Antibacterial activity Lactobacillus plantarum isolated from fermented vegetables and investigation of the plantaricin genes

1 Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, R.O.C. Taiwan. 2 Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, R.O.C. Taiwan. 3 Department of Food Science and Technology, Chia Nan University of Pharmacy and Science, Tainan, R.O.C. Taiwan. 4 Department of Hotel and Restaurant Management, Chia Nan University of Pharmacy and Science, Tainan, R.O.C. Taiwan.

. Many food-poisoning outbreaks of E. coli have been associated with contaminated food, such as beef, pork, chicken and water (Wang, 2008).S. aureus may produce a number of toxins, the most important ones with respect to foodborne illness belong to the family of heat-stable staphylococcal enterotoxins (SEs).Another public health concern is associated with the increased incidence of antibiotic-resistant strains isolated from poultry meat (Dan et al., 2015).Due to the widespread use of antimicrobials in chicken and pig growth units, the development of resistant strains that can infect humans via the food chain has increased (Mihaiu et al., 2014).As a result, contamination of pathogenic microorganism is recognized as a potential public health concern.As more bacteria become resistant to traditional antibiotics, this leads to emergence and re-emergence of multidrugresistant pathogens.
Lactic acid bacteria (LAB) have been used in the production of varieties of fermented dairy, vegetables and meat products for many centuries (Man et al., 2014).Recent research revealed that LAB can produce antibacterial substances including organic acids, hydrogen peroxide, diacetyl, inhibitory enzymes and bacteriocins (Herna´ndez et al., 2005) to inhibit the growth of a wide range of intestinal pathogens (García-Ruiz et al., 2013).Bacteriocins were defined as antimicrobial peptides or proteins have been observed in many genera of bacteria, including many strains of LAB, which are directed mainly to inhibit the growth of related species (Anyogu et al., 2014).Lactobacillus rhamnosus has been reported to interact with intestinal epithelium and prevent the internalization of enterohemorrhagic E. coli (Moorthy et al., 2007).Lactobacillus sake C2 which produced a bacteriocin strongly inhibited S. aureus ATCC 63589 and E. coli ATCC 25922 was isolated from traditional Chinese fermented cabbage (Gao et al., 2010).Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chourico, inhibited the growth of a number of Grampositive and Gram-negative meat spoilage bacteria (Todorov et al., 2010).L. plantarum B0105 isolated from traditional Taiwan fermented mustard, was found to produce bacteriocin inhibiting Streptococcus mutans BCRC 10793 (Chen et al., 2013).L. plantarum ST71KS was isolated from homemade goat feta cheese and displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 (Martinez et al., 2013).
LAB are very important in ensuring the safety of various foods by production of bacteriocins and other antimicrobial substances.Bacteriocins produced by L. plantarum are known as plantaricins (Omar et al., 2008).The genetic determinants for plantaricins were reported as shown for strains L. plantarum strain C11 (Diep et al., 2003), WCFS1 (Kleerebezem et al., 2003), NC8 (Maldonado et al., 2003) and J23 (Rojo-Bezares et al., 2008).The L. plantarum C11 plantaricin cluster contains five operons (Diep et al., 2003).Two of them (plnEFI and plnJKLR) code for bacteriocins and immunity proteins, the transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins; plnABCD for the signal-transducing pathway, and plnMNOP containing genes with unknown functions in bacteriocin synthesis (Diep et al., 2003).In this study, LAB isolated from traditional fermented products, were screened for the antibacterial activity and presence of plantaricin genes in strains were also investigated.

Bacterial strains
Lactic acid bacteria were isolated from traditional fermented mustard and vegetable samples collected from southern areas of Taiwan.The LAB isolates were characterized based on acid production, Gram stain and catalase test were tested.Lactococcus lactis subsp.lactis (ATCC 11454) and L. plantarum (ATCC 14917) with plantaricin genes as control strain, S. aureus (BCRC 12653, BCRC 12654, BCRC12658, BCRC13824 and BCRC 13829) and E. coli (BCRC 14825, BCRC 15375 and BCRC 41443), used as test microorganisms in determining antibacterial activity were obtained from Bioresource Collection and Research Center (BCRC), Hsin Chu, Taiwan.

Screening antibacterial activity of LAB
For screening the antibacterial activity of LAB isolates, 1% (v/v) of these cultures were inoculated into 50 ml de Man, Rogosa and Sharpe (MRS) broth individually and incubated at 35°C for 24 h without agitation.Bacterial cells were removed by centrifugation (17,000 g, 10 min, 4°C) and the resulting solution were subjected to filtration with 0.22 µm filter, then, the diameters of inhibition zones were measured using the agar diffusion assay method (Anyogu et al., 2014).Overnight test cultures of E. coli and S. aureus were diluted in saline solution into 10 8 CFU/ml and 100 µl of dilution were inoculated in nutrient agar medium.Briefly, 100 µl of spent cell-free supernatant (SCS) were placed into wells (10.0 mm in diameter) on nutrient agar plates seeded with the test pathogens.After incubation at 35°C for 14 h, the diameter of inhibition zones was determined.The pH of MRS broth was also adjusted to the same value as blank.The antimicrobial activity was lost after treatment with pepsin, indicating a peptide nature (Rojo-Bezares et al., 2007).The bacterial cell-free supernatants were incubated 37°C overnight with or without pepsin (Sigma, St. Louis, Missouri) at a final concentration of 2 mg/ml.L. lactis subsp.lactis (nisin producing strain, BCRC 11454) was used as the negative control and L. plantarum with plantaricin genes (ATCC 14917) as the positive control.As a blank control, aliquots of MRS broth treated as filtered supernatants were used.All of the tests were repeated 3 times.Diameters of inhibition zones were determined.Reduced inhibition zone (mm) = inhibition zone of SCS -inhibition zone of SCS treated with pepsin.

Strain identification
A polymerase chain reaction (PCR) assay was performed using genomic DNA from strains that showed antibacterial activity.Amplification of 16S rDNA sequences by PCR was performed using the primers 27F-AGAGTTTGATCMTGGCTCAG and 1492R-GGYTACCTTGTTACGACTT described by Tanner et al. (2000).For the PCR identification, genomic DNA was extracted using the  Genomic isolation kit (GeneMark, Georgin, USA) according to the manufacturer's instructions.Genomic DNA concentration was determined spectrophotometrically (Hitachi, U-2800A, Tokyo, Japan).PCR primers were used to amplify a 1484 bp DNA fragment.The reaction mixture contained 10 µl genomic DNA, 2.5 units of Taq polymerase (Promega, Madision, WI), 2 µl each of 10 mM dATP, dTTP, dCTP and dGTP, 5 µl of 10 X reaction buffer (10 mM Tris-HCl (pH 8.3 at 25°C) containing 50 mM KCl, 0.01% Triton X-100, 0.01% gelatin, 6.0 mM MgCl2), and 50 ρmol of each primers in a final volume of 50 µl.The DNA was denatured at 94°C for 2 min and amplified for 35 cycles at 94°C for 40 s, 45°C for 50 s and 72°C for 50 s.A final extension incubation of 2 min at 72°C was included.Amplification reactions were performed on a thermal cycler (Perkin-Elmer GeneAmp PCR System 2400, Foster city, CA).The PCR products were purified with Gel/PCR DNA fragments extraction kit (Geneaid, Taipei, Taiwan) and sequenced by automated sequencing core laboratory, National Cheng Kung University (Tainan, Taiwan).Sequence homologies were examined by comparing the obtained sequence with those in the DNA data bases (http//www.ncbi.nim.nih.gov/BLAST)(Todorov et al., 2010).

PCR amplification of plantaricin genes
A PCR assay was performed using total genomic DNA from strains that showed antibacterial activity against test pathogens.PCR amplification of plantaricin genes were carried out, the primers and conditions were specified in Table 1 (Sáenz et al., 2009), with initial

Screening antibacterial activity of LAB
A total of 50 presumptive lactic acid bacteria isolates obtained from traditional fermented mustard and vegetables were determined with phenotypical and physiological tests.Out of the 50 LAB isolates, only 7 isolates showed antagonistic effect against the test pathogens (S. aureus and E. coli) (Table 2).The diameters of inhibition zones against E. coli and S. aureus ranged between 12 and 29 mm.In some cases, antimicrobial activity reduced after addition of pepsin on the SCS of LAB, the reduction of diameters of inhibition zones against E. coli and S. aureus ranged between 2 and 7 mm (Table 3).

Strains identification
Amplification of 16S rDNA sequence by PCR was performed using the primers described by Tanner et al. (2000).The identification of LAB isolates with antibacterial activity revealed that strain B0039 was identified as Lactobacillus paracasei and other strains were identified as L. plantarum.The 16S rDNA nucleotide sequences of the test strains were 99 to 100% similarity with the GenBank access number assigned in Table 4.

Amplification of plantaricin genes
In an attempt to determine whether the strains that showed antibacterial activity against test pathogens carried genes for the production of known plantaricins.A PCR assay was performed using total genomic DNA from strains.Several strains carried at least one or more genes of the plantaricin cluster.L. plantarum strains B0013, B0055, B0126 tested positive for the plnABCD operon.The complete plnEFI operon was detected in L. plantarum B0013, B0055 and B0126, while B0105 was detected only plnEF.Genes encoding for the two-peptide plnJ/K were detected only in L. plantarum B0055 and B0126.Finally, the plnG/V, which is part of a large operon involved in plantaricin export, was also found in L. plantarum B0013, B0055, and B0126.Three strains gave all negative plantaricin genes in PCR analyses (Table 5).Among these positive results, B0055 was Lactobacillus plantarum HM058986 100 confirmed by sequencing.The plantarum gene sequences of B0055 were 97 to 100% similarity with the GenBank access number assigned in Table 6.

DISCUSSION
Bacteriocins of LAB are active against Grampositive bacteria, such as LAB (García-Ruiz et al., 2013) and S. aureus (Anyogu et al., 2014;Omar et al., 2008;Sebastià et al., 2011).However, plantaricins produced by L. plantarum strains had broad spectra of inhibition activity against Gram-negative bacteria including E. coli and Salmonella enterica (Anyogu et al., 2014;Omar et al., 2008).Strain L. sakei C2 producing a bacteriocin strongly inhibited S. aureus and E. coli (Gao et al., 2010).In our study, seven strains showed antibacterial activity against test pathogens.Rojo-Bezares et al. ( 2007) indicated that antimicrobial activity was lost after treatment with trypsin, α-chymotrypsin, papaine, protease, proteinase K, and acid proteases, indicating bacteriocin was peptide nature.Gao et al. (2010) indicated that after treatment by all the three kinds of protease, the antimicrobial activity of cell-free supernatant of strain L. sake C2 disappeared and the protein nature of this antimicrobial substance produced by strains C2 was verified.In this study, after treatment by pepsin, the inhibition zones of seven strains selected towards the tested pathogens reduced.It indicated that the substance  with antibacterial activity was sensitive to pepsin, where it might be considered that the bioactive compound produced by some strains might be the protein or peptide nature (Rojo-Bezares et al., 2007;Gao et al., 2010).The variation of the antibacterial activity of our test strains are almost similar to those shown by other works on the antibacterial activity of LAB (García-Ruiz et al., 2013;Anyogu et al., 2014;Gao et al., 2010).Previous studies on the microorganisms of traditional fermented vegetables and fruits had shown that L. plantarum was dominant among the isolates from fermented mustards (Chen et al., 2013).All test strains were identified as L. plantarum except B0039 as L. paracasei.L. plantarum is important in many food fermentations as a component of the natural microflora or as a starter culture (Gao et al., 2010;Omar et al., 2008).
Bacteriocin-producing strains of L. plantarum have been reported from many vegetable foods such as fermented cereal doughs, wara (Omar et al., 2008), fermented cassava (Kostinek et al., 2005) and fermented mustard (Chen et al., 2013).Bacteriocins of lactic acid bacteria are active against closely related bacteria.However, activity against Gram-negative bacteria has been described in several cases.Omar et al. (2008)showed that bacteriocins plnABCD encode for the signal-transducing pathway.Variations in this operon have also been reported for the gene clusters described in L. plantarum from poto poto with only plnC being not conserved (Omar et al., 2008)  Lactobacillus strains from poto poto also reported the absence of plnNC8 plantaricins, while plnN gene was absent in strains (Omar et al., 2008).However, the genes encoding for plnN and plnNC8 were not detected in any strain.Finally, plnG and plnV gene, which is part of a large operon involved in plantaricin transport operon (Sáenz et al., 2009) were found in L. plantarum strains B0013, B0055, B0126 strains.With antibacterial activity, but showed negative in plantaricin gene, these findings might explain that L. plantarum may contain other bacteriocin genes that were not detected in this study.However, L. plantarum B0125 and B0134 strains which gave negative results for the PCR amplification should be investigated for possible sequence heterogeneity in the plantaricin operons.Plantaricin gene sequences of B0055 were 97 and 100% similarity with the GenBank access number.The bactericidal effect might be from the production of organic acids and/or in combination with the production of bacteriocin (Lin et al., 2008).In our results, antibacterial activity of selected strains might be from carried genes for the production of plantaricins and production of organic acids.

Conclusion
LAB isolated from fermented vegetalbes seem to have high potentials for production of antimicrobial substances, and also seem to have variations in the plantaricin genes.
In this study, fermented vegetables were found to contain LAB and other pathogens.Since L. plantarum strains produce bacteriocins that inhibit food-borne pathogens such as E. coli and S. aureus, the use of these antimicrobial substances as food additives or use of the bacteriocin-producing strains as starters, might contribute to the production of a safer and healthier traditional fermented product (Omar et al., 2008).The findings in this study suggest that LAB producing bacteriocins can be used as alternate mechanism to inhibit the growth of drug-resistant pathogens.

Table 1 .
Plantaricin gene primers and conditions used in this study.

Table 2 .
Antibacterial activities of lactic acid bacteria spent cell supernatants (SCS) against test pathogens.
*All of the tests are repeated at least 3 times.

Table 3 .
Effect of pepsin treatment on the antibacterial activity of LAB SCS against indicators.

Table 4 .
Identification of LAB strains based on 16S rDNA sequence similarity.

Table 5 .
Plantaricin genes detected by PCR from lactic acid bacteria with antibacterial activity.
Omar et al. (2008)l., 2008)rape must, with only plnD being conserved(Rojo-Bezares et al., 2008).In this study, L. plantarum strains, B0013, B0055 and B0126 tested positive for the plnABCD operon, others are not detected.In other words, variations in plnABCD also exist in our strains.Two of plnEFI and plnJ/K code for bacteriocins to inhibit pathogens.Genes encoding for the plnEFI and plnJ/K were detected in L. plantarum B0055 and B0126, while L. plantantum B0013 only test positive for plnEFI.Omar et al. (2008)indicated that the plnEFI operon was detected in thirteen isolates from poto poto, while others only tested positive for plnEF or plnI.From our results, B0105 detected only positive of plnEF.