Next Article in Journal
Closing the Diagnostic Gap in Encephalitis and Acute Disseminated Encephalomyelitis through Digital Case Classification and Viral Metagenomics
Previous Article in Journal
Antagonistic Interactions in Onychomycosis: Antifungal Activity of Extracts from Pure and Mixed Cultures of Candida parapsilosis and Trichophyton spp.
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microbiology Research
Volume 15
Issue 2
10.3390/microbiolres15020058
microbiolres-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens

by
Ezequiel Hernandez-Mendoza
1,
Etna Aida Peña-Ramos
1,*,
Vijay K. Juneja
2,
Miguel Ángel Martínez-Téllez
1,
Humberto González-Ríos
1,
María de la Cruz Paredes-Aguilar
3,
Martin Valenzuela-Melendres
1 and
Emmanuel Aispuro-Hernández
1
1
Centro de Investigación en Alimentación y Desarrollo, A.C., Coordinación de Tecnología de Alimentos de Origen Animal, Gustavo Astiazaran Rosas 46, Hermosillo 83304, Sonora, Mexico
2
U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PL 19308, USA
3
Centro de Investigación en Alimentación y Desarrollo, A.C., Área de Inocuidad Alimentaria, Varadero Nacional km 6.6, Las Playitas, Guaymas 85480, Sonora, Mexico
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(2), 889-899; https://doi.org/10.3390/microbiolres15020058
Submission received: 5 April 2024 / Revised: 1 May 2024 / Accepted: 13 May 2024 / Published: 22 May 2024
Browse Figures
Review Reports Versions Notes

Abstract

:
Lactic acid bacteria (LAB) can produce peptides known as bacteriocins with antagonistic activity against foodborne pathogens. The potential of LAB isolated from the surface of jalapeno peppers to produce bacteriocins with antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium was evaluated. Previously isolated LAB strains were reactivated, and their cell-free supernatants (CFSs) were evaluated. Out of 390 reactivated strains, 60 produced bacteriocin-like inhibitory substances (BLIS) since their antagonistic activity was lost after proteases addition. Subsequently, 16 BLIS showed heat resistance (HR-BLIS), retaining their bioactivity after heat treatment (121 °C for 15 min). By 16S rRNA gene sequencing and antibiotic susceptibility tests, LAB strains producing HR-BLIS were identified as Enterococcus lactis. Four HR-BLIS exhibited a minimum inhibitory concentration (MIC) of 80 mg/mL against L. monocytogenes. MIC and minimum bactericidal concentration (MBC) of HR-BLIS-67 for S. aureus (MIC = 80 mg/mL; MBC = 320 mg/mL), S. Typhimurium (MIC = 150 mg/mL; MBC = 250 mg/mL), and E. coli O157:H7 (MIC = 250 mg/mL; MBC = 400 mg/mL) were determined. LAB isolated from the surface of jalapeno pepper produced HR-BLIS (possibly enterocin) that exhibited broad-spectrum antagonistic activity against foodborne pathogens; therefore, they are a promising source of natural antimicrobials to ensure food safety.

1. Introduction

Lactic acid bacteria (LAB) are a group of Gram-positive microorganisms widely distributed in nature, so they can be isolated from different food sources, including fruits and vegetables like cantaloupe, pickles, tomato, and peppers [1,2,3]. Native to Mexico, jalapeno peppers (Capsicum annuum) are grown throughout much of the country and account for 31% of all pepper production in Sonora, having an important impact on local economy. Within its microbiome, LAB are present, which may play a protective role against the spoilage of this type of pepper [4]. Among the most representative genera are Lactobacillus, Streptococcus, Pediococcus, and Enterococcus. In addition, LAB have been used in the food industry as natural biopreservatives due to the production of inhibition agents with antimicrobial activity, known as bacteriocins [5].
Bacteriocins are bioactive peptides with the ability to inhibit the growth of pathogenic and spoilage microorganisms, with a bactericidal and/or bacteriostatic effect [6]. Some bacteriocins are heat resistant and effective across a broad pH range, particularly in acidic media [7]; these characteristics make them ideal for use as biopreservatives in foods. Nisin and pediocin are the most studied and commercially available bacteriocins that have been approved for use as food additives by the food and drug administration. Nisin is produced by Lactococcus lactis, and pediocin PA-1/AcH from Pediococcus acidilactici. Both possess antagonistic activity against Gram-positive bacteria; however, they are not effective against Gram-negative bacteria when used alone, limiting their applications [8,9].
There are various criteria to consider when classifying bacteriocins, such as their genetic and molecular weight, as well as their physical, chemical, and phenotypic properties [10]. Bacteriocins produced by the genus Enterococci are named enterocins [11]. Enterocins act by inhibition of the synthesis of the cell membrane components or by pore formation leading to cell lysis [12]. In general, bacteriocins are more effective against Gram-positive bacteria than Gram-negative bacteria. However, new LAB-derived bacteriocins, including those from vegetable sources, have demonstrated antimicrobial activity against several pathogens of both types of bacteria [1,2,3,13,14]. Although these studies confirm the antimicrobial effects of LAB-bacteriocins from vegetables, there are vegetable sources that are yet to be studied, such as jalapeno peppers, that may also have the potential to contain bacteriocin-LAB producers with antagonistic effects that could unveil natural alternatives for food preservation.
Among the main pathogenic bacteria that compromise food safety are L. monocytogenes, S. aureus, S. Typhimurium, and E. coli O157:H7 [15,16,17,18]. Outbreaks of infections caused by these pathogens [19] have been reported to cost hundreds of billions of dollars per year in productivity losses and medical expenses related to contaminated food consumption [20]. L. monocytogenes stands out for its ability to persist in ready-to-eat products after surviving multiple hurdles like heat treatment, low pH, refrigeration conditions, and high salt concentrations [21]. Therefore, it is an enormous challenge for the food industry to control and ensure the safety of their products against all pathogens, especially L. monocytogenes. Hence, the search for antimicrobial alternatives, preferably of natural origin with broad-spectrum antimicrobial bioactivity is very important. For these reasons, the objective of this investigation was to identify LAB isolated from the surface of jalapeno peppers with the ability to produce bacteriocins with antagonistic effect against foodborne pathogens.

2. Materials and Methods

2.1. Bacteria Strains: Conditions and Activation

A total of 390 LAB strains previously isolated from jalapeno pepper collected from commercial fields in Sonora, Mexico, belonging to the strain collection of the Molecular Plant Physiology Laboratory of the Research Center for Food and Development (CIAD) were used during this experiment. LAB strains were cryopreserved at −80 °C and were reactivated by inoculation in Man, Rogosa, and Sharpe (MRS) broth (DIFCO, Detroit, MI, USA) pH 7.0 ± 0.2 at 37 °C for 18 h. Two transfers were made under the same conditions as the previous inoculum and used in further experiments. All LAB strains were numerically labelled consecutively from 1 to 390.
Cryopreserved pathogenic bacteria (Table 1) at −80 °C in Brain-Heart Infusion (BHI) Broth (DIFCO, USA) with 15% glycerol were reactivated by transferring 0.1 mL of the inoculum to BHI broth and incubated at 37 °C for 20 h. Cultures were maintained in BHI broth at 8 °C throughout the study with monthly transfers. One day before the experiment, the inoculum of each strain was prepared by transferring 0.1 mL of the stock culture to 35 mL of BHI broth in 50 mL conical centrifuge tubes and incubated at 37 °C for 18 h with constant stirring. Overnight cultures were centrifuged (10,000× g, 10 min, 4 °C), and the resulting pellet of each strain was washed with 20 mM sodium phosphate buffer pH 6.5 ± 0.2, adjusted to an optical density at 600 nm of 0.1 and used during experiments.

2.2. Screening for LAB from Jalapeno Pepper with Antagonistic Activity against L. monocytogenes

The antagonistic activity of the reactivated LAB isolates from jalapeno peppers was tested against L. monocytogenes using the spot-on-lawn method described by Hilal Cadi and Citak (2005) [22]. A volume of 20 μL of each LAB was spotted on the surface of MRS agar plates and allowed to dry. Next, the microbial inoculum (O.D. 0.1) was mixed in BHI (DIFCO, NJ, USA) soft agar medium and overlayed onto the MRS plates which contained the LAB strains. After solidification, the plates were incubated at 37 °C for 24 h. Positive results were those LAB strains which presented a clear inhibition zone around the LAB culture.

2.3. Preparation and Antagonistic Activity Evaluation of Cell-Free Supernatants (CFSs)

Cell-free supernatants of selected LAB were obtained from fresh overnight (18 h growth) cultures, centrifuged (10,000× g, 10 min, 4 °C), frozen at −80 °C, and freeze-dried (Labconco Freezone 4.5, Kansas City, MO, USA) in order to concentrate the bioactive compounds in the supernatants. Freeze-dried CFSs were resuspended (1:10 w:v) in 20 mM sodium phosphate buffer pH 7.0 ± 0.2, sterilized by microfiltration (Durapore®, 0.22 µm size; Millipore Co., St. Louis, MO, USA), and used for subsequent analyses. The antagonistic activity against L. monocytogenes, S. aureus, S. Typhimurium, and E. coli O157:H7 was tested by the spot-on-lawn test. After incubation at 37 °C for 24 h, clear growth zones around the CFS drop were visually detected.

2.4. Identification of the Antagonistic Compound in CFSs and Heat Stability Test

Using the method described by Cruz-Guerrero et al. (2014) [23], the chemical nature of antimicrobial compounds within each CFS was identified. Briefly, this technique consisted of first adding NaOH (0.1 M) to adjust pH to 7 to neutralize the effect of CFSs’ organic acids (lactic acid); then adding 1 mg/mL of catalase (90 min at 25 °C and 10 min at 65 °C) to prevent the bactericide effect of H2O2; and finally, to confirm the production of protein compounds, a pool of proteases (protease, proteinase K, and trypsin at 1 mg/mL) was added, incubated for 2 h at 37 °C, and inactivated at 65 °C for 10 min, followed by the antagonistic test against L. monocytogenes between each step. For further discrimination, the thermal stability evaluation was performed on those CFSs containing antibacterial protein compounds by submitting them to different thermal treatments (80 or 100 °C for 10 min or 121 °C for 15 min), followed by the inhibition test against L. monocytogenes.

2.5. LAB 16S rRNA Sequence Analysis and Identification

For molecular identification, genomic DNA from selected LAB, based on their ability to produce heat-resistant CFS (HR-CFS) were extracted by alkaline lysis according to the Molecular Cloning Laboratory Manual 2012 [24] and used as template for standard PCR reactions using GoTaq® Flexi DNA Polymerase (Promega, Madison, WI, USA). Universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1301R (5′-TACTAGCGATTCCGACTTC-3′) were used. The PCR conditions were as follows: initial denaturation at 95 °C for 10 min; 30 denaturation cycles at 95 °C for 1 min each; primer alignment at 55 °C for 1 min and primer extension at 72 °C for 2 min; and a final extension step at 72 °C for 10 min. Next, for the sequence analysis of the gene 16S rRNA, the PCR products were purified with GFX columns (Amersham Bio-sciences, Piscataway, NJ, USA) and Sanger sequenced at Macrogen (Seoul, Korea) using the 518F universal primer (5′-CCAGCAGCCGCGGTAATACG-3′). Sequences were analyzed against the GenBank database with the Blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 10 October 2023), and the DNA sequences of the top hit matches were used as reference organisms for phylogenetic analysis. DNA sequence alignments were performed with the Clustal W function, and a phylogenetic tree was constructed with MEGA-X software v. 10.2.6. using the maximum-likelihood method and the general time-reversible model with gamma distribution to estimate the evolutionary distances (1000 bootstrap replicates).
Furthermore, to discriminate between LAB strains, antibiotics susceptibility of identified isolates was carried out by advanced colorimetric tests using the VITEK® 2 Compact equipment. The equipment applied a total of 43 biochemical tests, including 17 enzymatic tests for up to 8 h [25].

2.6. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Antimicrobial activity of freeze-dried HR-CFSs was evaluated following the method described by Morales-Figueroa et al. (2022) [26] using the broth microdilution technique in a 96-well microplate (COSTAR). Each well was inoculated with 5 μL of adjusted microbial inoculum (O.D. 0.1. approximately 1 × 108 CFU/mL) and mixed with 295 μL of diluted filter-sterilized freeze-dried HR-CFSs (10–400 mg/mL). Furthermore, three wells with only BHI broth and the other three with pathogen + BHI were used as negative and positive controls, respectively. Microplates were incubated for 24 h at 37 °C and the next day, 20 μL from each well were plated into TSA and incubated under the same conditions. MIC was established as the lowest concentration of each resuspended HR-CFS where growth inhibition was unveiled; MBC, as the lowest concentration required to inactivate 99.9% of the pathogen where no visible growth was detected in TSA plates. Experiments were executed in triplicates, and the results were expressed as mg/mL. HR-CFSs with the lowest MICs for L. monocytogenes were selected for further experiments.

2.7. Growth Inhibition Assay against L. monocytogenes

The effect of different concentrations (40, 80, 160, and 320 mg/mL) of selected HR-CFSs against L. monocytogenes growth was carried out using a 96-well microplate. Bacteria (5 μL; O.D. 0.1) were treated with different concentrations of HR-CFSs in BHI broth, achieving a final volume of 300 μL per well. The microplate was placed into a FLUOstar Omega microplate reader (BMG LabTech, Ortenberg, Germany) for 18 h at 37 °C. Optical density (600 nm) was recorded every 30 min. Positive and negative controls were prepared using BHI broth with and without L. monocytogenes, respectively. Subsequently, the most effective HR-CFS, with the longest lag phase and lowest growth rate of L. monocytogenes at the lowest concentration, was selected and its MIC and MBC against S. aureus, S. Typhimurium, and E. coli O157:H7 were established following the procedure described in the previous section.

2.8. Statistical Analysis

To estimate lag time (h) and maximal growth rate (µmax; OD/h), optical density values of selected HR-CFSs against L. monocytogenes growth were plotted against time. Growth curves were fitted using DMFit add-in version 3.5 (Baranyi and Roberts, 1994) in Excel (Microsoft Office excel 2019). The most appropriate model was selected considering the determination coefficient or R2.

3. Results

3.1. Jalapeno Pepper’s LAB Antagonistic Activity against L. monocytogenes

Out of 390 LAB isolates from jalapeno pepper collected from Sonora, Mexico, a total of 72 isolates showed antimicrobial activity against L. monocytogenes, evidenced by their clear inhibition zones after the spot-on-lawn assay (Figure 1). The CFSs of these bioactive LAB isolates were freeze-dried for further in vitro inhibition assays.

3.2. In Vitro Assays of CFSs against Pathogens

Freeze-dried CFSs obtained from the 72 LAB isolates with antagonistic effect against L. monocytogenes were tested against different Gram-positive and Gram-negative pathogens. After adjusting the pH to 7 and adding catalase to the media, with the purpose of searching for the nature of the antagonistic compound, a total of 60 CFSs retained their antimicrobial activity against L. monocytogenes and S. aureus with clear inhibition zones, while no antimicrobial activity was detected for S. Typhimurium or E. coli O157:H7 at the evaluated concentration (100 mg/mL). The inhibitory effect of these 60 bioactive CFSs can be attributed to bacteriocin-like inhibitory substances (BLIS), since its antagonistic activity was completely inactivated after treating bioactive CFSs with different proteolytic enzymes (protease, trypsin, and proteinase K). These 60 BLIS were subjected to a thermal evaluation in order to select those able to resist heat, and 16 BLIS retained their antagonistic activity against the Gram-positive bacteria after being treated at sterilization conditions of 121 °C for 15 min.
The MIC values of the 16 heat-resistant BLIS (HR-BLIS) against L. monocytogenes ranged between 80 and >100 mg/mL. Samples from LAB strains 67, 144, 172, and 205 showed the lowest MIC values of 80 mg/mL. The ability of different concentrations of these four samples to inhibit the growth of L. monocytogenes is depicted in Figure 2. Control samples without HR-BLIS showed a normal growth curve for L. monocytogenes with characteristic lag, exponential, and stationary phases. Then, by treating L. monocytogenes with HR-BLIS at 80, 160, or 320 mg/mL, their inhibitory and bactericide effects can be noticed by prolonging the lag phase, preventing the beginning of the exponential phase for over 18 h. Figure 2 also depicts that the growth of L monocytogenes was affected depending on the source of the HR-BLIS, when samples were added at only 40 mg/mL (0.5 MIC). Therefore, growth parameters were determined in order to establish which HR-BLIS was the most effective (Table 2). In general, the estimated maximum growth rate of L. monocytogenes decreased by 83, 78, 61, and 70% when HR-BLIS from LAB strains 67, 144, 172, and 205, respectively, were added at 40 mg/mL. HR-BLIS of strain 67 (HR-BLIS-67) achieved the highest estimated lag phase time of 13 h, in comparison to the other evaluated HR-BLIS, and it was selected for further inhibition assays (MIC and MBC) against other pathogens (S. aureus, S. Typhimurium, and E. coli O157:H7).
MIC and MBC of HR-BLIS-67 for L. monocytogenes, S. aureus, S. Typhimurium, and E. coli O157:H7 are shown in Table 3. Gram-positive bacteria (L. monocytogenes and S. aureus) had an MIC of 80 mg/mL and an MBC of 320 mg/mL to exert a bactericide effect. However, for Gram-negative bacteria (S. Typhimurium and E. coli O157:H7), the MIC required for an inhibitory effect was 150 and 250 mg/mL, and to achieve the MBC, it was 250 and 400 mg/mL for S. Typhimurium and E. coli O157:H7, respectively.

3.3. LAB 16S rRNA Sequencing and Identification

The alignment of genomic sequences allowed the classification of 16 HR-BLIS-producer LAB as members of the genus Enterococcus spp., with high similarity scores to E. lactis, E. faecium, E. durans, and E. faecalis according to the BLAST algorithm. Further, the phylogenetic analysis showed that three isolates (67, 87, and 172) were more related (53%) to the Enterococcus lactis IS05 strain. However, for the remaining LAB isolates (12, 13, 64, 78, 109, 122, 131, 138, 144, 155, 205, 220, and 166), this molecular approach was not enough to discern between E. lactis and E. faecium (Figure 3), which are closely related species with a high 16S rRNA gene sequence identity. Therefore, after analyzing the antibiograms of these LAB strains, samples were categorized into five groups according to their antibiotic susceptibility profiles (Table 4). Samples 64, 12, 144, and 109 have unique profiles, while samples 13, 67, 78, 87, 122, 131, 138, 155, 166, 172, 205, and 220 all share the same profile.

4. Discussion

The analysis performed to identify LAB in jalapeno pepper suggested different Enterococcus spp., specifically E. lactis and E. faecium, as the LAB responsible for producing the antimicrobial compounds (Figure 3). The lack of distinction between these two species might be caused by the similarity in their nucleotide sequences in the analyzed gene (16S rRNA). Currently, there is controversy between the classification of E. faecium and E. lactis species and how to distinguish between these two bacteria. Li and Gu (2021) [27] confirmed that E. faecium and E. lactis were different species. Conversely, other reports found that some E. faecium strains belong, in fact, to E. lactis species [28,29]. Nevertheless, in an earlier study by Morandi et al., [30] Enterococcus lactis was introduced in 2012 after sequencing specific genes and applying biochemical and antibiotic tests. They showed that E. lactis, unlike E. faecium, was susceptible to vancomycin, an important antibiotic effective against a broad spectrum of multi-drug-resistant pathogens. Susceptibility to this antibiotic is an important feature for LAB strains with the potential to be used as biopreservatives. Table 4 shows that, with the exception of strain 109, 15 of the 16 bioactive strains isolated from jalapeno peppers were susceptible to vancomycin; therefore, they could be classified as E. lactis strains. These strains may play a protective role against human foodborne pathogens by competition for space and nutrients and preventing pathogen adhesion due to the production of stable inhibitory compounds, such as bacteriocins [31].
Since the antagonistic effect shown by the CFSs from E. lactis isolated from jalapeño pepper was lost after adding proteases to the media, their CFSs’ bioactivity can be attributed to bacteriocin-like inhibitory substances (BLIS), possibly enterocins [32]. Various studies have shown the ability of Enterococcus strains to produce different kinds of enterocins, including enterocins A, B, and/or P with antagonistic activity against pathogenic bacteria and fungi [33,34]. BLIS from Enterococcus spp., including those isolated from vegetables sources, possess a higher in vitro spectrum activity against pathogenic bacteria (including Gram-negative bacteria) in comparison to BLIS reported from other LAB from other food sources, such as dairy and meat products [35,36].
Thermostability is an important and desirable property for new food biopreservatives intended to be used in the food industry, especially if they are going to be used as part of a hurdle system where a thermal processing is commonly required. A total of 16 CFSs of Enterococcus lactis from jalapeno peppers were identified as HR-BLIS (121 °C for 15 min) since they were able to maintain their ability to inhibit the growth of pathogenic bacteria. This thermoresistant property has also been reported for a few other enterocins (As-48 and Gr17, LD3, and mudticin) produced by E. faecalis, E. hirae, and E. mundtii [37,38] and some E. faecium subspecies isolated from different vegetable sources, such as Chinese pickles [39] and black olives [40].
It is worth pointing out that an MIC of 80 mg/mL of HR-BLIS from selected Enterococcus isolated from jalapeno peppers for L. monocytogenes is significantly lower than previously reported MICs for nisin of 740 or 14,800 mg/mL against L. monocytogenes ATCC 7644 and ATCC 7644K, respectively [41]. The bactericide effect shown by HR-BLIS against Gram-positive bacteria has also been reported for other enterocins [37,42,43]. Enterocins’ function by destabilizing the bacterial cell wall or the cytoplasmatic membrane, causing leakage of intracellular content or by inhibiting gene expression, leading to cell death [44].
The addition of different concentrations of HR-BLIS (equal and above the MIC) into the growth media of L. monocytogenes confirmed their inhibitory effects (Figure 2). Furthermore, evaluating the addition of only 0.5 MIC (40 mg/mL) helped to identify HR-BLIS-67 as the most effective sample since it was more efficient in increasing the lag phase and decreasing the growth of L. monocytogenes in comparison with the other HR-BLIS evaluated. The antagonistic activity of HR-BLIS-67 against Gram-positive bacteria (L. monocytogenes and S. aureus) was expected, since bacteriocins are reported to be more effective against this type of bacteria [45].
Conversely, bactericide and inhibitory effects against Gram-negative bacteria (S. Typhimurium and E. coli O157:H7) (Table 3) have only been reported for a few other bacteriocins from LAB isolated from vegetable sources. However, MICs of 150 and 250 mg/mL of HR-BLIS-67 for Salmonella and E. coli O157:H7, respectively, are considerably higher when compared to those reported for CFS from Pediococcus pentosaceus (CM175) isolated from cantaloupe [1], crude bacteriocin from Lactobacillus plantarum from molasses [13], or L. pentosus DZ35 from pickles [14] where they use concentrations as low as μg/mL to inhibit the growth of Gram-negative bacteria (S. Typhimurium, S. Saintpaul, and/or E. coli O157:H7). Differences found between HR-BLIS-67 and these previous reports regarding their ability to inhibit Gram-negative bacteria may be due to differences of several environmental factors, which may impact their native microbiome [4]. As a response to harsh environmental factors, LAB can generate different antagonistic compounds, including BLIS, which can be more bioactive when compared with the BLIS obtained in the present study from E. lactis. These bioactivity differences enhance the necessity to continue the screening, isolation, and BLIS characterization of LAB from different food sources and to study their antagonistic capabilities against foodborne pathogens.

5. Conclusions

Enterococcus lactis strain 67 isolated from the surface of jalapeno peppers from Sonora, Mexico possess the ability to produce HR-BLIS (possibly enterocin) that exhibit broad-spectrum antagonistic activity against L. monocytogenes, S. aureus, S. Typhimurium, and E. coli O157:H7. Therefore HR-BLIS-67, is a promising natural antimicrobial alternative for the control of foodborne pathogens and promote food safety. Further analysis to purify and characterize HR-BLIS-67 should be performed to elucidate its stability and mechanism of action, as well as to perform in situ assays to confirm that its bioactivity is maintained once used in a food matrix, such as meat.

Author Contributions

Conceptualization, E.A.P.-R. and M.Á.M.-T.; methodology, E.H.-M. and E.A.-H.; software, E.A.-H.; formal analysis, M.V.-M. and E.A.-H.; investigation, E.H.-M.; resources, M.d.l.C.P.-A.; writing—original draft preparation, E.H.-M. and E.A.P.-R.; writing—review and editing, H.G.-R. and V.K.J.; visualization, E.H.-M., E.A.P.-R., M.Á.M.-T., H.G.-R. and E.A.-H.; supervision, E.A.P.-R. and V.K.J.; project administration, E.A.P.-R.; funding acquisition, M.Á.M.-T. and M.d.l.C.P.-A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are available upon request.

Acknowledgments

Author Hernandez-Mendoza wants to acknowledge CONAHCYT for the granted doctoral fellowship.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. González-Pérez, C.J.; Vargas-Arispuro, I.; Aispuro-Hernández, E.; Aguilar-Gil, C.L.; Aguirre-Guzmán, Y.E.; Castillo, A.; Hernández-Mendoza, A.; Ayala-Zavala, J.; Martínez-Téllez, M. Potential Control of Foodborne Pathogenic Bacteria by Pediococcus pentosaceus and Lactobacillus graminis Isolated from Fresh Vegetables. Microbiol. Biotechnol. Lett. 2019, 47, 183–194. [Google Scholar] [CrossRef]
  2. Todorov, S.D.; Prévost, H.; Lebois, M.; Dousset, X.; LeBlanc, J.G.; Franco, B.D.G.M. Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya)—From isolation to application: Characterization of a bacteriocin. Food Res. Int. 2011, 44, 1351–1363. [Google Scholar] [CrossRef]
  3. Sadiq, S.; Imran, M.; Hassan, M.N.; Iqbal, M.; Zafar, Y.; Hafeez, F.Y. Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants. LWT Food Sci. Technol. 2014, 59, 204–210. [Google Scholar] [CrossRef]
  4. Abdelfattah, A.; Freilich, S.; Bartuv, R.; Zhimo, V.Y.; Kumar, A.; Biasi, A.; Salim, S.; Feygenberg, O.; Burchard, E.; Dardick, C.; et al. Global analysis of the apple fruit microbiome: Are all apples the same? Environ. Microbiol. 2021, 23, 6038–6055. [Google Scholar] [CrossRef] [PubMed]
  5. Gao, Z.; Daliri, E.B.-M.; Wang, J.; Liu, D.; Chen, S.; Ye, X.; Ding, T. Inhibitory effect of lactic acid bacteria on foodborne pathogens: A review. J. Food Prot. 2019, 82, 441–453. [Google Scholar] [CrossRef] [PubMed]
  6. Verma, D.K.; Thakur, M.; Singh, S.; Tripathy, S.; Gupta, A.K.; Baranwal, D.; Patel, A.R.; Shah, N.; Utama, G.L.; Niamah, A.K.; et al. Bacteriocins as antimicrobial and preservative agents in food: Biosynthesis, separation and application. Food Biosci. 2022, 46, 101594. [Google Scholar] [CrossRef]
  7. Juturu, V.; Wu, J.C. Microbial production of bacteriocins: Latest research development and applications. Biotechnol. Adv. 2018, 36, 2187–2200. [Google Scholar] [CrossRef] [PubMed]
  8. Khorshidian, N.; Khanniri, E.; Mohammadi, M.; Mortazavian, A.M.; Yousefi, M. Antibacterial Activity of Pediocin and Pediocin-Producing Bacteria Against Listeria monocytogenes in Meat Products. Front. Microbiol. 2021, 12, 709959. [Google Scholar] [CrossRef] [PubMed]
  9. Hernandez-Mendoza, E.; Aida Peña-Ramos, E.; Juneja, V.K.; Valenzuela-Melendres, M.; Susana Scheuren-Acevedo, M.; Osoria, M. Optimizing the effects of nisin and NaCl to thermal inactivate Listeria monocytogenes in ground beef with chipotle sauce during sous-vide processing. J. Food Prot. 2023, 86, 100086. [Google Scholar] [CrossRef]
  10. Zimina, M.; Babich, O.; Prosekov, A.; Sukhikh, S.; Ivanova, S.; Shevchenko, M.; Noskova, S. Overview of Global Trends in Classification, Methods of Preparation and Application of Bacteriocins. Antibiotics 2020, 9, 553. [Google Scholar] [CrossRef]
  11. Simons, A.; Alhanout, K.; Duval, R.E. Bacteriocins, antimicrobial peptides from bacterial origin: Overview of their biology and their impact against multidrug-resistant bacteria. Microorganisms 2020, 8, 639. [Google Scholar] [CrossRef] [PubMed]
  12. Hernández-González, J.C.; Martínez-Tapia, A.; Lazcano-Hernández, G.; García-Pérez, B.E.; Castrejón-Jiménez, N.S. Bacteriocins from Lactic Acid Bacteria. A Powerful Alternative as Antimicrobials, Probiotics, and Immunomodulators in Veterinary Medicine. Animals 2021, 11, 979. [Google Scholar] [CrossRef] [PubMed]
  13. Todorov, S.D.; Dicks, L.M.T. Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria. Enzyme Microb. Technol. 2005, 36, 318–326. [Google Scholar] [CrossRef]
  14. Yi, L.; Qi, T.; Hong, Y.; Deng, L.; Zeng, K. Screening of bacteriocin-producing lactic acid bacteria in Chinese homemade pickle and dry-cured meat, and bacteriocin identification by genome sequencing. LWT Food Sci. Technol. 2020, 125, 109177. [Google Scholar] [CrossRef]
  15. Saludes, M.; Troncoso, M.; Figueroa, G. Presence of Listeria monocytogenes in Chilean food matrices. Food Control 2015, 50, 331–335. [Google Scholar] [CrossRef]
  16. Soto Varela, Z.; Pérez Lavalle, L.; Estrada Alvarado, D. Bacterias causantes de enfermedades transmitidas por alimentos: Una mirada en Colombia. Rev. Salud Uninorte 2016, 32, 105–122. [Google Scholar] [CrossRef]
  17. Contreras-Soto, M.B.; Medrano-Félix, J.A.; Ibarra-Rodríguez, J.R.; Martínez-Urtaza, J.; Chaidez, Q.C.; Castro-del Campo, N. The last 50 years of Salmonella in Mexico: Sources of isolation and factors that influence its prevalence and diversity. Revista Bio Ciencias. 2019, 6, 540. [Google Scholar] [CrossRef]
  18. CDC Foodborne Germs and Illnesses|CDC 2023. Available online: https://www.cdc.gov/foodsafety/foodborne-germs.html (accessed on 8 November 2022).
  19. CDC Foodborne Outbreaks|CDC 2023. Available online: https://www.cdc.gov/foodsafety/outbreaks/index.html (accessed on 8 November 2022).
  20. World Bank. Food-borne Illnesses Cost US$ 110 Billion Per Year in Low- and Middle-Income Countries 2018. Available online: https://www.worldbank.org/en/news/press-release/2018/10/23/food-borne-illnesses-cost-us-110-billion-per-year-in-low-and-middle-income-countries (accessed on 9 November 2022).
  21. Townsend, A.; Strawn, L.K.; Chapman, B.J.; Dunn, L.L. A systematic review of Listeria species and Listeria monocytogenes prevalence, persistence, and diversity throughout the fresh produce supply chain. Foods 2021, 10, 1427. [Google Scholar] [CrossRef] [PubMed]
  22. Hilal Cadi, B.; Citak, S. A Comparison of Two Methods Used for Measuring Antagonistic Activity of Lactic Acid Bacteria. Pak. J. Nutr. 2005, 4, 237–241. [Google Scholar] [CrossRef]
  23. Cruz-Guerrero, A.; Hernández-Sánchez, H.; Rodríguez-Serrano, G.; Gómez-Ruiz, L.; García-Garibay, M.; Figueroa-González, I. Commercial probiotic bacteria and prebiotic carbohydrates: A fundamental study on prebiotics uptake, antimicrobials production and inhibition of pathogens. J. Sci. Food Agr. 2014, 94, 2246–2252. [Google Scholar] [CrossRef]
  24. Sambrock, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning, A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 1989; Volumes 1–3, ISBN 0-87969-309-6. [Google Scholar]
  25. Funke, G.; Funke-Kissling, P. Performance of the New VITEK 2 GP Card for Identification of Medically Relevant Gram-Positive Cocci in a Routine Clinical Laboratory. J. Clin. Microbiol. 2005, 43, 84–88. [Google Scholar] [CrossRef] [PubMed]
  26. Morales-Figueroa, G.G.; Pereo-Vega, G.D.; Reyna-Murrieta, M.E.; Pérez-Morales, R.; López-Mata, M.A.; Sánchez-Escalante, J.J.; Tapia-Rodriguez, M.R.; Ayala-Zavala, J.F.; Juárez, J.; Quihui-Cota, L. Antibacterial and Antioxidant Properties of Extracts of Yucca Baccata, a Plant of Northwestern Mexico, against Pathogenic Bacteria. Biomed. Res. Int. 2022, 2022, 9158836. [Google Scholar] [CrossRef] [PubMed]
  27. Li, Y.Q.; Gu, C.T. Proposal of enterococcus Xinjiangensis Ren et al. 2020 as a later heterotypic synonym of enterococcus Lactis Morandi et al. 2012. Int J Syst Evol Microbiol 2021, 71, 004716. [Google Scholar] [CrossRef] [PubMed]
  28. Belloso Daza, M.V.; Cortimiglia, C.; Bassi, D.; Cocconcelli, P.S. Genome-based studies indicate that the enterococcus faecium clade b strains belong to enterococcus lactis species and lack of the hospital infection associated markers. Int. J. Syst. Evol. Microbiol. 2021, 71, 004948. [Google Scholar] [CrossRef] [PubMed]
  29. Kim, E.; Kim, D.S.; Yang, S.M.; Kim, H.Y. The accurate identification and quantification of six Enterococcus species using quantitative polymerase chain reaction based novel DNA markers. LWT Food Sci. Technol. 2022, 166, 113769. [Google Scholar] [CrossRef]
  30. Morandi, S.; Cremonesi, P.; Povolo, M.; Brasca, M. Enterococcus lactis sp. nov., from Italian raw milk cheeses. Int. J. Syst. Evol. Microbiol. 2012, 62(Pt_8), 1992–1996. [Google Scholar] [CrossRef] [PubMed]
  31. Martín, I.; Rodríguez, A.; Delgado, J.; Córdoba, J.J. Strategies for biocontrol of Listeria monocytogenes using lactic acid bacteria and their metabolites in ready-to-eat meat- and dairy-ripened products. Foods 2022, 11, 542. [Google Scholar] [CrossRef] [PubMed]
  32. Hanchi, H.; Mottawea, W.; Sebei, K.; Hammami, R. The genus Enterococcus: Between probiotic potential and safety concerns-an update. Front. Microbiol. 2018, 9, 1791. [Google Scholar]
  33. Ben Braïek, O.; Morandi, S.; Cremonesi, P.; Smaoui, S.; Hani, K.; Ghrairi, T. Biotechnological potential, probiotic and safety properties of newly isolated enterocin-producing Enterococcus lactis strains. LWT Food Sci. Technol. 2018, 92, 361–370. [Google Scholar] [CrossRef]
  34. Ankaiah, D.; Esakkiraj, P.; Perumal, V.; Ayyanna, R.; Venkatesan, A. Probiotic characterization of Enterococcus faecium por1: Cloning, over expression of Enterocin-A and evaluation of antibacterial, anti-cancer properties. J. Funct. Foods 2017, 38, 280–292. [Google Scholar] [CrossRef]
  35. Da Costa, R.J.; Voloski, F.L.S.; Mondadori, R.G.; Duval, E.H.; Fiorentini, Â.M. Preservation of Meat Products with Bacteriocins Produced by Lactic Acid Bacteria Isolated from Meat. J. Food Qual. 2019, 2019, 4726510. [Google Scholar] [CrossRef]
  36. Gutiérrez-Cortés, C.; Suarez, H.; Buitrago, G.; Nero, L.A.; Todorov, S.D. Characterization of bacteriocins produced by strains of Pediococcus pentosaceus isolated from Minas cheese. Ann. Microbiol. 2018, 68, 383–398. [Google Scholar] [CrossRef]
  37. Kasimin, M.E.; Shamsuddin, S.; Molujin, A.M.; Sabullah, M.K.; Gansau, J.A.; Jawan, R. Enterocin: Promising biopreservative produced by Enterococcus sp. Microorganisms 2022, 10, 684. [Google Scholar] [CrossRef] [PubMed]
  38. Gupta, A.; Tiwari, S.K.; Netrebov, V.; Chikindas, M.L. Biochemical Properties and Mechanism of Action of Enterocin LD3 Purified from Enterococcus hirae LD3. Probiotics Antimicrob. Proteins 2016, 8, 161–169. [Google Scholar] [CrossRef] [PubMed]
  39. Du, R.; Ping, W.; Ge, J. Purification, characterization and mechanism of action of enterocin HDX-2, a novel class IIa bacteriocin produced by Enterococcus faecium HDX-2. LWT Food Sci. Technol. 2022, 153, 112451. [Google Scholar] [CrossRef]
  40. Franz, C.M.A.P.; Schillinger, U.; Holzapfel, W.H. Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives. Int. J. Food Microbiol. 1996, 29, 255–270. [Google Scholar] [CrossRef] [PubMed]
  41. Benkerroum, N.; Sandine, W.E. Inhibitory Action of Nisin Against Listeria monocytogenes. J. Dairy. Sci. 1988, 71, 3237–3245. [Google Scholar] [CrossRef] [PubMed]
  42. Qiao, X.; Du, R.; Wang, Y.; Han, Y.; Zhou, Z. Purification, characterization and mode of action of enterocin, a novel bacteriocin produced by Enterococcus faecium TJUQ1. Int. J. Biol. Macromol. 2020, 144, 151–159. [Google Scholar] [CrossRef] [PubMed]
  43. Line, J.E.; Svetoch, E.A.; Eruslanov, B.V.; Perelygin, V.V.; Mitsevich, E.V.; Mitsevich, I.P.; Levchuk, V.P.; Svetoch, O.E.; Seal, B.S.; Siragusa, G.R.; et al. Isolation and purification of enterocin E-760 with broad antimicrobial activity against Gram-positive and Gram-negative bacteria. Antimicrob. Agents Chemother. 2008, 52, 1094–1100. [Google Scholar] [CrossRef]
  44. Wu, Y.; Pang, X.; Wu, Y.; Liu, X.; Zhang, X. Enterocins: Classification, synthesis, antibacterial mechanisms and food applications. Molecules 2022, 27, 2258. [Google Scholar] [CrossRef]
  45. Pérez-Ramos, A.; Madi-Moussa, D.; Coucheney, F.; Drider, D. Current knowledge of the mode of action and immunity mechanisms of LAB-bacteriocins. Microorganisms 2021, 9, 2107. [Google Scholar] [CrossRef] [PubMed]
Microbiolres 15 00058 g001
Figure 1. Representative plate showing the inhibition zones of LAB strains 87, 109, 122C, 131, 138, and 144.
Figure 1. Representative plate showing the inhibition zones of LAB strains 87, 109, 122C, 131, 138, and 144.
Microbiolres 15 00058 g001
Microbiolres 15 00058 g002
Figure 2. Growth curves of L. monocytogenes with different amounts (40–320 mg/mL) of heat-resistant bacteriocin-like inhibitory substances. Strains (A) 67, (B) 172, (C) 144, and (D) 205, isolated from the surface of jalapeno peppers. Data are expressed as means ± SE (n = 3).
Figure 2. Growth curves of L. monocytogenes with different amounts (40–320 mg/mL) of heat-resistant bacteriocin-like inhibitory substances. Strains (A) 67, (B) 172, (C) 144, and (D) 205, isolated from the surface of jalapeno peppers. Data are expressed as means ± SE (n = 3).
Microbiolres 15 00058 g002
Microbiolres 15 00058 g003
Figure 3. Tree phylogenetic relationship amongst LAB isolated from jalapeno peppers. Maximum likelihood tree based on 16S rRNA gene sequences. Bootstrap support values are displayed at the three nodes, and the branch lengths and scale bar represent the number of substitutions per site. ● LAB isolates from Sonora, Mexico.
Figure 3. Tree phylogenetic relationship amongst LAB isolated from jalapeno peppers. Maximum likelihood tree based on 16S rRNA gene sequences. Bootstrap support values are displayed at the three nodes, and the branch lengths and scale bar represent the number of substitutions per site. ● LAB isolates from Sonora, Mexico.
Microbiolres 15 00058 g003
Table 1. Pathogenic strains used with their designation.
Table 1. Pathogenic strains used with their designation.
StrainsDesignation
Listeria monocytogenesATCC 7644
Staphylococcus aureusATCC 6538
Escherichia coli O157:H7K3999 (FDA/CFSAN)
Salmonella TyphimuriumATCC 14028
Table 2. Kinetic parameters of L. monocytogenes with 40 mg/mL of heat-resistant bacteriocin-like inhibitory substances from Enterococcus spp.
Table 2. Kinetic parameters of L. monocytogenes with 40 mg/mL of heat-resistant bacteriocin-like inhibitory substances from Enterococcus spp.
EnterococcusGrowth Rate (OD/h)Lag (h)R2
670.125612.990.99
1440.16166.260.99
1720.289112.170.99
2050.22126.590.99
L. monocytogenes0.73555.150.99
Table 3. Minimum inhibitory concentration (MIC) and bactericide concentration (MBC) of heat-resistant bacteriocin-like inhibitory substances from strain 67.
Table 3. Minimum inhibitory concentration (MIC) and bactericide concentration (MBC) of heat-resistant bacteriocin-like inhibitory substances from strain 67.
PathogenMIC
(mg/mL)		MBC
(mg/mL)
L. monocytogenes80320
S. aureus80320
S. Typhimurium150250
E. coli O157:H7250400
Table 4. Antibiotic susceptibility profiles of Enterococcus spp. isolated from the surface of jalapeno peppers.
Table 4. Antibiotic susceptibility profiles of Enterococcus spp. isolated from the surface of jalapeno peppers.
EnterococcusAntibiotic
AMPGENSTRCIPLEXERILZDVANDOXTCYTIGNIT
12SSSSSISSIISI
13SSSSSISSSSSI
64SSSSSISSSSSS
67SSSSSISSSSSI
78SSSSSISSSSSI
87SSSSSISSSSSI
109SSSSSIRRRISI
122SSSSSISSSSSI
131SSSSSISSSSSI
138SSSSSISSSSSI
144SRSSSIRSRISI
155SSSSSISSSSSI
166SSSSSISSSSSI
172SSSSSISSSSSI
205SSSSSISSSSSI
220SSSSSISSSSSI
S: Sensitive, I: Intermedium, R: Resistant. AMP: Ampicillin, GEN: Gentamicin, STR: Streptomycin, CIP: Ciprofloxacin, LEX: Levofloxacin, ERI: Erythromycin, LZD: Linezolid, VAN: Vancomycin, DOX: Doxycycline, TCY: Tetracycline, TIG: Tigecycline, NIT: Nitrofurantoin.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Hernandez-Mendoza, E.; Peña-Ramos, E.A.; Juneja, V.K.; Martínez-Téllez, M.Á.; González-Ríos, H.; Paredes-Aguilar, M.d.l.C.; Valenzuela-Melendres, M.; Aispuro-Hernández, E. Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens. Microbiol. Res. 2024, 15, 889-899. https://doi.org/10.3390/microbiolres15020058

AMA Style

Hernandez-Mendoza E, Peña-Ramos EA, Juneja VK, Martínez-Téllez MÁ, González-Ríos H, Paredes-Aguilar MdlC, Valenzuela-Melendres M, Aispuro-Hernández E. Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens. Microbiology Research. 2024; 15(2):889-899. https://doi.org/10.3390/microbiolres15020058

Chicago/Turabian Style

Hernandez-Mendoza, Ezequiel, Etna Aida Peña-Ramos, Vijay K. Juneja, Miguel Ángel Martínez-Téllez, Humberto González-Ríos, María de la Cruz Paredes-Aguilar, Martin Valenzuela-Melendres, and Emmanuel Aispuro-Hernández. 2024. "Antagonistic Activity of Bacteriocin-like Inhibitory Substances from Enterococcus lactis Isolated from the Surface of Jalapeno Pepper against Foodborne Pathogens" Microbiology Research 15, no. 2: 889-899. https://doi.org/10.3390/microbiolres15020058

Article Metrics

Back to TopTop