamycin
10 μg 100 μg 10 μg 50 μg
– + – –
24 mm 8 mm 28 mm 17 mm
– – – +
Next, we analyzed the effect of CFCS on DNA. E. coli 0157:H7 plasmid was isolated and treated with different concentrations of CFCS. As presented in Fig. 3D, CFCS shows a dose-dependent potential to interfere with the DNA integrity, in a manner similar to H2O2 treated samples. DNA damage after H2O2 treatment (positive control) was observed to increase by 0.55 ± 0.11 fold, and a similar increase was observed after exposure to 2%, 4%, 8%, and 16% CFCS, where the fold changes recorded were 0.16 ± 0.11, 0.28 ± 0.06, 0.34 ± 0.06, and 0.42 ± 0.16, respectively. This result clearly indicates that CFCS of DD_A7 disturbs not only the membrane-bound proteins but also damages the DNA integrity, which further promotes cell death (Khan & Kang, 2016).
+ – + –
B. Enzymatic activity
Amylolytic
Proteolytic
W. confusa DD_A7
+
+
Next, we validated the inhibitory effect of CFCS (50%), by studying its effect on the viability of E. coli 0157:H7 using the AO/EtBr dual stain. Our results indicate 43 ± 0.3% cell death after 24 h treatment, which is a significant increase (P < 0.001) as compared to the control (13 ± 0.5%). This study proves that E. coli 0157:H7 cells were less viable after exposure, as compared to untreated control cells (Fig. 2). We further hypothesized that disruption in the bacterial cell membrane allows EtBr to permeate inside the cell, resulting in subsequent cell death. We, therefore, studied the effect of CFCS (50%) on bacterial membrane permeability using the Rh 123 staining method. We observed that the membrane integrity of E. coli 0157:H7 was decreased after treatment when compared with the untreated control bacterial cells (Hu et al., 2016; Wei et al., 2011). Exposure to CFCS resulted in the targeted cells losing their membrane integrity and becoming leaky or ruptured (Fig. 3A). Due to the rupture in the cell membrane, intracellular Ca++ ions were released, and subsequently, the Fura-2AM fluorescence ratio decreased significantly by 67 ± 2% (P < 0.001) (Fig. 3B). These findings were supported by the results of SEM analysis (Fig. 3C). Our results are similar to the finding of Werthén and Lundgren (2001), where they suggest that more Ca++ ions are found in a bacterium with an intact cell membrane.
3.4. Validation of safety concerns associated with the usage of DD_A7 3.4.1. Antibiotic susceptibility test The present study also has shown the antibiogram susceptibility of DD_A7 against the antibiotics in Table 4. Our results indicate that DD_A7 is susceptible against gentamycin (10 μg/mL) and ampicillin (10 μg/mL), showing 24 mm and 28 mm of clear zone of inhibition, respectively. Intermediate susceptibility was observed against kanamycin (50 μg/mL; 17 mm clear zone of inhibition), whereas the strain was considered to be resistant against spectinomycin (100 μg/mL; 8 mm clear zone of inhibition). The criterion used here for the interpretation of results is adapted from CLSI M100-S20 (2010). The results proved the sensitivity of DD_A7 towards the commonly prescribed antibiotics at their minimum concentrations, thereby imparting the strain to be more acceptable for further use. 3.4.2. Cytotoxic assessment of CFCS In order to investigate the toxic effects of CFCS in the host body, we evaluated the effect of CFCS in vitro on two different cell lines: colon cancer HCT-116 and RAW 264.7 macrophages cell line. Fig. 4A and B depicts, non-significant morphological changes of those treated cell; in addition, a non-significant decrease was observed in the cell density of
Fig. 4. Cytotoxic effect of CFCS was evaluated in, (A) HCT-116, (B) and RAW 264.7 cell lines. Representative images of morphological changes were presented, which was observed under the inverted microscope. The effect of CFCS on the cell density was analyzed by MTT assay, where untreated cells were considered as control. Scale bar = 0.1 mm. Experiments were performed in triplicates and data are presented as mean ± SD. (C) Non-toxic effects of CFCS on the morphological zebrafish larvae. The arrow represents the spinal cord curvature upon LPS treatment. The mortality rate upon treatment was also recorded. 669
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Fig. 5. (A) Biofilm formation ability of DD_A7 at 74 h. The image is quantified by ImageJ software, version 1.50i, under the 3D surface plot, at a resolution of 100X as viewed under an inverted microscope. The results shown in the figure are the mean of three independent experiments. (B) Adhesion of DD_A7 on HCT-116 cells after 2 h of incubation, where green fluorescence (upper left) represented the bacteria stained with FITC, and HCT-116 cells were stained with DAPI (upper right) and MTDR (lower left). The images obtained at different filters were finally merged and presented (lower right). (C) Cell surface hydrophobicity of DD_A7 was analyzed using BATH assay. (D) Analysis of time-dependent auto-aggregation ability of DD_A7. Experiments were performed in triplicates and data are presented as mean ± SD. (E) The growth of DD_A7 in MRS broth with different concentration of bile salts was evaluated for 24 h at 37 °C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
10% (w/v) skim milk by using these ingredients as a primary carbon source (Table 2B). These characteristics of the strain make it more useful for its further use in the industry involved in the isolation of amylolytic and proteolytic enzymes.
both cell lines (90.3 ± 3.0% and 89.4 ± 2.1% in HCT-116 and RAW 264.7 cells, respectively) after treatment with the 50% of CFCS for 24 h, as compared to the untreated control cells. The assay shows similar results in both the cell lines. Further, we have determined the non-toxic effect of CFCS on the morphological and survival rate of zebrafish larvae and compared with the LPS treated larval group. As the result depicted in Fig. 4C, after LPS treatment, morphological development of larvae was altered. LPS treatment triggered the spinal cord curving, which, however, was recovered after treatment of CFCS. Moreover, LPS treatment was found to affect the mortality rate of the zebrafish larvae, which was not the case in CFCS treated larval group. The observation validates the non-toxic effect of CFCS to the host cells. Thus, the safety concern of DD_A7 is validated and supports its further use for imparting health benefits.
3.6. DD_A7 forms a biofilm due to its adhesive nature As previously reported in our in vitro study (Dey et al., 2019), W. confusa has the potential to reduce inflammation induced by pathogens. However, in order to survive in the gastrointestinal tract and exert its benefits, colonization on the gut surface is essential. Biofilm plays a crucial role in maintaining the relationship between the residence and microbes. Our study revealed that DD_A7 strain has the ability to form a biofilm at 74 h (Fig. 5A). The colonization of DD_A7 was observed clearly. The adherence density of the probiotic was further estimated by ImageJ software, version 1.50i, which shows a dense bacterial population in root mean square (RMS) value (140–180 pixels). The density of the bacterial population was gradually observed to increase with increasing incubation time (data not shown). In addition, microscopic observation proved that the W. confusa DD_A7 has a potential adhesion property to the surface of HCT-116 cells (Fig. 5B). It is evident from several studies that bacterial cell surface plays a critical role in the colonization of bacteria on the human gut surface, and this process is critically linked with the cell surface property. The first phase of biofilm formation is due to its hydrophobic surface potential which facilitates the colonization of the beneficial micro-organism and modulates the host immune system (Tresse et al., 2006).
3.5. Enzymatic activity of DD_A7 Since W. confusa are used as a starter species in various traditional fermentation processes (Vogel et al., 2011), we decided to evaluate the amylolytic and proteolytic activity of DD_A7. These enzymes play crucial roles in hydrolyzing starch into disaccharide or trisaccharide and converting peptides into free amino acids (De Souza & Nobre, 2017). Assessing the secreting ability of these enzymes could, therefore, add value to its usage as a starter species. The aim of the experiment was also to identify whether the strain has the potential to utilize starch and agar as a primary carbon source for growth. As expected, DD_A7 has the ability to grow on agar plates containing 1% (w/v) starch and 670
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For adhesion to the intestinal cells or any solid surface, bacteria must possess a hydrophobic surface. Hence, BATH assay was performed using three different solvents to evaluate bacterial adhesion which would help us to determine the cell surface potential: xylene (polar solvent), ethyl acetate (monopolar and a strong Lewis-basic solvent), and chloroform (monopolar but a Lewis-acidic solvent). This experiment revealed that DD_A7 showed 82.11% hydrophobicity towards xylene, indicating that the bacterial cell possesses a greater hydrophobic surface. The affinity towards ethyl acetate was 24.7%, which is higher than that of chloroform (3.8%) (Fig. 5C). These results indicate that DD_A7 has the strongest electron donating characteristic. Further, the results were justified by examining the auto-aggregation ability, where DD_A7 shows an increasing auto-aggregation depending on the time of incubation (Fig. 5D). The highest auto-aggregation shown by DD_A7 was 23% at 24 h. The findings of our experiment are in accordance with those reported by Xu et al. (2009).
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J., Han, N. S., & Kim, J. H. (2012). Probiotic properties of Weissella strains isolated from human faeces. Anaerobe, 18, 96–102. Lomax, A. R., & Calder, P. C. (2009). Probiotics, immune function, infection, and inflammation: A review of the evidence from studies conducted in humans. Current Pharmaceutical Design, 15, 1428–1518. Mohanram, R., Jagtap, C., & Kumar, P. (2016). Isolation, screening, and characterization of surface-active agent-producing, oil-degrading marine bacteria of Mumbai Harbor. Marine Pollution Bulletin, 105, 131–138. Montoro, B. P., Benomar, N., Lerma, L. L., Gutierrez, S. C., Galvez, A., & Abriouel, H. (2016). Fermented Alorena table olives as a source of potential probiotic Lactobacillus pentosus strains. Frontiers in Microbiology, 7, 1–13. Nuño, G., Alberto, M. R., Arena, M. E., Zampini, I. C., & Isla, M. I. (2018). Effect of Zuccagnia punctata Cav. (Fabaceae) extract on pro-inflammatory enzymes and on planktonic cells and biofilm from Staphylococcus aureus. 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3.7. Bile salts tolerance of DD_A7 W. confusa DD_A7 possesses tolerance against bile salts. The strain is not only tolerant, but its cell numbers were even increased after 6-h exposure to 0.3% bile salts in MRS broth like its control. However, a fall in the cell density of DD_A7 was also observed at 12 h in 0.3% bile salts when compared with the control (Fig. 5E). 4. Conclusion The present study describes, for the first time the polymorphism in the conserved region of the 16S rRNA of Weissella. The study analyzed the SNPs among the 19 other strains which are isolated from food and the human intestine. These SNPs provided more intra-specific information which could be effectively used to define the evolutionary history of the species in the future. The information would be helpful to design the set of primers or specific probe to identify the candidate prebiotic strain. The finding of the study also demonstrated the antimicrobial activity of non-pathogenic DD A7 strain of W. confusa. Moreover, the study also validates the contemporary safety demands of DD_A7 and its CFCS for its further usage. To validate its future for host immunomodulating characteristic is ongoing. Author contributions and acknowledgements DDK conceived most of the experiments with the help of KBG. CS conducted the bioinformatics analysis. DDK prepared the manuscript, which was edited and finalized by KSC. All the authors have read and approved the manuscript before submission. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.05.089. References Al-Shabib, N. A., Husain, F. M., Khan, R. A., Khan, M. S., Alam, M. Z., Ansari, F. A., et al. (2018). Interference of phosphane copper (I) complexes of β-carboline with quorum sensing regulated virulence functions and biofilm in foodborne pathogenic bacteria: A first report. Saudi Journal of Biological Sciences (in press). Chhabriaa, S., & Desai, K. (2018). Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin. Saudi Journal of Biological Sciences, 25, 1429–1438. Choi, S. M., Jeon, Y. S., Rhee, S. H., et al. (2002). Fermentation characteristics and antimutagenicity of kimchi that prepared with a different ratio of seed in red pepper powder. Journal of Korean Association of Cancer Prevention, 7, 51–59. De Souza, J. V., & Nobre, F. S. D. (2017). Protective, technological, and functional properties of select autochthonous lactic acid bacteria from goat dairy products. Current Opinion in Food Science, 13, 1–9. Dey, D. K., Khan, I., & Kang, S. C. (2019). Anti-bacterial susceptibility profiling of Weissella confusa DD_A7 against the multidrug-resistant ESBL-positive E. coli.
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53–62. Vogel, R. F., Hammes, W. P., Habermeyer, M., Engel, K. H., Knorr, D., & Eisenbrand, G. Senate commission on food safety (SKLM) of the German Research foundation. (2011). Microbial food cultures–opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG). Molecular Nutrition & Food Research, 55(4), 654–662. Warnes, S. L., Caves, V., & Keevil, C. W. (2012). Mechanism of copper surface toxicity in Escherichia coli 0157:H7:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria. Environmental Microbiology, 14, 1730–1743. Wei, Z., Yang, X., Yin, S., et al. (2011). Efficacy of Bacillus-fortified organic fertiliser in
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Characterization of Weissella confusa DD_A7 isolated from kimchi 1
1
Debasish Kumar Dey , Bon Gyo Koo , Chanchal Sharma, Sun Chul Kang
∗
T
Department of Biotechnology, Daegu University, Gyeongsan, Gyeongbuk, 38453, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Weissella confusa DD_A7 Non-pathogenic bacteria 16S-rRNA SNPs Antibacterial activityAbbreviations: NCBI National center for biotechnology information ML Maximum likelihood method MCL Maximum composite likelihood MRS De man Rogosa and sharpe medium SNP Single nucleotide polymorphism RMS Root mean square PBS Phosphate buffer saline BATH Bacteria adhesion to hydrocarbon CFCS Cell-free culture supernatant AO Acridine orange EtBr Ethidium bromide Ca++ Calcium SEM Scanning electron microscope SD Standard deviation NCCP National culture collection for pathogens ATCC American type culture collection
Foodborne pathogenic bacteria like, Escherichia coli 0157:H7, Bacillus cereus and Salmonella typhimurium are the biggest threat to food industries. Their growth in the environment and transmission of deadly diseases has become a challenge. The present study confirmed the genetic identity of DD_A7 is similar to the reported probiotic strains of Weissella confusa by analyzing the polymorphism in the conserved region of 16S-rRNA sequence. The Kimchi isolated, non-pathogenic W. confusa DD_A7 strain has shown the potential to control the growth of foodborne pathogens. Furthermore, the cell-free culture supernatant of DD_A7 shows the potential to rapture the membrane integrity of E. coli 0157:H7 by influencing the Ca++ ion channels. The safety concern associated with the usage of DD_A7 strain was also determined by testing its antibiotic susceptibility against different antibiotics and confirmed its non-toxic behavior on mammalian cells. Nevertheless, the strain has also encompassed several applications such as isolation of amylolytic and proteolytic enzymes. Collectively, the study will help to control the growth of foodborne pathogenic bacteria using the studied probiotic strain. In addition, the study will also help to construct a species-specific probe in future for the identification of the non-pathogenic strain of W. confusa for its application in food industries.
∗ Corresponding author. Department of Biotechnology, College of Engineering, Daegu University Jillyang, Naeri-ri, Gyeongsan, Gyeongbuk, 38453, Republic of Korea. E-mail addresses: [email protected] (D.K. Dey), [email protected] (B.G. Koo), [email protected] (C. Sharma), [email protected] (S.C. Kang). 1 Authors contributed equally.
https://doi.org/10.1016/j.lwt.2019.05.089 Received 23 February 2019; Received in revised form 9 May 2019; Accepted 18 May 2019 Available online 20 May 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
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1. Introduction
this study. The pathogenic bacteria were grown in Luria-Bertani Broth (LB) media at 37 °C.
Food is a necessity for the survival and presences of pathogens in it have been the biggest culprit for food industries. Numerous government agencies and educational institutions are working to bring advancement in the field of food science and technology (Kim, Parka, & Kang, 2018). The field of study importantly deals with the quality of food and safety aspects. However, despite of numerous advancements in the field, food safety is still a concern. As these pathogens are associated with several virulence factors which cause severe disease and illness such as, listeriosis, diarrhea, hemolytic-uremic syndrome, and so on in human. Currently, the demands of functional foods are increasing tremendously because of their beneficial properties (Montoro et al., 2016; Shangpliang, Sharma, Rai, & Tamang, 2017). Among the several other functional foods, Korean cuisine, kimchi, has gained importance due to its health benefits (Khan & Kang, 2016). Researchers have shown that microbes isolated from Kimchi possess an inherent ability to modulate the host immune response by producing a large number of secretory metabolites such as proteins, enzymes, antimicrobial compounds, and vitamins (Dey, Khan, & Kang, 2019; Lee et al., 2014). Additionally, several pieces of evidence have underscored the importance of Kimchi and the non-pathogenic microbes isolated from Kimchi regulating apoptosis in cancer cell (Choi et al., 2002), healing infections (RestaLenert & Barrett, 2003), having anti-aging potential (Kim et al., 2002), and alleviating inflammatory associated diseases by inhibiting the growth of pathogens (Dey et al., 2019; Lomax & Calder, 2009; Yadav, Jain, & Sinha, 2007). Thus, functional foods and the microbes present in it have demonstrated positive health effects on the host and are thereby considered beneficial (Domingos-Lopes, Stanton, Ross, Dapkevicius, & Silva, 2017). A Gram-positive bacterium, the genus Weissella is closely related to lactic acid bacteria (Lee et al., 2012). Several reports highlight the presence of Weissella confusa in fermented foods such as sugar cane, moth bean, meat, rice wine, and sausage, and discuss its beneficial contributions (Fairfax, Lephart, & Salimnia, 2014; Sharma, Kandasamy, Kavitake, Prathap, & Shetty, 2018). As per the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), W. confusa has traditionally and to date been used for food fermentation processes (Jeong et al., 2018; Sturino, 2018; Vogel et al., 2011). However, some species of Weissella are also reported as an opportunistic pathogen (Fairfax & Salimnia, 2012; Lee, Huang, Liao, Lai, & Lee, 2011), although the criteria for the identification of pathogenic strain is not yet identified (Fairfax et al., 2014). Thus, identification and isolation of a potent probiotic strain of W. confusa are imperative to use further in the food industry and its co-cultures may improve the technological properties of the other fermented food to confer the human health in several other ways. Hence, the present study was undertaken to identify a potent starter strain of Weissella from Kimchi by analyzing the 16S-rRNA sequence. We identified the single nucleotide polymorphisms (SNPs) in the conserved region of 16S-rRNA sequence. The study has shown the potential of DD_A7 inhibiting the growth of foodborne pathogenic bacteria which further justified the prophylactic potential of the strain. In addition, we have shown its future industrial application to extract amylolytic and proteolytic enzymes.
2.2. Isolation, purification, and culture condition of kimchi isolated probiotic A total of 20 Kimchi samples were obtained from 15 different local restaurants of Daegu, South Korea. The samples were serially diluted in saline, spread on de Man, Rogosa and Sharpe (MRS) agar (Sigma), and incubated aerobically at 37 °C for 24 h. 2.2.1. Identification of isolates In the study, 18 pure colonies were isolated from the Kimchi sample and further subjected to Gram staining, according to the method suggested by Kozaki, Uchimura, and Okada (1992). Once the strain was identified as Gram-positive bacteria, we processed the sample for 16S ribosomal RNA sequencing provided by Genotech Crop (Pohang, South Korea) using the Sanger dideoxy sequencing (Sequencing kit, Applied Biosystems, Foster City), according to the method of Hellberg et al. (2013). Accession numbers were obtained by depositing the 16S-rRNA sequence of the strains at the National Center for Biotechnology Information (NCBI), GenBank database. The isolates were stored at −80 °C, supplemented with 50% glycerol for further use. In total, we identified three different bacteria, Lactobacillus Plantarum (2 strain) and W. confusa (1 strain) which was reported in our previous study (Khan, I., and Kang, S.C., 2016; Dey et al., 2019). 2.2.2. Estimation of genetic diversity To compare possible similarity or diversity, nBLAST (NCBI) was performed using the 16S-rRNA sequences of Kimchi isolated W. confusa DD_A7 against the published database sequences. The top 19 strains selected and multiple sequence alignment was performed with reference to DD_A7 using CLUSTALW version 1.6 (Kyoto University Bioinformatics Center, Japan), and incorporated in MEGA version 7.026 (Center for Evolutionary Medicine and Informatics, Biodesign Institute, McAllister Ave, USA). The nucleotide substitution matrix and transition-transversion bias (R) were estimated for the best model by applying the maximum likelihood method. The pattern of nucleotide substitution and transition-transversion bias was also estimated using the Maximum Composite Likelihood algorithm with the Tamura-Nei model. The evolutionary history was inferred using the Maximum likelihood method (ML); additionally, positions with a gap and missing data were eliminated. Phylogenetic analyses were conducted using the MEGA version 7.026 (Center for Evolutionary Medicine and Informatics, Biodesign Institute, McAllister Ave, USA) by applying the UPGMA method. 2.2.3. Single nucleotide polymorphism (SNP) analysis Next, we performed SNP analysis to identify the species-specific mutation and similarly of DD_A7 with the pre-reported probiotic strain of W. confusa. To this aim, we analyzed the 16S-rRNA conserved sequences among the 19 different strains of W. confusa and compared with DD_A7. Multiple sequence alignment was critically visualized for detecting any polymorphism present in both the forward and reverse strand, which was further confirmed by plotting the substitution matrix in the study. The substitution matrix characterized the exchange rate of one character to another character over time.
2. Material and methods 2.3. Preparation of cell-free culture supernatant 2.1. The bacterial strain used and culture condition The cell-free culture supernatant (CFCS) of DD_A7 was prepared as described by Chhabriaa and Desai (2018), with some minor modifications. Briefly, the overnight grown culture of DD_A7 in MRS broth at 37 °C was centrifuged at 6000 rpm for 15 min at 4 °C, the supernatant was collected and further filter-sterilized. The filtered supernatant was then freeze-dried to collect the dry mass and stored in −80 °C until
The pathogenic bacteria Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889) and Extended-spectrum beta-lactamase (ESBL)-E. coli 4 (NCCP, KBN10P03347), and ESBL-E. coli 5 (NCCP, KBN10P03452) were used in 664
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bacterial cells were treated with or without CFCS (as described above). After treatment, cells were washed three times using 50 mM PBS (pH 7.3), followed by centrifugation at 4000 rpm for 5 min. The centrifuged cells were resuspended in PBS and thinly smeared on a glass slide, followed by fixation with glutaraldehyde (2.5 g/100 ml; Electron Microscopy Science, USA) for 2 h. Fixed cells were subsequently dehydrated using graded ethanol (ranging from 50% to 100%), followed by CO2 drying. The dried cells were coated with gold in a sputter coater and observed under a scanning electron microscope (Hitachi-S4300, Japan).
used. 2.4. Influence of CFCS on the pathogenic bacteria CFCS of DD_A7 was tested against seven different pathogenic bacterial strains after neutralizing the pH. The pathogenic bacteria used in the study include Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889), ESBL-E. coli 4 (NCCP, KBN10P03347), and ESBL-E. coli 5 (NCCP, KBN10P03452). To investigate the antagonistic activity, we applied the agar well dilution assay as described by Fatima, Ahmada, and Chaudhrya (2017), with some minor changes. The well-grown bacterial culture (107 CFU/ml) was spread on the specific agar plate with 50% (v/v) of CFCS in the center well; plates were incubated at 37 °C for 24 h, and the clear zone of inhibition around the well was measured to evaluate the antibacterial activity.
2.6. DNase activity of CFCS The DNase activity of the CFCS was evaluated using the E. coli 0157:H7 plasmid. The protocol was followed as described by Jakhar et al. (2014), with some modifications. The plasmid was incubated with different concentrations of CFCS (2%, 4%, 8%, and 16%) to evaluate its effect on DNA, and resolved on 1% agarose gel to visualize and observe its effect. Where the plasmid subjected to 2 mM H2O2 treatment followed by exposure to UV light for 1 min was considered as a positive control in the experiment.
2.5. Effect of CFCS on the foodborne pathogenic Escherichia coli 0157:H7 2.5.1. Determination of bacterial cell viability The bacterial cell viability was evaluated for the 24 h CFCS (50%) pre-treated E. coli 0157:H7 culture in the presence of acridine orange (AO)/ethidium bromide (EtBr) (50 μg/ml; vol/vol) dual stain under the fluorescence microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification, as described by Hameed et al. (2016). AO stains both the live and dead cell population, whereas EtBr stains only the cells with disintegrated membrane and permits EtBr to enter the cell. Hence, live cells appear green and dead cells appear red in color. The fluorescence intensity was measured and analyzed using ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).
2.7. Safety assessment of DD_A7 2.7.1. Antibiotic susceptibility test Resistance towards antibiotics such as gentamycin (10 μg/ml), spectinomycin (100 μg/ml), ampicillin (10 μg/ml) and kanamycin (50 μg/ml) was evaluated by the disc overlay diffusion method as described by Odeyemi and Ahmad (2017), with some minor modification. Paper discs overlaid with antibiotics were placed on the agar surface seeded with the micro-organism (107 CFU/ml) and incubated for 24 h at 37 °C. The clear zone of inhibition was measured around the discs to analyze the resistivity.
2.5.2. Measurement of membrane integrity Rhodamine 123 (Rh 123, 10 μg/ml) staining was used to analyze the effect on the membrane integrity, as described by Warnes, Caves, and Keevil (2012). Pathogenic E. coli 0157:H7 were pre-treated for 24 h with 50% CFCS, followed by staining with Rh 123, and observed under a microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification to measure the intensity of green fluorescence. In the experiment, untreated cells were considered as a control. The green fluorescence indicates accumulation of the dye on the inner surface of the intact cell membrane. Images were further analyzed under the 3D surface plot using ImageJ 1.50i version (National Institute of Health, Bethesda, MD, USA).
2.7.2. Cytotoxic assessment on mammalian cells In order to estimate the toxic effect of CFCS, morphological changes of HCT-116 (ATCC® CCL-247™) and RAW 264.7 (ATCC® TIB-71™) cell were evaluated after exposure to varying concentrations of CFCS (0%, 10%, 30%, and 50%) for 24 h; morphological variations were assessed under the inverted microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at the magnification of 20×. Additionally, the effect on cell density after treatment was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Briefly, 1 × 105 cells/well were seeded in a 96-well tissue culture plate and treated with the above-mentioned concentrations of CFCS; to evaluate the toxic effect, cell viability was compared with untreated control cells.
2.5.3. Effect on intracellular Ca++ ions In order to estimate the intracellular Ca++ ion, Fura-2AM a cellpermeable fluorescent probe was used, as described by Werthén and Lundgren (2001), with some modification. Briefly, pathogenic E. coli 0157:H7 cells were treated with or without CFCS (50%) for 24 h. After 24 h incubation at 37 °C, the bacterial culture was centrifuged at 4000 rpm for 5 min. The bacterial pellets were then incubated with 5 μM of Fura 2-AM for 60 min at 37 °C in dark, followed by washing with 50 mM phosphate buffer saline (PBS, pH 7.3) and perfused in HBSS (Hank's balanced salt solution) buffer. The fluorescence intensity of cells was analyzed under a fluorescence microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 100× magnification. Fluorescence intensity was quantified using ImageJ 1.50i version (National Institute of Health, Bethesda, MD, USA), after nullifying the background staining.
2.7.3. Affect of CFCS on zebrafish larvae The non-toxic behavior of CFCS was determined by morphological analysis in the zebrafish model system. Briefly, CFCS (10, 30, and 50%) and LPS (0.1%) treatment was given to 6 days post fertilization (dpf) zebrafish larva (n = 10) in a 6 well plate and were maintained in E3 medium for 24 h. To prepare a 60X stock E3 medium, take 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 and dissolve the ingredients in distilled water to a final volume of 2 L (adjust the pH to 7.2 with NaOH). To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L and add 0.00003% methylene blue (Sigma-Aldrich) to the solution. 2.8. Application of DD_A7
2.5.4. Scanning electron microscopy (SEM) To further support the membrane permeability findings, E. coli 0157:H7 were examined under the scanning electron microscope (SEM) and images obtained were compared with the untreated sample, as described by Nuño, Alberto, Arena, Zampini, and Isla (2018). Briefly,
2.8.1. Enzymatic activity of DD_A7 The amylolytic and proteolytic activities were evaluated using the streaking method, as reported by Sharma et al. (2018). Bacterial cultures were streaked on the starch (1% w/v) and skim milk (10% w/v) agar plates, respectively, followed by incubation at 37 °C for 24 h. 665
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Table 1 Various strains of W. confusa isolated from different food sources and human gastrointestinal tract, and reported from different geographical areas. Strain
NCBI accession number
Query cover
E value
Ident
Isolation matrix
Geographic origin
Genome size
DD_A7 BSR5 JCM 1093 NH 02 SM32 SM10 MMZ50B HYM105 HBUAS54173 HBUAS54147 IMAU50297 IMAU50289 IMAU50206 IMAU50203 HBUAS52116 HA12 HBUAS54202 HBUAS54166 HBUAS54121 HBUAS54114
MH341584.1 KY203672.1 LC063164.1 AB425970.1 KU060296.1 KU060300.1 EU157913.1 KT982252.1 MH701962.1 MH701936.1 MG547239.1 MG547233.1 MF623270.1 MF623233.1 MH392877.1 MF148168.1 MH701991.1 MH701955.1 MH701912.1 MH701906.1
98% 97% 97% 97% 97% 97% 97% 96% 96% 96% 96% 96% 96% 96% 97% 96% 96% 96% 96%
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%
Kimchi Fermented batter of Kodomillet and Moth bean Sugar cane Thai fermented sausage sugarcane stalk after cutting sugarcane stalk after cutting Gueddid an artisanal Tunisian dry fermented meat Huyumei broad bean paste Young peoples intestinal tract Young peoples intestinal tract human intestine human intestine rice wine rice wine Zha-Chili larvae and adults of sheep abomasal nematode parasite Young peoples intestinal tract Young peoples intestinal tract Young peoples intestinal tract Young peoples intestinal tract
South Korea India Japan Thailand South Africa South Africa Tunisia China China China China China China China China New Zealand China China China China
1558 1547 1538 1534 1537 1537 1548 1513 1499 1499 1499 1499 1499 1499 1499 1516 1499 1499 1499 1499
Bacterial cells were suspended at the MOI of 1:100 ratio to HCT116 cells and allowed the bacterial cell to adhered on the colon cells for 2 h. After 2 h of incubation, cells was fixed with the antifade solution (Sigma) and observed under the fluorescent microscope.
2.8.2. Bacterial adhesion to different solvents and auto-aggregation ability The Bacteria Adhesion To Hydrocarbon (BATH) test was used to evaluate the cell surface properties of strain DD_A7. Briefly, bacterial cells were incubated in different solvents (xylene, ethyl acetate, and chloroform) for 4 h, as proposed by Dias, Duarte, and Schwan (2013) and Mohanram, Jagtap, and Kumar (2016). The auto-aggregation ability of strain DD_A7 was also analyzed as described by Khan and Kang (2016). To calculate the auto-aggregation ability, OD was recorded for the next 24 h at intervals of every 4 h, using a spectrophotometer at 600 nm (UV-2120 Optizen, Mecasys, South Korea). The below-mentioned equation was then applied for calculations:
Auto − aggregation % =
bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp bp
2.8.5. Bile-salt tolerance of DD_A7 Tolerance of DD_A7 against bile-salt was examined by measuring the absorbance values of the culture at 600 nm in MRS broth with different concentrations of bile salts [0.05%, 0.1%, 0.3% (w/v)] during 24 h of incubation at 37 °C with the initial turbidity of 0.2 OD600 and compared with its control.
1−At x 100 A0
2.9. Statistical analysis
where, At indicates the reading after incubation of bacteria at different time points, and A0 indicates the readings at the initial point (0h) at 600 nm.
All results are expressed as the mean ± standard deviation (SD) and the statistical significance is calculated using Student's “t” tests.
2.8.3. Biofilm formation ability The biofilm formation ability of DD_A7 was analyzed in 30 mm flat bottom plates, as described by Al-Shabib et al. (2018), with little changes in the protocol. Briefly, 1% well-grown cultures of DD_A7 were inoculated at initial turbidity of 0.2 at OD600 nm, in freshly prepared MRS broth. The cultures were allowed to grow without shaking for the next 72 h at 37 °C. The plates were then washed twice with PBS to remove non-adherent cells from the surface. Plates with the adherent colonies were stained with crystal violet for 20 min, rinsed with PBS to remove the extra stain, and images were captured under an inverted microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at a resolution of 100X. Images were then analyzed under the 3D surface plot using ImageJ version 1.50i (National Institute of Health, Bethesda, MD, USA).
3. Result and discussion 3.1. Concordant phylogeny clade of DD_A7 with pre-reported prebiotic W. confusa The 16S-rRNA sequence of DD_A7 was submitted to GenBank against the accession ID (MH341584.1), and FASTA sequence was retrieved for further analysis (https://www.ncbi.nlm.nih.gov/nuccore/ 1389426126). Next, we selected 19 sequences from the public database which exhibits 99% similarity with identical genotypic identity and covered the maximum query in the range between 96 and 98% with a minimum error value (0.00) in respect to DD_A7 sequence. The strains of 19 different sequences were either isolated from food or found in a healthy human gastrointestinal tract (Table 1). We analyzed the root centered phylogenic tree with maximum likelihood and built the gene cluster (Fig. 1) based on the 16S-rRNA sequencing among the chosen strains. This group of 19 strains is further referred to as the W. confusa group. The result of the evolutionary divergence reveals an overall mean distance of 0.001 (p distance) (Supplementary Table 1). The evolutionary history was inferred using the Maximum Likelihood method. Initial tree(s) for the heuristic search were obtained by Neighbor-Join algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. Further topology was selected with superior log-likelihood value. All positions containing gap (s) and missing data were eliminated. The Phylogenetic tree revealed
2.8.4. Assessment for adhesion property of W. confusa DD_A7 on colon cells The adhesive property of DD_A7 was evaluated using HCT-116 cell line model as described by Jayashree et al., 2018, with some little modification. Briefly, the overnight grown culture of DD_A7 was centrifuged at 5000 rpm for 3 min. After centrifuge, cells were washed with PBS for three times and suspended in 1 ml of 0.1 M sodium carbonate buffer containing 2 μg/ml of Fluorescein isothiocyanate (FITC). The cells were incubated at 25 °C for 1 h. After incubation, cells were washed thrice with PBS to remove the excess stain. On the other hand, HCT-116 cells were stained with Mitotracker Deep Red (MTDR) (1 μg/ ml) and DAPI (1 μg/ml), incubated for 30 min at 37 °C in a 5% CO2. 666
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Table 3 Antibacterial potential of DD_A7 against pathogenic bacteria. Results are expressed as the diameter of the clear zone of inhibition (in mm). The zone diameter interpretive criterion was followed as described by CLSI M100-S20 (2010). Experiments were performed in triplicates and data are presented as mean ± SD. Strain name
ATCC number
Zone of inhibition (in mm)
Listeria monocytogenes Staphylococcus aureus Salmonella typhimurium Bacillus cereus Escherichia coli 0157:H7 ESBL-E. coli 4 ESBL-E. coli 5
ATCC 7644 ATCC 12600 ATCC 43174 ATCC 13061 ATCC 43889 NCCP, KBN10P03347 NCCP, KBN10P03452
22 9 17 20 28 17 24
alignment, as shown in Supplementary Fig. 1. Next, polymorphism was differentiated based on their types, i.e., transition and transversion (Table 2). Transition polymorphisms are the interchange either between two purines or two pyrimidines, whereas transversion polymorphisms are the interchanges of purine to pyrimidine or vice versa. In the current study, transversion was found to be more prevalent, where 40 sites were detected with a change in the nucleotide G to nucleotide T, and 30 sites were found for T to G transversion. On the other hand, only 36 sites were detected with a transition, where G was replaced by A. This result of the SNP analysis indicates that transversion is the commonly occurring polymorphism, especially the transversion of G/T or T/G. In addition, the site 1529(G/T) and 1530(T/G) were the predominant sites located in 18 sequences out of 19 when aligned with the query sequence. To further validate the results of evolution, nucleotide substitution pattern (Supplementary Table 2) and substitution matrix (Supplementary Table 3) were built using the MEGA software, version 7.026.
Fig. 1. The full-length 16S-rRNA sequence was aligned, and a phylogenetic tree was constructed by the UPGMA method using the MEGA software, version 7.026. The tree represents the relationship with other bacterial species. The data represents the similarity of DD_A7 with W. confusa.
that all native strains fit into an evolutionary cluster comprising the members of W. confusa strains, and other species are grouped into a separate cluster according to their relatedness found if any. A total of 201 nodes were thus identified (Fig. 1). The tree is drawn to scale of 0.005, with branch lengths measured in the number of substitutions per site. Further, we critically analyzed the sequence of the W. confusa group, and polymorphic sites were evaluated where two different nucleotide bases were detected at a particular position. The detected polymorphic sites in the sequences were analyzed on sequence
3.2. CFCS of DD_A7 as a potent antibacterial agent The isolated DD_A7 strain under evaluation was found to have
Table 2 Different types of polymorphisms observed in the multiple sequence alignment performed using CLUSTALW, version 1.6 (Kyoto University Bioinformatics Center, Japan). Accession no.
Transition
Transversion
KY203672.1
4(G/A), 5(G/A), 6(G/A), 15(T/C), 16(C/T), 1531(G/A), 1533(G/A), 1535(T/C) 4(G/A), 5(G/A), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/A) 14(T/C), 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/A) 5(A/G), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 111(G/A), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1547(G/A) 5(A/G), 6(G/A), 14(T/C), 15(T/C), 16(C/T), 36(C/T), 1054(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1547(G/ A) 15(T/C), 16(C/T), 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A), 1550(A/G), 1551(C/T), 1555(G/A) 1531(G/A), 1533(A/G), 1535(T/C), 1542(G/A), 1546(G/A) – – – – – – – 1145(C/T), 1168(C/T), 1207(A/G), 1531(G/A), 1533(A/G), 1535(T/ C), 1542(G/A), 1546(G/A) 1168(C/T) 331(A/G) 41(T/C) –
7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T)
LC063164.1 AB425970.1 KU060296.1 KU060300.1
EU157913.1 KT982252.1 MH701962.1 MH701936.1 MG547239.1 MG547233.1 MF623270.1 MF623233.1 MH392877.1 MF148168.1 MH701991.1 MH701955.1 MH701912.1 MH701906.1
7(T/G), 10(G/T), 17(T/G), 1529(G/T), 1530(G/T), 1534(G/T), 1356(G/C), 1545(G/T), 1548(G/C), 1549(A/C) 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(G/C), 1545(G/T), 1548(G/C) 7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1546(G/T), 1548(G/C), 1549(A/C) 7(T/G), 10(G/T), 12(T/A), 17(T/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1548(G/C), 1549(A/C) 17(T/G), 214(A/C), 1444(C/G), 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/ T), 1548(G/C), 1549(A/C), 1552(C/A), 1553(C/G), 1554(C/G), 1556(T/G), 1559(A/C) 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T), 1548(G/C), 1549(A/C) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G), 1534(G/T), 1536(C/G), 1545(G/T) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 1529(G/T), 1530(T/G) 101(T/G), 1529(G/T), 1530(T/G)
667
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Fig. 2. Microscopic assessment of pathogenic E. coli 0157:H7 (ATCC 43889) after treatment with 50% CFCS. Bacterial cell death was examined by the AO/EtBr dual staining platform. The fluorescent intensity was recorded under the inverted microscope (Scale bar = 10 μm).
Fig. 3. Potential of 50% CFCS effecting the necessary biomolecules of pathogenic E. coli 0157:H7. (A) Effect on the membrane integrity was assessed by Rh 123 staining. Increase in bright green fluorescence intensity indicates the intact membrane of pathogenic bacteria due to the accumulation of dye on the cell surface. (B) Intracellular Ca++ accumulation was quantified using the ImageJ software after staining with Fura-2AM. (C) Scanning electron microscope analysis of E. coli O157: H7: untreated control cells show a regular and intact cell morphology, whereas CFCS treated cells show a destroyed cell membrane morphology. Scale bar = 2.5 μm. (D) DNase activity of CFCS was visualized on 1% agarose gel. From left to right → M, 1 Kb DNA marker; Lane 1, Control (E. coli plasmid); Lane 2, DNA damage induced by H2O2 treatment followed by 1 min UV radiation was exposed to the plasmid; Lane 3, CFCS (2%) treated plasmid; Lane 4, CFCS (4%) treated plasmid; Lane 5, CFCS (8%) treated plasmid; Lane 6, CFCS (16%) treated plasmid. The ratio of Nicked: supercoiled DNA was estimated. Statistical significance was determined with the help of Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
antagonism varied among different strains, with inhibition ranging from 9 to 28 mm, as presented in Table 3. The CFCS was observed to be most effective against the Escherichia coli 0157:H7 (ATCC 43889) with 28 mm zone of inhibition, whereas Staphylococcus aureus (ATCC 12600) showed the least susceptibility (9 mm of clear zone of inhibition). Since E. coli 0157:H7 was found to be the most susceptible strain, we decided to use it in all further experiments.
antimicrobial activity against pathogenic bacteria such as Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 12600), Salmonella typhimurium (ATCC 43174), Bacillus cereus (ATCC 13061), Escherichia coli 0157:H7 (ATCC 43889), ESBL-E. coli 4 (NCCP, KBN10P03347) and ESBL-E. coli 5 (NCCP, KBN10P03452). We observed that 50% CFCS of DD_A7 has antagonistic effects against all the tested pathogens, but the results revealed that the degrees of 668
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3.3. Influence of CFCS on the DNA integrity
Table 4 (A) Antibiogram susceptibility test of DD_A7 against the commonly prescribed antibiotics, (B) Enzymatic activity of W. confusa DD_A7. Experiments were performed in triplicates and data are presented as mean ± SD. A. Antibiotic
Concentration Zone of inhibition
Resistant Intermediate Susceptible
Gentamycin Spectinomycin Ampicillin Kanamycin
10 μg 100 μg 10 μg 50 μg
– + – –
24 mm 8 mm 28 mm 17 mm
– – – +
Next, we analyzed the effect of CFCS on DNA. E. coli 0157:H7 plasmid was isolated and treated with different concentrations of CFCS. As presented in Fig. 3D, CFCS shows a dose-dependent potential to interfere with the DNA integrity, in a manner similar to H2O2 treated samples. DNA damage after H2O2 treatment (positive control) was observed to increase by 0.55 ± 0.11 fold, and a similar increase was observed after exposure to 2%, 4%, 8%, and 16% CFCS, where the fold changes recorded were 0.16 ± 0.11, 0.28 ± 0.06, 0.34 ± 0.06, and 0.42 ± 0.16, respectively. This result clearly indicates that CFCS of DD_A7 disturbs not only the membrane-bound proteins but also damages the DNA integrity, which further promotes cell death (Khan & Kang, 2016).
+ – + –
B. Enzymatic activity
Amylolytic
Proteolytic
W. confusa DD_A7
+
+
Next, we validated the inhibitory effect of CFCS (50%), by studying its effect on the viability of E. coli 0157:H7 using the AO/EtBr dual stain. Our results indicate 43 ± 0.3% cell death after 24 h treatment, which is a significant increase (P < 0.001) as compared to the control (13 ± 0.5%). This study proves that E. coli 0157:H7 cells were less viable after exposure, as compared to untreated control cells (Fig. 2). We further hypothesized that disruption in the bacterial cell membrane allows EtBr to permeate inside the cell, resulting in subsequent cell death. We, therefore, studied the effect of CFCS (50%) on bacterial membrane permeability using the Rh 123 staining method. We observed that the membrane integrity of E. coli 0157:H7 was decreased after treatment when compared with the untreated control bacterial cells (Hu et al., 2016; Wei et al., 2011). Exposure to CFCS resulted in the targeted cells losing their membrane integrity and becoming leaky or ruptured (Fig. 3A). Due to the rupture in the cell membrane, intracellular Ca++ ions were released, and subsequently, the Fura-2AM fluorescence ratio decreased significantly by 67 ± 2% (P < 0.001) (Fig. 3B). These findings were supported by the results of SEM analysis (Fig. 3C). Our results are similar to the finding of Werthén and Lundgren (2001), where they suggest that more Ca++ ions are found in a bacterium with an intact cell membrane.
3.4. Validation of safety concerns associated with the usage of DD_A7 3.4.1. Antibiotic susceptibility test The present study also has shown the antibiogram susceptibility of DD_A7 against the antibiotics in Table 4. Our results indicate that DD_A7 is susceptible against gentamycin (10 μg/mL) and ampicillin (10 μg/mL), showing 24 mm and 28 mm of clear zone of inhibition, respectively. Intermediate susceptibility was observed against kanamycin (50 μg/mL; 17 mm clear zone of inhibition), whereas the strain was considered to be resistant against spectinomycin (100 μg/mL; 8 mm clear zone of inhibition). The criterion used here for the interpretation of results is adapted from CLSI M100-S20 (2010). The results proved the sensitivity of DD_A7 towards the commonly prescribed antibiotics at their minimum concentrations, thereby imparting the strain to be more acceptable for further use. 3.4.2. Cytotoxic assessment of CFCS In order to investigate the toxic effects of CFCS in the host body, we evaluated the effect of CFCS in vitro on two different cell lines: colon cancer HCT-116 and RAW 264.7 macrophages cell line. Fig. 4A and B depicts, non-significant morphological changes of those treated cell; in addition, a non-significant decrease was observed in the cell density of
Fig. 4. Cytotoxic effect of CFCS was evaluated in, (A) HCT-116, (B) and RAW 264.7 cell lines. Representative images of morphological changes were presented, which was observed under the inverted microscope. The effect of CFCS on the cell density was analyzed by MTT assay, where untreated cells were considered as control. Scale bar = 0.1 mm. Experiments were performed in triplicates and data are presented as mean ± SD. (C) Non-toxic effects of CFCS on the morphological zebrafish larvae. The arrow represents the spinal cord curvature upon LPS treatment. The mortality rate upon treatment was also recorded. 669
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Fig. 5. (A) Biofilm formation ability of DD_A7 at 74 h. The image is quantified by ImageJ software, version 1.50i, under the 3D surface plot, at a resolution of 100X as viewed under an inverted microscope. The results shown in the figure are the mean of three independent experiments. (B) Adhesion of DD_A7 on HCT-116 cells after 2 h of incubation, where green fluorescence (upper left) represented the bacteria stained with FITC, and HCT-116 cells were stained with DAPI (upper right) and MTDR (lower left). The images obtained at different filters were finally merged and presented (lower right). (C) Cell surface hydrophobicity of DD_A7 was analyzed using BATH assay. (D) Analysis of time-dependent auto-aggregation ability of DD_A7. Experiments were performed in triplicates and data are presented as mean ± SD. (E) The growth of DD_A7 in MRS broth with different concentration of bile salts was evaluated for 24 h at 37 °C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
10% (w/v) skim milk by using these ingredients as a primary carbon source (Table 2B). These characteristics of the strain make it more useful for its further use in the industry involved in the isolation of amylolytic and proteolytic enzymes.
both cell lines (90.3 ± 3.0% and 89.4 ± 2.1% in HCT-116 and RAW 264.7 cells, respectively) after treatment with the 50% of CFCS for 24 h, as compared to the untreated control cells. The assay shows similar results in both the cell lines. Further, we have determined the non-toxic effect of CFCS on the morphological and survival rate of zebrafish larvae and compared with the LPS treated larval group. As the result depicted in Fig. 4C, after LPS treatment, morphological development of larvae was altered. LPS treatment triggered the spinal cord curving, which, however, was recovered after treatment of CFCS. Moreover, LPS treatment was found to affect the mortality rate of the zebrafish larvae, which was not the case in CFCS treated larval group. The observation validates the non-toxic effect of CFCS to the host cells. Thus, the safety concern of DD_A7 is validated and supports its further use for imparting health benefits.
3.6. DD_A7 forms a biofilm due to its adhesive nature As previously reported in our in vitro study (Dey et al., 2019), W. confusa has the potential to reduce inflammation induced by pathogens. However, in order to survive in the gastrointestinal tract and exert its benefits, colonization on the gut surface is essential. Biofilm plays a crucial role in maintaining the relationship between the residence and microbes. Our study revealed that DD_A7 strain has the ability to form a biofilm at 74 h (Fig. 5A). The colonization of DD_A7 was observed clearly. The adherence density of the probiotic was further estimated by ImageJ software, version 1.50i, which shows a dense bacterial population in root mean square (RMS) value (140–180 pixels). The density of the bacterial population was gradually observed to increase with increasing incubation time (data not shown). In addition, microscopic observation proved that the W. confusa DD_A7 has a potential adhesion property to the surface of HCT-116 cells (Fig. 5B). It is evident from several studies that bacterial cell surface plays a critical role in the colonization of bacteria on the human gut surface, and this process is critically linked with the cell surface property. The first phase of biofilm formation is due to its hydrophobic surface potential which facilitates the colonization of the beneficial micro-organism and modulates the host immune system (Tresse et al., 2006).
3.5. Enzymatic activity of DD_A7 Since W. confusa are used as a starter species in various traditional fermentation processes (Vogel et al., 2011), we decided to evaluate the amylolytic and proteolytic activity of DD_A7. These enzymes play crucial roles in hydrolyzing starch into disaccharide or trisaccharide and converting peptides into free amino acids (De Souza & Nobre, 2017). Assessing the secreting ability of these enzymes could, therefore, add value to its usage as a starter species. The aim of the experiment was also to identify whether the strain has the potential to utilize starch and agar as a primary carbon source for growth. As expected, DD_A7 has the ability to grow on agar plates containing 1% (w/v) starch and 670
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For adhesion to the intestinal cells or any solid surface, bacteria must possess a hydrophobic surface. Hence, BATH assay was performed using three different solvents to evaluate bacterial adhesion which would help us to determine the cell surface potential: xylene (polar solvent), ethyl acetate (monopolar and a strong Lewis-basic solvent), and chloroform (monopolar but a Lewis-acidic solvent). This experiment revealed that DD_A7 showed 82.11% hydrophobicity towards xylene, indicating that the bacterial cell possesses a greater hydrophobic surface. The affinity towards ethyl acetate was 24.7%, which is higher than that of chloroform (3.8%) (Fig. 5C). These results indicate that DD_A7 has the strongest electron donating characteristic. Further, the results were justified by examining the auto-aggregation ability, where DD_A7 shows an increasing auto-aggregation depending on the time of incubation (Fig. 5D). The highest auto-aggregation shown by DD_A7 was 23% at 24 h. The findings of our experiment are in accordance with those reported by Xu et al. (2009).
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3.7. Bile salts tolerance of DD_A7 W. confusa DD_A7 possesses tolerance against bile salts. The strain is not only tolerant, but its cell numbers were even increased after 6-h exposure to 0.3% bile salts in MRS broth like its control. However, a fall in the cell density of DD_A7 was also observed at 12 h in 0.3% bile salts when compared with the control (Fig. 5E). 4. Conclusion The present study describes, for the first time the polymorphism in the conserved region of the 16S rRNA of Weissella. The study analyzed the SNPs among the 19 other strains which are isolated from food and the human intestine. These SNPs provided more intra-specific information which could be effectively used to define the evolutionary history of the species in the future. The information would be helpful to design the set of primers or specific probe to identify the candidate prebiotic strain. The finding of the study also demonstrated the antimicrobial activity of non-pathogenic DD A7 strain of W. confusa. Moreover, the study also validates the contemporary safety demands of DD_A7 and its CFCS for its further usage. To validate its future for host immunomodulating characteristic is ongoing. Author contributions and acknowledgements DDK conceived most of the experiments with the help of KBG. CS conducted the bioinformatics analysis. DDK prepared the manuscript, which was edited and finalized by KSC. All the authors have read and approved the manuscript before submission. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.05.089. References Al-Shabib, N. A., Husain, F. M., Khan, R. A., Khan, M. S., Alam, M. Z., Ansari, F. A., et al. (2018). Interference of phosphane copper (I) complexes of β-carboline with quorum sensing regulated virulence functions and biofilm in foodborne pathogenic bacteria: A first report. Saudi Journal of Biological Sciences (in press). Chhabriaa, S., & Desai, K. (2018). Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin. Saudi Journal of Biological Sciences, 25, 1429–1438. Choi, S. M., Jeon, Y. S., Rhee, S. H., et al. (2002). Fermentation characteristics and antimutagenicity of kimchi that prepared with a different ratio of seed in red pepper powder. Journal of Korean Association of Cancer Prevention, 7, 51–59. De Souza, J. V., & Nobre, F. S. D. (2017). Protective, technological, and functional properties of select autochthonous lactic acid bacteria from goat dairy products. Current Opinion in Food Science, 13, 1–9. Dey, D. K., Khan, I., & Kang, S. C. (2019). Anti-bacterial susceptibility profiling of Weissella confusa DD_A7 against the multidrug-resistant ESBL-positive E. coli.
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