Diversity of Endophytic Bacteria in a Fern Species Dryopteris uniformis (Makino) Makino and Evaluation of Their Antibacterial Potential Against Five Foodborne Pathogenic Bacteria

Abstract

The fern plant Dryopteris uniformis has traditionally been used in herbal medicine and possesses many biological activities. This study was conducted to explore the endophytic bacterial diversity associated with D. uniformis and evaluate their antibacterial potential against foodborne pathogenic bacteria (FPB). Among 51 isolated endophytic bacteria (EB), 26 EB were selected based on their morphological characteristics and identified by 16S rRNA gene analysis. The distribution of EB was diverse in the leaf and the stem/root tissues. When the EB were screened for antibacterial activity against five FPB, Listeria monocytogenes, Salmonella Typhimurium, Bacillus cereus, Staphylococcus aureus, and Escherichia coli O157:H7, four EB Bacillus sp. cryopeg, Paenibacillus sp. rif200865, Staphylococcus warneri, and Bacillus psychrodurans had a broad spectrum of antibacterial activity (9.58 ± 0.66 to 21.47 ± 0.27 mm inhibition zone). The butanol solvent extract of B. sp. cryopeg and P. sp. rif200865 displayed effective antibacterial activity against the five FPB, which was evident from the scanning electron microscopy with irregular or burst cell morphology in the EB-treated bacteria compared to smooth and regular cells in case of the control bacteria. The minimum inhibitory concentration and minimum bactericidal concentration values ranged between 250–500 μg/mL and 500–100 μg/mL, respectively. The above outcomes signify the huge prospective of the selected EB in the food industry. Overall, the above results suggested that D. uniformis contains several culturable EB that possess effective antibacterial compounds, and that EB can be utilized as a source of natural antibacterial agents for their practical application in food industry to control the spread of FPB as a natural antibacterial agent.

Introduction

Endophytes are ubiquitous in the environment, interacting with their host plants by producing resources that afford protection and facilitate survival of the plant (Lodewyck et al., 2002). Awareness regarding the profusion and composition of bacterial endophytes is a vital requirement for improved research regarding their interactions with host plants (Sun et al., 2008). Even though the associations between endophytes and crop plants have been studied extensively (Sun et al., 2008), there are little data available regarding the composition of endophytic bacterial species in the fern plant (Chen et al., 2012). Information about the profusion and arrangement of bacterial endophytes is essential to improve knowledge of their relationships with host plants (Chen et al., 2012).

Many studies have shown that endophytes are a novel source of biological products with antagonistic activities that can be utilized by the drug and agricultural industry (Yu et al., 2010; Nair and Padmavathy, 2014; Golinska et al., 2015). Because endophytes remain in a stable environment within the living plant tissue, they have higher bioactivity than microbes isolated from plant surfaces or soil (Selim et al., 2014). Endophytes live inside plants with a great potential source of antimicrobial substances as the plant provides a normal selection system to antibiotics and endophytes (Seo et al., 2010; El-Deeb et al., 2013). To date, a large number of novel biological products with antagonistic activity have been isolated from endophytes (Strobel and Daisy, 2003; Yu et al., 2010). These compounds belong to various structural groups, including steroids, flavonoids, peptides, and terpenoids (Yu et al., 2010). It is widely believed that finding biological products produced by endophytic bacteria (EB) can resolve the problems associated with currently available antibiotics and lead to development of highly effective, low-toxicity, and low environmental impact antibiotics effective against resistant bacterial species (Yu et al., 2010).

Although food is capable of providing nutrients to consumers, it can also support the growth of infectious microorganisms. Indeed, foodborne pathogens comprise one of the most severe threats to human health worldwide (Voravuthikunchai et al., 2006). It has been observed that ferns are not infected by microbial pathogens (Sen and Nandi, 1951; Mandal and Mondal, 2011), and this group of plants has tremendous medicinal and ecological significance. Indeed, ferns are extensively used to treat skin tumefaction, hepatitis, diarrhea, burns, and trauma. The fern plants Polypodium interjectum Shivas, Polystichum woronowii Fomin, Polystichum aculeatum (L.) Roth., Dryopteris affinis (Lowe) Fraser-Jenk, Athyrium filix-femina (L.) Roth, Asplenium scolopendrium L., Asplenium adiantum-nigrum L., and Pteris cretica L. are also known to have antimicrobial, antioxidant, antiviral, antitumor, and even anti-HIV properties (Bahadori et al., 2015). The fern plant Dryopteris uniformis (Makino) Makino is widely known as an ornamental plant, while its young branches are consumed as food in Asia. Although some researchers have identified endophytic fungi from fern plants (Fisher, 1996), the presence of EB internalized in ferns has not yet been investigated in depth (Barros et al., 2010). Moreover, almost no studies have investigated the diversity of EB in ferns or their antibacterial activity. Therefore, this study was conducted to evaluate the diversity of EB in the fern plant, D. uniformis, and to evaluate its antibacterial potential against foodborne pathogenic bacteria (FPB).

Materials and Methods

Collection and isolation of EB

A number of fern plant D. uniformis samples were collected randomly from different locations of the Bohyun Mountain, Yeongchun, Republic of Korea, in May of 2014 and identified by Dr. Seonjoo Park, a plant taxonomist (Department of Life Sciences, Yeungnam University, Republic of Korea). The plant was washed under running tap water, after which 2 g each from leaves (5–10) or stems/roots (5–10 pieces of 1 cm each) were sterilized using 70% ethanol for 60 s followed by 2% sodium hypochlorite for 90 s, 100% ethanol for 30 s, and then three rinses with sterilized distilled water. Sterilized tissues were ground with 6 mL of 0.9% NaCl using a sterile mortar and pestle and incubated to allow the complete release of EB from the host tissues at room temperature for 3 h. For isolation of EB, the tissue extracts were diluted 10- to 100-fold in 0.9% NaCl and plated on Petri dishes having yeast extract and nutrient agar media in three replicates for each dilution (10⁻¹ and 10⁻²). Finally, samples were incubated for up to 15 days at 28°C.

Identification of EB by morphology and 16S rRNA sequences

The EB isolated from D. uniformis were classified according to their morphological characteristics of the bacterial colony. The EB were finally identified by 16S rRNA gene sequencing. Phylogenetic tree analysis of the isolated EB was conducted using the 16S rRNA sequences. The sequences were aligned and a neighbor-joining tree was constructed. The overhanging ends were detached from both ends to ensure that all sequences were of the same length and the tree was built by the MEGA6 software (version 6) using the neighbor-joining method. Bootstrap tests were performed using 1000 replicates (Tamura et al., 2013; Susilowati et al., 2015).

Screening of antibacterial activity of the isolated EB

Five FPB were investigated in this study, Listeria monocytogenes ATCC 7644, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 12600, Escherichia coli O157:H7 ATCC 43890, and Salmonella Typhimurium DT104. These strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The evaluation for antibacterial potential was conducted using the patch plate method described by Roh et al. (2009). All the experiments were carried out in triplicate.

Growth kinetics and fractionation by solvent extracts

The EB were cultured in YNB media while shaking at 150 rpm and at 28°C. The absorbance at 600 nm was measured at 24-h intervals from 0 to 96 h using a spectrophotometer (ASP 3700; ACT Gene, Inc., Piscataway, NJ) to evaluate the growth of EB (Araujo et al., 2010). The fractionation of EB extracts was conducted successively using different polarity-based solvents (n-hexane, chloroform, ethyl acetate, and butanol) based on the standard protocol (Zubair et al., 2011). All separated extracts were dried in a rotary evaporator at 55°C, after which 5% Dimethyl sulphoxide (DMSO) was added to the extracts at 0.1 g/mL.

Antibacterial activity of solvent extracts

The antibacterial activity of different solvent extracts was evaluated using the standard disc diffusion method (Diao et al., 2013). The butanol extracts of the two selected EB Bacillus sp. cryopeg and Paenibacillus sp. rif200865 were evaluated for their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against five different pathogenic foodborne bacteria by the twofold dilution method (Kubo et al., 2004).

Analysis by scanning electron microscopy

The effects of butanol fraction of the two selected EB, B. sp. cryopeg and P. sp. rif200865, on the morphology of the five FPB were determined by scanning electron microscopy (SEM) (Patra et al., 2015). The foodborne bacteria were then treated with 5% DMSO as the control or with the MIC of butanol faction as the treatment. Immediately before SEM analysis, the specimens were sputter coated with platinum using an ion coater for 2 min before analysis and then examined under a scanning electron microscope (S-4100; Hitachi, Japan).

Statistical analyses

The results of this study are expressed as mean ± standard deviation (SD). Statistical analyses consisted of one-way ANOVA and Duncan's multiple range tests, with p < 0.05 considered to indicate significance. Statistical Analysis Software (SAS) version 9.4 (SAS, Inc., Cary) and Molecular Evolutionary Genetics Analysis (MEGA) version 6.06 were used for all analyses.

Results

Distribution of EB isolated from different tissues of D. uniformis

A total of 51 types of EB were isolated from different samples of the plants D. uniformis collected from different locations of Bohyun Mountain, Yeongchun, Republic of Korea (Fig. 1A), based on their morphological characteristics. Out of these, 29 and 22 bacteria species were isolated from leaves and from stems/roots, respectively. The morphological characteristics of isolated bacteria are described in detail in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/fpd). The population levels of EB in the leaves and the stems/roots together were 1.2 ± 0.42 × 10⁵ and 1.5 ± 0.56 × 10⁵ cfu/g, respectively. Based on this morphology, a total of 26 EB were selected and identified by 16S rRNA gene analysis (Table 1 and Supplementary Table S2). The EB found only in leaf tissues belonged to five families, Rhizobiaceae, Paenibacillaceae, Sphingomonadaceae, Micrococcaceae, and Burkholderiaceae, whereas those found only in stems/roots only belonged to Xanthomonadaceae. The EB isolated from both leaves and stems/roots belonged to Bacillaceae and Staphylococcaceae (Fig. 1B).

FIG. 1.

(A) Photograph of Dryopteris uniformis. (B) Distribution of EB in different tissues of D. uniformis. EB, endophytic bacteria.

Table 1.

Identification of Endophytic Bacteria Isolated from Dryopteris uniformis

Original name	Tissue	Closest relative	Maximum score	Identity%
DUL10	Leaf	Staphylococcus epidermidis	2652	99
DUS12	Stem/root	Agrobacterium rhizogenes	2516	99
DUS14	Stem/root	Burkholderia	2573	99
DUL44	Leaf	Burkholderia	2320	95
DUL50	Leaf	Staphylococcus epidermidis	2673	99
DUS52	Stem/root	Staphylococcus saprophyticus	2673	99
DUS56	Stem/root	Staphylococcus	2669	99
DUS59	Stem/root	Bacillus sp. Cryopeg	2604	99
DUL76	Leaf	Sphingomonas	2547	99
DUL88	Leaf	Staphylococcus saprophyticus	2651	99
DUS118	Stem/root	Agrobacterium rhizogenes	2529	99
DUL119	Leaf	Bacillus	1515	87
DUL120	Leaf	Paenibacillus	1801	91
DUL128	Leaf	Paenibacillus sp. rif200865	1707	95
DUS130	Stem/root	Staphylococcus warneri	1688	88
DUS131	Stem/root	Bacillus psychrodurans	2617	99
DUS134	Stem/root	B. psychrodurans	2638	99
DUL146	Leaf	Kocuria palustris	2610	99
DUL148	Leaf	Staphylococcus caprae	2447	97
DUS183	Stem/root	Luteibacter	2569	99
DUL185	Leaf	Agrobacterium rhizogenes	2527	99
DUL190	Leaf	Luteibacter	2569	99
DUL192	Leaf	Agrobacterium rhizogenes	2516	99
DUL198	Leaf	Staphylococcus epidermidis	2660	99
DUS202	Stem/root	Psychrobacillus psychrodurans	2617	99
DUS203	Stem/root	B. psychrodurans	2612	99

Identity%, highest percent of similarity between the query and subject sequences over the length of the coverage area. Maximum score, the highest alignment score (bit-score) between the query sequence and the database sequence segment.

Evaluation of antibacterial activity

The EB isolated from D. uniformis were evaluated for their antibacterial effects against five FPB. Of all EB tested, four showed broad-spectrum antibacterial activity against the five investigated FPB. Specifically, B. sp. cryopeg (DUS59), P. sp. rif200865 (DUL128), Staphylococcus warneri (DUS130), and Bacillus psychrodurans (DUS131) showed inhibition zones of 13.85 ± 0.30, 11.06 ± 0.36, 11.86 ± 0.77, and 11.36 ± 0.53 mm, respectively, against L. monocytogenes ATCC 7644. However, they showed inhibition zones of 12.56 ± 0.46, 11.60 ± 0.79, 12.59 ± 1.09, and 9.58 ± 0.66 mm against S. Typhimurium, EB B. sp. cryopeg, P. sp. rif200865, S. warneri, and B. psychrodurans, respectively in NA plates. The zones of inhibition of B. sp. cryopeg, P. sp. rif200865, S. warneri, and B. psychrodurans were 15.88 ± 0.09, 14.23 ± 0.10, 12.70 ± 0.33, and 10.75 ± 0.09 mm, respectively, against B. cereus, while they were 23.07 ± 0.11, 19.81 ± 0.03, 18.64 ± 0.07, and 21.47 ± 0.27 mm, respectively, against S. aureus, and 15.47 ± 0.27, 10.11 ± 0.18, 14.33 ± 0.12, and 12.60 ± 0.04 mm, respectively, against E. coli O157:H7. Among the EB, B. sp. cryopeg (DUS59) exhibited the highest inhibitory activity against S. aureus (23.07 ± 0.11 mm inhibition zone), while B. psychrodurans (DUS131) exhibited the lowest activity against S. Typhimurium (9.58 ± 0.66 mm inhibition zone; Table 2 and Fig. 2a). The four EB were assessed for their growth, and the optical density of EB DUL128 was highest at 48 h, while that of the remaining three EB (DUS59, DUS130, and DUS131) was highest at 98 h. Once reaching maximum growth, the growth started declining for all species (Fig. 2b).

FIG. 2.

(a) Antibacterial activity of EB against five FPB. A, Listeria monocytogenes ATCC 7644; B, Salmonella Typhimurium DT104; C, Bacillus cereus ATCC 10876; D, Staphylococcus aureus ATCC 12600; E, Escherichia coli O157:H743890; I, Bacillus sp. cryopeg (DUS59); II, Paenibacillus sp. rif200865 (DUL128); III, Staphylococcus warneri (DUS130); IV, Bacillus psychrodurans (DUS131). (b) Growth kinetics of the four EB with antimicrobial activity against five foodborne pathogens. (c) Antibacterial activity of the butanol extracts of the two selected EB against five FPB. A, B. sp. cryopeg (DUS59); B, P. sp. rif200865 (DUL128); I, L. monocytogenes ATCC 7644; II, S. Typhimurium DT104; III, B. cereus ATCC 10876; IV, S. aureus ATCC 12600; V, E. coli O157:H7 ATCC 43890. EB, endophytic bacteria; FPB, foodborne pathogenic bacteria.

Table 2.

Antibacterial Potential of Endophytic Bacteria Isolated from Dryopteris uniformis Against Foodborne Pathogenic Bacteria

Inhibition zone (mm)^*
Endophytic bacteria	Listeria monocytogenes ATCC 7644	Salmonella Typhimurium DT104	Bacillus cereus ATCC 10876	Staphylococcus aureus ATCC 12600	Escherichia coli O157:H7 ATCC 43890
DUS59	13.85 ± 0.30^f ^**	12.56 ± 0.46^gh	15.88 ± 0.09^e	23.07 ± 0.11^a	15.47 ± 0.27^e
DUL128	11.06 ± 0.36^if	11.6 ± 0.79ⁱ	14.23 ± 0.10^f	19.81 ± 0.03^c	10.11 ± 0.18^kl
DUS130	11.86 ± 0.77^hi	12.59 ± 1.09^gh	12.7 ± 0.33^g	18.64 ± 0.07^d	14.33 ± 0.12^f
DUS131	11.36 ± 0.53^ij	9.58 ± 0.66^l	10.75 ± 0.09^jk	21.47 ± 0.27^b	12.60 ± 0.04^gh

Diameter of zone of inhibition expressed as mean ± SD.

Different superscript letters represent significant differences at p < 0.05.

SD, standard deviation.

Evaluation of antibacterial activity of solvent extracts

B. sp. cryopeg (DUS59) isolated from leaf tissue and Paenibacillus sp. (DUL128) isolated from stem/root tissue of D. uniformis were selected for further solvent extraction. The antibacterial activities of these two selected EB solvent extracts were tested against the five FPB. The solvent extracts of concentrated n-hexane, chloroform, and ethyl acetate showed no activity against any of the five pathogenic bacteria, whereas butanol extract exerted different degrees of antibacterial activity against the test organisms. Specifically, the butanol fraction of DUS59 produced zones of inhibition of 10.38–19.09 mm against the five FPB, with the highest inhibition activity being against E. coli 0157:H7 and the lowest against L. monocytogenes (10.38 mm). The DUL128 butanol fraction exhibited zones of inhibition of 9.39–14.35 mm against five tested FPB (Table 3 and Fig. 2c), with the highest being observed against S. aureus and the lowest against B. cereus. The 5% DMSO, which was taken as the solvent control, did not show any inhibition activity against any of the tested pathogens. Phylogenetic tree analysis of the two selected EB was conducted using their 16S rRNA sequences. The neighbor-joining tree showed these two EB to be different genera, with DUS59 and DUL128 being closely related to B. sp. cryopeg and P. sp. rif200865, respectively (Fig. 3).

FIG. 3.

Phylogenetic tree exhibiting similarity among 16S gene sequences of Bacillus sp. cryopeg (DUS59) and Paenibacillus sp. rif200865 with the most closely related species. Evolutionary distances were computed using the maximum composite likelihood method. Bootstrap values (1000 tree interactions) are indicated at the nodes.

Table 3.

Antibacterial Activity, Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Values of the Butanol Fraction of Two Endophytic Bacteria, Bacillus sp. cryopeg and Paenibacillus sp. rif200865, Against Five Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria	B. sp. cryopeg (BuOH Fr.)^*	MIC (μg/mL)	MBC (μg/mL)	P. sp. rif200865 (BuOH Fr.)^*	MIC^* (μg/mL)*	MBC^* (μg/mL)*
Listeria monocytogenes	10.38 ± 0.05^f ^***	500	1000	11.41 ± 0.19^e ^***	500	1000
Salmonella Typhimurium	13.59 ± 0.23^d	500	1000	11.81 ± 0.08^e	500	1000
Bacillus cereus	13.40 ± 0.56^d	500	500	9.39 ± 0.23^f	500	1000
Staphylococcus aureus	16.20 ± 0.27^b	250	500	14.35 ± 0.19^c	250	500
Escherichia coli 0157:H7	19.09 ± 0.12^a	250	500	11.65 ± 0.46^e	500	1000

Diameter of zone of inhibition in mm expressed as mean ± SD.

Each experiment is carried out in triplicate.

***

Different superscript letters represent significant differences at p < 0.05.

MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; SD, standard deviation.

MIC and MBC of solvent extract

The MIC values of the butanol extract of B. sp. cryopeg were 250 μg/mL against S. aureus and E. coli O157:H7, while they were 500 μg/mL against L. monocytogenes, S. Typhimurium, and B. cereus. The MBC values were 500 μg/mL against B. cereus, S. aureus, and E. coli O157:H7, while they were 1000 μg/mL against L. monocytogenes and S. Typhimurium. In the case of P. sp. rif200865, the MIC values of the butanol extract were 250 μg/mL against S. aureus ATCC 12600, and 500 μg/mL against L. monocytogenes, S. Typhimurium, B. cereus, and E. coli O157:H7. The MBC values were 500 μg/mL against S. aureus and 1000 μg/mL against L. monocytogenes, S. Typhimurium, B. cereus, and E. coli O157:H7. The MIC values of both B. sp. cryopeg and P. sp. rif200865 solvent extracts were 250 μg/mL, while their MBC values were 500 μg/mL against S. aureus ATCC 12600 (Table 3). In the case of B. sp. cryopeg, the MIC value was 250 μg/mL and the MBC value was 500 μg/mL against E. coli O157:H7.

SEM analysis

The effects of butanol extract of B. sp. cryopeg (DUS59) and Paenibacillus sp. (DUL128) on the morphology of five tested pathogenic bacteria were monitored by SEM analysis. The five tested FPB treated with the butanol extract of B. sp. cryopeg and Paenibacillus sp. varied in morphology, having irregular and disrupted cell structures compared with the control bacteria treated with 5% DMSO, which had smooth and regular structures (Fig. 4). The bacteria treated with the butanol extracts of B. sp. cryopeg and Paenibacillus sp. had less in density compared with the control bacteria (Fig. 4).

FIG. 4.

Scanning electron microscopy of the butanol solvent extract of Bacillus sp. cryopeg (DUS59) and Paenibacillus sp. rif200865 (DUL128) against five FPB. (A) Bacillus cereus ATCC 10876 (Control with 5% DMSO); (B) Listeria monocytogenes ATCC 7644 (Control with 5% DMSO); (C) Salmonella Typhimurium DT104 (Control with 5% DMSO); (D) Staphylococcus aureus ATCC 12600 (Control with 5% DMSO); (E) Escherichia coli O157:H743890 (Control with 5% DMSO). The control bacteria showed high density and smooth and regular structures, whereas the bacteria treated with the butanol extract of B. sp. cryopeg and Paenibacillus sp. displayed very low density with varied morphology, irregular, and disrupted cell structures. FPB, foodborne pathogenic bacteria.

Discussion

To explore the potential applications of EB from D. uniformis, it was necessary to determine the distribution of different EB associated with the host plants. We identified 51 culturable EB associated with leaf (5–10 leaves) and stem/root (5–10 of 1 cm cut) tissues of a number of fern plants, D. uniformis samples collected randomly from different locations of the Bohyun Mountain, Yeongchun, Republic of Korea. There have been many reports of diversity of EB from various other traditional medicinal plants (El-Deeb et al., 2012; Li et al., 2012; Bhore et al., 2013; Sivasankari et al., 2013; Min et al., 2014; Zhao et al., 2015); however, to the best of our knowledge, this is the first study to show diverse types and the applications of EB in D. uniformis. EB can produce valuable metabolites that can be utilized by the pharmaceutical industry because they produce the same metabolites as their hosts (Strobel and Daisy, 2003; Ebrahimi et al., 2010; Mehanni and Safwat, 2010; Preveena and Bhore, 2013).

The presence of microbes in a host plant can be affected by the compounds contained in the host plants (Strobel and Daisy, 2003; Sulistiyani et al., 2014). For these reasons, bacterial endophytes have attracted a great deal of attention owing to their potential for use in isolation of bioactive antimicrobial metabolites (Menpara and Chanda, 2013). The extracts from D. uniformis are well known to have antimicrobial activity (Preveena and Bhore, 2013); therefore, the isolated culturable EB of D. uniformis might produce novel antibiotics. The population diversity of EB varies based on the habitat, stage, and tissues of the host plants. Bacillus, Enterobacter, Microbacterium, Pseudomonas, Stenotrophomonas, Klebsiella, Mycobacterium, Pantoea are the most abundant EB found in plants (Seghers et al., 2004; Sulistiyani et al., 2014). Thirty different bacterial genera from 63 isolates have been identified from the interior of ginseng root (Cho et al., 2007), and 20 different endophytic bacterial genera have been isolated from the meristematic tissues of three varieties of strawberry (Dias et al., 2009). In addition, 49 actinobacteria were isolated from surface-sterilized wheat roots (Justin et al., 2003). These reports confirm that EB populations are noticeably divergent in different plant species and that the diversity of the populations may differ considerably for different locations.

The present study was conducted to isolate EB from the fern plant D. uniformis (Fig. 1A and Supplementary Table S1). The results revealed that D. uniformis is enriched with 51 EB, and the density of EB was higher in leaf tissues than stems/roots, after which 26 EB were identified based on their 16S rRNA sequences (Table 1). When compared with the bacterial distribution in various plant species that were not Pteridophytes found by Seghers et al. (2004) and Sulistiyani et al. (2014), the EB population of D. uniformis is very different, possibly due to it being a Pteridophyte.

FPB are a cause for concern worldwide (Scott, 2003). The major FPB, E. coli, L. monocytogenes, B. cereus, S. aureus, and S. Typhimurium (Hall et al., 2005; Jimenez et al., 2005; Voravuthikunchai et al., 2006; Zarei et al., 2014), have been found to be extremely important to human health and disease. S. aureus and B. cereus are also responsible for many health-related problems (Sofia et al., 2007). The majority of EB are producers of different types of antibiotics (Christina et al., 2013), and the results of the present study indicate that B. sp. cryopeg (DUS59), P. sp. rif200865 (DUL128), S. warneri (DUS130), and B. psychrodurans (DUS131) exerted a broad spectrum of antibacterial activity against L. monocytogenes, B. cereus, S. aureus, E. coli, and S. Typhimurium (Fig. 2 and Table 2). The above four EB were active against Gram-positive and Gram-negative FPB, which is similar to the results reported by Roy and Banerjee (2010).

Among the four EB, two EB, P. sp. rif200865 and B. sp. cryopeg, were further characterized for their antimicrobial capacity by the disk diffusion method using solvent extraction fractions. Basing on literatures (Wise et al., 2012; Kumar et al., 2013; Dutta et al., 2014), butanol was used for extraction of metabolites from the EB. The butanol extract of the two EB showed antibacterial activity against the five FPB (Table 3 and Fig. 2c). The neighbor-joining tree showed that DUS59 and DUL128 belong to two different genera (Fig. 3). SEM analysis revealed that the foodborne pathogens treated with the MIC of butanol extract go through various changes, a loss of cellular integrity and deformations on the surface of the bacterial cell (Fig. 4). When tied with the earlier study, these conclusions proposed that the active compounds of butanol extract might have bound to the surface of bacterial cells, entered the cytoplasmic membrane, and resulted in cell disruption (Fig. 4) (Zhu et al., 2005).

Conclusions

The results of this study demonstrate that D. uniformis contain diverse culturable EB and that some EB produce antibiotic substances that were capable of preventing the growth of FPB. Specifically, the butanol extract of P. sp. rif200865 and B. sp. cryopeg effectively inhibited L. monocytogenes, B. cereus, S. aureus, E. coli, and S. Typhimurium. Accordingly, the isolated EB can be used by the food industry as a natural method to protect food or as sources of novel antimicrobial substances by the pharmaceutical industry. With the increased attention that natural foods and remedies are receiving, the EB isolated from D. uniformis could be a promising source of natural antibacterial substitutes.

Footnotes

Acknowledgment

This work was supported by grants from the Systems and Synthetic Agro-biotech Center through the Next-Generation Bio-Green 21 Program (PJ011117), Rural Development Administration, Republic of Korea.

Disclosure Statement

No competing financial interests exist.

References

Araujo

, Luiz

, Lucio

. The effect of different growth regimes on the endophytic bacterial communities of the fern, Dicksonia Sellowiana Hook (DICKSONIACEAE). Braz J Microbiol, 2010; 41:956–965.

Bahadori

, Mahmoodi

, Ali

, Bahadori

, Valizadeh

. Antibacterial evaluation and preliminary phytochemical screening of selected ferns from Iran. Res J Pharmacogn, 2015; 2:53–59.

Barros

, Araujo

, Azevedo

. The effect of different growth regimes on the endophytic bacterial communities of the fern, Dicksonia sellowiana Hook (Dicksoniaceae). Braz J Microbiol, 2010; 41:956–965.

Bhore

, Komathi

, Kandasamy

. Diversity of endophytic bacteria in medicinally important Nepenthes species. J Nat Sci Biol Med, 2013; 4:431–434.

Chen

, Luo

, Chen

, Wan

, Liu

, Pang

, Lai

, Zeng

. Diversity of endophytic bacterial populations associated with Cd-hyper accumulator plant Solanum nigrum L. grown in mine tailings. Appl Soil Ecol, 2012; 62:24–30.

Cho

, Su

, Lee

, Kim

, Kahng

, Lim

, Kim

, Han

. Endophytic bacterial communities in Ginseng and their antifungal activity against pathogens. Microb Ecol, 2007; 54:341–351.

Christina

, Christapher

, Bhore

. Endophytic bacteria as a source of novel antibiotics: An overview. Pharmacogn Rev, 2013; 7:11–16.

Diao

, Hu

, Feng

, Li

, Xu

. Chemical composition and antibacterial activity of the essential oil from green huajiao (Zanthoxylum schinifolium) against selected foodborne pathogens. J Agric Food Chem, 2013; 61:6044–6049.

Dias

ACF

, Costa

FEC

, Andreote

, Lacava

, Teixeira

, Assumpcao

, Araujo

, Azevedo

, Melo

. Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion. World J Microbiol Biotechnol, 2009; 25:189–195.

10.

Dutta

, Puzari

, Gogoi

, Dutta

. Endophytes: Exploitation as a tool in plant protection. Braz Arch Biol Technol, 2014; 57:621–629.

11.

Ebrahimi

, Asgharian

, Habibian

. Antimicrobial activities of isolated endophytes from some Iranian native medicinal plants. Iranian J Pharmac Sci, 2010; 6:217–222.

12.

El-Deeb

, Fayez

, Gherbawy

. Isolation and characterization of endophytic bacteria from Plectranthus tenuiflorus medicinal plant in Saudi Arabia desert and their antimicrobial activities. J Plant Interac, 2013; 8:56–64.

13.

Fisher

. Survival and spread of the endophyte Stagonospora pteridiicola in Pteridium aquilinum; other ferns and some flowering plants. New Phytol, 1996; 132:119–122.

14.

Golinska

, Wypij

, Agarkar

, Rathod

, Dahm

, Rai

. Endophytic actinobacteria of medicinal plants: Diversity and bioactivity. Antonie Van Leeuwenhoek, 2015; 108:267–289.

15.

Hall

, Kirk

, Becker

, Gregory

, Unicomb

, Millard

, Stafford

, Lalor

; Ozfoodnet Working Group. Estimating foodborne gastroenteritis, Australia. Emerg Infect Dis, 2005; 11:1257–1264.

16.

Jimenez

, Soler

, Venanzi

, Cante

, Varela

, Martinez-Navarro

. An outbreak of Campylobacter jejuni enteritis in a school of Madrid, Spain. Euro Surveill, 2005; 10:118–121.

17.

Justin

, Christopher

MMF

. Isolation and identification of actinobacteria from surface-sterilized wheat roots. Appl Environ Microbiol, 2003; 69:5603–5608

18.

Kubo

, Fujita

, Kubo

, Nihei

, Ogura

. Antibacterial activity of coriander volatile compounds against Salmonella choleraesuis . J Agric Food Chem, 2004; 52:29–32.

19.

Kumar

, Kaushik

, Proksch

. Identification of antifungal principle in the solvent extract of an endophytic fungus Chaetomium globosum from Withania somnifera . Springer Plus, 2013; 2:37.

20.

, Sinkko

, Montonen

, Wei

, Lindstrom

, Rasanen

. Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China. FEMS Microbiol Ecol, 2012; 79:46–68.

21.

Lodewyck

, Vangronsveld

, Porteous

, Moore

ERB

, Taghavi

, Mezgeay

, Van Der Lelie

. Endophytic bacteria and their potential application. Crit Rev Plant Sci, 2002; 86:583–606.

22.

Mandal

, Mondal

. Studies on antimicrobial activities of some selected ferns and lycophytes in Eastern India with special emphasis on ethno-medicinal uses. African J Plant Sci, 2011; 5:412–420.

23.

Mehanni

, Safwat

. Endophytes of medicinal plants. Acta Hort (ISHS) XIII Int Conference Medicin Aromatic Plants 2010; 854:31–39.

24.

Menpara

, Chanda

. Endophytic bacteria-unexplored reservoir of antimicrobials for combating microbial pathogens. In: Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education. Méndez-Vilas

(ed.). Formatex, Extremadura, Spain: 2013, pp. 1095–1103.

25.

Min

, Park

, Fong

, Quan

, Jung

, Lim

. Diversity and saline resistance of endophytic fungi associated with Pinus thunbergii in coastal shelterbelts of Korea. J Microbiol Biotechnol, 2014; 24:324–333.

26.

Nair

, Padmavathy

. Impact of endophytic microorganisms on plants, environment and humans. Sci World J, 2014; 2014:250693.

27.

Patra

, Hwang

, Choi

, Baek

. Bactericidal mechanism of bio-oil obtained from fast pyrolysis of Pinus densiflora against two foodborne pathogens, Bacillus cereus and Listeria monocytogenes . Foodborne Patho Dis, 2015; 12:529–535.

28.

Preveena

, Bhore

. Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn. Anc Sci Life, 2013; 32:173–177.

29.

Roh

, Lee

, Ra

, Choi

, Moon

, Heu

. Diverse antibacterial activity of Pectobacterium carotovorum subsp. Carotovorum isolated in Korea. J Microbiol Biotechnol, 2009; 19:42–50.

30.

Roy

, Banerjee

. Isolation of antimicrobial compound by endophytic bacteria from Vinca Rosea . Int J Curr Res, 2010; 5:47–51.

31.

Scott

. Food safety and foodborne disease in 21st century homes. Can J Infect Dis, 2003; 14:277–280.

32.

Seghers

, Wittebolle

, Top

, Verstraete

, Siciliano

. Impact of agricultural practices on the Zea mays L. endophytic community. Appl Environ Microbiol, 2004; 70:1475–1482.

33.

Selim

HMM

, Gomaa

, Essa

AMM

. Comparative in vitro study of the antimicrobial activities of some endophytic bacteria. Wulfinia J, 2014; 21:1–11.

34.

Sen

, Nandi

. Antibiotics from the pteridophytes. Sci Cult, 1951; 16:328–329.

35.

Seo

, Lim

, Kim

, Yun

, Lee

, Cho

. Endophytic bacterial diversity in the young radish and their antimicrobial activity against pathogens. J Korean Soc Appl Biol Chem, 2010; 53:493–503.

36.

Sivasankari

, Pitchaimani

, Anandharaj

. A study on traditional medicinal plants of Uthapuram, Madurai District, Tamilnadu, South India. Asian Pac J Trop Biomed, 2013; 3:975–979.

37.

Sofia

, Prasad

, Vijay

, Srivastava

. Evaluation of antibacterial activity of Indian spices against common foodborne pathogens. Int J Food Sci Technol, 2007; 42:910–915

38.

Strobel

, Daisy

. Bioprospecting for microbial endophytes and their natural products. Microbiol Mol Biol Rev, 2003; 67:491–502.

39.

Sulistiyani

, Lisdiyanti

, Lestari

. Population and diversity of endophytic bacteria associated with medicinal plant Curcuma zedoaria . Microbiol Indones, 2014; 8:65–72.

40.

Sun

, Qiu

, Zhang

, Dai

, Dong

, Song

. Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microb Ecol, 2008; 55:415–424.

41.

Susilowati

, Sabdono

, Widowati

. Isolation and characterization of bacteria associated with brown algae Sargassum spp. From Panjang Island and their antibacterial activities. Procedia Envir Sci, 2015; 23:240–246.

42.

Tamura

, Stecher

, Peterson

, Filipski

, Kumar

. MEGA6: Molecular evolutionary genetic analysis version 6.0. Mol Biol Evol, 2013; 30:2725–2729.

43.

Voravuthikunchai

, Limsuwan

, Supapol

, Subhadhirasakul

. Antibacterial activity of extracts from family zingiberaceae against foodborne pathogens. J Food Safety, 2006; 26:325–334.

44.

Wise

, Novitsky

, Tsopmo

, Avis

. Production and antimicrobial activity of 3-hydroxypropionaldehyde from Bacillus subtilis strain CU12. J Chem Ecol, 2012; 38:1521–1527.

45.

, Zhang

, Linli Zheng

, Guo

, Li

, Sun

, Qin

. Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol Res, 2010; 165:437–449.

46.

Zarei

, Jamnejad

, Khajehali

. Antibacterial effect of silver nanoparticles against four foodborne pathogens. Jundishapur J Microbiol, 2014; 7:e8720.

47.

Zhao

, Xu

, Lai

X-H

, Shan

, Deng

, Ji

. Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters. Braz J Microbiol. 2015; 46:977–989

48.

Zhu

, Yang

, Yu

, Ying

, Zou

. Chemical composition and antimicrobial activity of the essential oils of Chrysanthemum indicum . J Ethnopharmacol, 2005; 96:151–158.

49.

Zubair

, Rizwan

, Rasool

, Afshan

, Shahid

, Ahmed

. Antimicrobial potential of various extract and fractions of leaves of Solanum nigrum . Int J Phytomed, 2011; 3:63–67.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.04 MB

0.03 MB